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Genistein Prevents Development of Spontaneous Ovarian Cancer and Inhibits
Tumor Growth in Hen Model
Kazim Sahin1 Engin Yenice
2 Birdal Bilir
3 Cemal Orhan
1 Mehmet Tuzcu
4 Nurhan Sahin
1
Ibrahim H Ozercan5 Nashwa Kabil
6 Bulent Ozpolat
67and Omer Kucuk
89
1Department of Animal Nutrition Faculty of Veterinary Science Firat University Elazig Turkey
2Poultry Research Institute Ankara Turkey
3Department of Pathology and Laboratory Medicine Emory University Atlanta GA USA
4Division of Biology Faculty of Science Firat University Elazig Turkey
5Department of Pathology Faculty of Medicine Firat University Elazig Turkey
6Department of Experimental Therapeutics Unit 422 The University of Texas MD Anderson
Cancer Center Houston Texas USA
7Center for RNA Interference and Non-Coding RNA The University of Texas MD Anderson
Cancer Center Houston Texas USA
8Department of Hematology and Medical Oncology Emory University Atlanta Georgia USA
9Winship Cancer Institute Emory University Atlanta Georgia USA
Corresponding authors Bulent Ozpolat MD PhD Associate Professor Department of
Experimental Therapeutics Unit 422 The University of Texas MD Anderson Cancer Center 1515
Holcombe Boulevard Houston TX 77030 Phone 713-563-0166 Fax 713-792-0362 E mail
Bozpolatmdandersonorg Omer Kucuk MD Professor of Hematology-Oncology and Urology
Winship Cancer Institute of Emory University1365 Clifton Road NE Room C-2110
Atlanta GA 30322 Phone(404) 778-3460 Fax (404) 778-5520
Running title Genistein prevents development of ovarian cancer
Keywords Genistein ovarian cancer spontaneous treatment chicken hen model
Chemoprevention
Conflict of interest The authors have no conflicts of interest to disclose
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ABSTRACT
Genistein the major isoflavone in soybean has been reported to exert anticancer effects
on various types of cancer including ovarian cancer however its chemopreventive effects and
mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously
developing ovarian cancer models In the present study we demonstrated the preventive effects
and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer
at high incidence rates Laying hens were randomized to three groups control (301 mghen n =
100) or low (5248 mghen n=100) and high genistein supplementation (10626 mghen per day
per group) At the end of 78 weeks hens were euthanized and ovarian tumors were collected and
analyzed We observed that genistein supplementation significantly reduced the ovarian tumor
incidence (p = 0002) as well as the number and size of the tumors (p= 00001) Molecular
analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde
(MDA) a marker for oxidative stress and the expression of NF-κB Bcl-2 and whereas it
upregulated Nrf2 HO-1 and Bax expression at protein level in ovarian tissues Moreover
genistein intake decreased the activity of mTOR pathway as evidenced by reduced
phosphorylation of mTOR p70S6K1 and 4E-BP1 Taken together our findings strongly support
the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of
the genistein on the molecular pathways involved in ovarian tumorigenesis
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INTRODUCTION
Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of
cancer-related mortality among women in the United States An estimated 22280 new cases of
ovarian cancer and 14240 deaths (accounting for 5 of cancer deaths among women) are
expected in the US in 2016 (1) Ovarian cancer is a molecularly and histopathologically
heterogeneous disease associated with risk factors including family history of breast or ovarian
cancer age at diagnosis race and smoking (2-4) The current standard of care for ovarian cancer
involves cytoreductive surgery followed by adjuvant chemotherapy (5) However the lack of
reliable screening tests for detection of ovarian cancer at early stages high rate of recurrence
after surgery and resistance to available chemotherapeutic drugs lead to poor prognosis and high
mortality with an overall 5-year survival rate of 46 (1 6-8) Therefore there is an urgent need
for novel therapeutic agents that target specific molecular defects and have the potential to
prevent ovarian cancer and improve outcomes for ovarian cancer patients
Epidemiological studies have demonstrated that the incidence of ovarian cancer as those
of breast and prostate cancers is much lower in Asian countries where soy foods are consumed
in larger amounts compared to Western countries suggesting the association between high
dietary intake of soy isoflavones and reduced risk of ovarian cancer (9-13) As the major
biologically active isoflavone in the soy diet genistein has been extensively investigated for its
chemopreventive and chemotherapeutic potential in various types of cancer Genistein is a
naturally occurring nonsteroidal plant compound with a structural similarity to the steroid
hormone estradiol (17β-estradiol) that functions as selective estrogen receptor modulators (14-
16) Several studies have shown that genistein inhibits ovarian carcinogenesis through
pleiotropic molecular mechanisms by targeting multiple signaling pathways associated with the
hormonal activity cell cycle apoptosis angiogenesis and metastasis (17-22) In addition
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genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in
ovarian cancer cells (23 17)
Although a number of mouse models for human ovarian cancer have been developed the
non-spontaneous nature of these models and the dissimilarities in the histopathology of ovarian
cancer between mouse and human limit the clinical relevance leading to an inappropriate animal
model to study human ovarian cancer (24) On the other hand the laying hen which is the only
non-human animal that spontaneously develops ovarian cancer with a high prevalence provides
a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25)
The key similarities between the ovarian cancer in the hen model and the one in human include
epidemiological histological and molecular characteristics supporting the laying hen as a
relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset
and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects
of novel agents on the disease (25-30) In the light of these recent findings we investigated the
effects of genistein on spontaneous ovarian cancer using the laying hen model providing further
mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian
cancer
MATERIALS AND METHODS
Animals and experimental design
A total of 300 brown laying hens (104 weeks old ATAK-S hybrid Gallus domesticus)
were used for the study in accordance with animal welfare regulations and under the Guide for
the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station
Turkey (Elazig Turkey) The animal protocol was approved by the Institutional Animal Care
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and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal
diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy
(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of
400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein
contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products
Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in
control low and high genistein groups respectively Animals received genistein 301 5248 and
10626 mghen per day in control low genistein and high genistein groups respectively The
dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient
composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in
batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was
set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the
experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein
and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and
ovaries and surrounding tissues were removed and the morphological and histological changes
were evaluated and compared Tumor incidence and sizes and were measured Tumor types were
determined by histological examination using hematoxylin and eosin staining of tissue sections
Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor
rates were presented in the present study
Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC
until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed
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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
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cancer Cancer Res 2010 704005-4014
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
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cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
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149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
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Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
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tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
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Figures 3-6
ABSTRACT
Genistein the major isoflavone in soybean has been reported to exert anticancer effects
on various types of cancer including ovarian cancer however its chemopreventive effects and
mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously
developing ovarian cancer models In the present study we demonstrated the preventive effects
and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer
at high incidence rates Laying hens were randomized to three groups control (301 mghen n =
100) or low (5248 mghen n=100) and high genistein supplementation (10626 mghen per day
per group) At the end of 78 weeks hens were euthanized and ovarian tumors were collected and
analyzed We observed that genistein supplementation significantly reduced the ovarian tumor
incidence (p = 0002) as well as the number and size of the tumors (p= 00001) Molecular
analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde
(MDA) a marker for oxidative stress and the expression of NF-κB Bcl-2 and whereas it
upregulated Nrf2 HO-1 and Bax expression at protein level in ovarian tissues Moreover
genistein intake decreased the activity of mTOR pathway as evidenced by reduced
phosphorylation of mTOR p70S6K1 and 4E-BP1 Taken together our findings strongly support
the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of
the genistein on the molecular pathways involved in ovarian tumorigenesis
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INTRODUCTION
Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of
cancer-related mortality among women in the United States An estimated 22280 new cases of
ovarian cancer and 14240 deaths (accounting for 5 of cancer deaths among women) are
expected in the US in 2016 (1) Ovarian cancer is a molecularly and histopathologically
heterogeneous disease associated with risk factors including family history of breast or ovarian
cancer age at diagnosis race and smoking (2-4) The current standard of care for ovarian cancer
involves cytoreductive surgery followed by adjuvant chemotherapy (5) However the lack of
reliable screening tests for detection of ovarian cancer at early stages high rate of recurrence
after surgery and resistance to available chemotherapeutic drugs lead to poor prognosis and high
mortality with an overall 5-year survival rate of 46 (1 6-8) Therefore there is an urgent need
for novel therapeutic agents that target specific molecular defects and have the potential to
prevent ovarian cancer and improve outcomes for ovarian cancer patients
Epidemiological studies have demonstrated that the incidence of ovarian cancer as those
of breast and prostate cancers is much lower in Asian countries where soy foods are consumed
in larger amounts compared to Western countries suggesting the association between high
dietary intake of soy isoflavones and reduced risk of ovarian cancer (9-13) As the major
biologically active isoflavone in the soy diet genistein has been extensively investigated for its
chemopreventive and chemotherapeutic potential in various types of cancer Genistein is a
naturally occurring nonsteroidal plant compound with a structural similarity to the steroid
hormone estradiol (17β-estradiol) that functions as selective estrogen receptor modulators (14-
16) Several studies have shown that genistein inhibits ovarian carcinogenesis through
pleiotropic molecular mechanisms by targeting multiple signaling pathways associated with the
hormonal activity cell cycle apoptosis angiogenesis and metastasis (17-22) In addition
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genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in
ovarian cancer cells (23 17)
Although a number of mouse models for human ovarian cancer have been developed the
non-spontaneous nature of these models and the dissimilarities in the histopathology of ovarian
cancer between mouse and human limit the clinical relevance leading to an inappropriate animal
model to study human ovarian cancer (24) On the other hand the laying hen which is the only
non-human animal that spontaneously develops ovarian cancer with a high prevalence provides
a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25)
The key similarities between the ovarian cancer in the hen model and the one in human include
epidemiological histological and molecular characteristics supporting the laying hen as a
relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset
and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects
of novel agents on the disease (25-30) In the light of these recent findings we investigated the
effects of genistein on spontaneous ovarian cancer using the laying hen model providing further
mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian
cancer
MATERIALS AND METHODS
Animals and experimental design
A total of 300 brown laying hens (104 weeks old ATAK-S hybrid Gallus domesticus)
were used for the study in accordance with animal welfare regulations and under the Guide for
the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station
Turkey (Elazig Turkey) The animal protocol was approved by the Institutional Animal Care
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and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal
diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy
(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of
400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein
contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products
Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in
control low and high genistein groups respectively Animals received genistein 301 5248 and
10626 mghen per day in control low genistein and high genistein groups respectively The
dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient
composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in
batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was
set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the
experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein
and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and
ovaries and surrounding tissues were removed and the morphological and histological changes
were evaluated and compared Tumor incidence and sizes and were measured Tumor types were
determined by histological examination using hematoxylin and eosin staining of tissue sections
Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor
rates were presented in the present study
Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC
until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed
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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
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45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
INTRODUCTION
Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of
cancer-related mortality among women in the United States An estimated 22280 new cases of
ovarian cancer and 14240 deaths (accounting for 5 of cancer deaths among women) are
expected in the US in 2016 (1) Ovarian cancer is a molecularly and histopathologically
heterogeneous disease associated with risk factors including family history of breast or ovarian
cancer age at diagnosis race and smoking (2-4) The current standard of care for ovarian cancer
involves cytoreductive surgery followed by adjuvant chemotherapy (5) However the lack of
reliable screening tests for detection of ovarian cancer at early stages high rate of recurrence
after surgery and resistance to available chemotherapeutic drugs lead to poor prognosis and high
mortality with an overall 5-year survival rate of 46 (1 6-8) Therefore there is an urgent need
for novel therapeutic agents that target specific molecular defects and have the potential to
prevent ovarian cancer and improve outcomes for ovarian cancer patients
Epidemiological studies have demonstrated that the incidence of ovarian cancer as those
of breast and prostate cancers is much lower in Asian countries where soy foods are consumed
in larger amounts compared to Western countries suggesting the association between high
dietary intake of soy isoflavones and reduced risk of ovarian cancer (9-13) As the major
biologically active isoflavone in the soy diet genistein has been extensively investigated for its
chemopreventive and chemotherapeutic potential in various types of cancer Genistein is a
naturally occurring nonsteroidal plant compound with a structural similarity to the steroid
hormone estradiol (17β-estradiol) that functions as selective estrogen receptor modulators (14-
16) Several studies have shown that genistein inhibits ovarian carcinogenesis through
pleiotropic molecular mechanisms by targeting multiple signaling pathways associated with the
hormonal activity cell cycle apoptosis angiogenesis and metastasis (17-22) In addition
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genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in
ovarian cancer cells (23 17)
Although a number of mouse models for human ovarian cancer have been developed the
non-spontaneous nature of these models and the dissimilarities in the histopathology of ovarian
cancer between mouse and human limit the clinical relevance leading to an inappropriate animal
model to study human ovarian cancer (24) On the other hand the laying hen which is the only
non-human animal that spontaneously develops ovarian cancer with a high prevalence provides
a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25)
The key similarities between the ovarian cancer in the hen model and the one in human include
epidemiological histological and molecular characteristics supporting the laying hen as a
relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset
and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects
of novel agents on the disease (25-30) In the light of these recent findings we investigated the
effects of genistein on spontaneous ovarian cancer using the laying hen model providing further
mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian
cancer
MATERIALS AND METHODS
Animals and experimental design
A total of 300 brown laying hens (104 weeks old ATAK-S hybrid Gallus domesticus)
were used for the study in accordance with animal welfare regulations and under the Guide for
the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station
Turkey (Elazig Turkey) The animal protocol was approved by the Institutional Animal Care
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and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal
diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy
(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of
400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein
contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products
Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in
control low and high genistein groups respectively Animals received genistein 301 5248 and
10626 mghen per day in control low genistein and high genistein groups respectively The
dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient
composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in
batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was
set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the
experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein
and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and
ovaries and surrounding tissues were removed and the morphological and histological changes
were evaluated and compared Tumor incidence and sizes and were measured Tumor types were
determined by histological examination using hematoxylin and eosin staining of tissue sections
Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor
rates were presented in the present study
Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC
until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed
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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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Figure 4 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in
ovarian cancer cells (23 17)
Although a number of mouse models for human ovarian cancer have been developed the
non-spontaneous nature of these models and the dissimilarities in the histopathology of ovarian
cancer between mouse and human limit the clinical relevance leading to an inappropriate animal
model to study human ovarian cancer (24) On the other hand the laying hen which is the only
non-human animal that spontaneously develops ovarian cancer with a high prevalence provides
a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25)
The key similarities between the ovarian cancer in the hen model and the one in human include
epidemiological histological and molecular characteristics supporting the laying hen as a
relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset
and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects
of novel agents on the disease (25-30) In the light of these recent findings we investigated the
effects of genistein on spontaneous ovarian cancer using the laying hen model providing further
mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian
cancer
MATERIALS AND METHODS
Animals and experimental design
A total of 300 brown laying hens (104 weeks old ATAK-S hybrid Gallus domesticus)
were used for the study in accordance with animal welfare regulations and under the Guide for
the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station
Turkey (Elazig Turkey) The animal protocol was approved by the Institutional Animal Care
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and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal
diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy
(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of
400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein
contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products
Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in
control low and high genistein groups respectively Animals received genistein 301 5248 and
10626 mghen per day in control low genistein and high genistein groups respectively The
dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient
composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in
batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was
set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the
experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein
and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and
ovaries and surrounding tissues were removed and the morphological and histological changes
were evaluated and compared Tumor incidence and sizes and were measured Tumor types were
determined by histological examination using hematoxylin and eosin staining of tissue sections
Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor
rates were presented in the present study
Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC
until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed
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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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cancer Cancer Res 2010 704005-4014
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
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149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
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in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
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Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
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epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
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with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
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Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal
diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy
(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of
400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein
contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products
Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in
control low and high genistein groups respectively Animals received genistein 301 5248 and
10626 mghen per day in control low genistein and high genistein groups respectively The
dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient
composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in
batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was
set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the
experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks
Sample collection
Blood samples were collected at the end of the study from the hens via the axillary vein
and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and
ovaries and surrounding tissues were removed and the morphological and histological changes
were evaluated and compared Tumor incidence and sizes and were measured Tumor types were
determined by histological examination using hematoxylin and eosin staining of tissue sections
Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor
rates were presented in the present study
Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC
until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed
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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)
were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based
on the histopathology classification system listed in Table 1 (26 33 34)
Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)
At the end of the study blood samples were collected from 12 birds randomly chosen
from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were
collected Sera samples were kept on the ice and protected from light until they were processed
to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until
analysis Serum genistein and daidzein concentrations were measured by high-performance
liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10
AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein
and daidzein) levels were measured by the method of our previous study (35) To 200 microl of
serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)
in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of
sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated
with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s
sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The
supernatants were evaporated We then dissolved the sample with 80 methanol at the same
volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear
gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by
vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)
Chemical analyses of the diet samples were performed using procedures of Association of
Official Analytical Chemists (36)
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Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
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cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
Detection of serum malondialdehyde concentrations using HPLC
Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were
measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-
visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and
CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)
were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-
tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection
volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +
175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm
Western blot analysis
Western blot analysis was performed as described previously (38) Proteins were
extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl
buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M
soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent
centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were
mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of
protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)
electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell
BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered
saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior
to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-
were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were
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striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
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301 5248 10626
0
1
2
3
4a
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c
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Genistein levels mghen per day
Ova
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mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
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a
b
c
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-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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301 5248 10626
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c
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a
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-actin
HO-1
Genistein levels mghen per day
HO
-1 p
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f co
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Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
striped and used blotted for with other antibodies All antibodies were purchased from Abcam
(Cambridge UK)
The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western
blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen
peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)
Samples were analyzed in quadruplicate under each experimental condition and protein levels
were measured densitometrically using the image analysis software program ImageJ (National
Institutes of Health)
Statistical analysis Tumor incidences in the control and experimental groups were
evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the
general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects
of genistein supplementation on tumor size protein expressions and serum metabolites When a
significant F statistic (P le 005) in the analysis of variance was noted the least squares mean
procedure was performed to separate means that were significantly different (P lt 005) Linear
and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three
dosages of genistein administered to the animals for serum metabolites
RESULTS
Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens
To investigate the effects of genistein supplementation on the development of
spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to
three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein
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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
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48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
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hen Gynecol Oncol 2009 112166-170
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pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
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cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
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Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary
Figure 1) At the end of 78 weeks administration of genistein the study was terminated and
necropsy was performed for the examination of gross pathology and microscopy of the tumors
(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of
spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed
ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly
decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control
group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the
histopathological assessment of the tumors two subtypes of ovarian cancer including serous and
mucinous carcinomas were observed in these hens representative images are illustrated in Figure
2B-C The histopathology grading system based on mitotic developments and cellular
differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence
of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the
control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them
had mucinous carcinoma However there was no significant difference in the incidence of each
subtype of ovarian tumors between control and treatment groups (Table 2)
Additionally genistein treatment significantly reduced both the number and size of
ovarian tumors compared to the control group (Table 2) Hens in the control group had an
average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high
genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors
were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)
At the end of 78 weeks administration of genistein we also observed that overall
survival rates in genistein groups were higher compared to that of control group although the
differences were not statistically significant (Table 2) In control group 83 of the hens stayed
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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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analysis Nucleic Acids Res 2010 385718-5734
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2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and
HG groups respectively
Genistein supplementation results in enhanced genistein levels in serum and reduced MDA
in the ovary of laying hens
To demonstrate that the lower ovarian cancer incidence in the treatment groups was
specifically due to the genistein intervention we measured serum levels of genistein and
daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or
high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and
35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and
the increase in the level of serum genistein was dose-dependent However genistein
supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure
3B)
Several studies have shown that oxidative stress is involved in a wide variety of cancers
including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the
context of oxidative stress we analyzed the levels of MDA which is a widely used marker of
oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed
that treatment of hens with genistein significantly and dose-dependently decreased the level of
MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the
control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed
groups respectively These results confirm that genistein treatment could ameliorate oxidative
stress in the hen ovary
Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of
Bax in the ovary of laying hens
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To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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Figure 4 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
To characterize the molecular mechanisms underlying the genistein-induced changes in
the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear
factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival
pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high
genistein treatments significantly reduced the protein expression level of NF-κB indicating that
genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In
addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are
transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed
that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading
to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis
Genistein suppresses the mTOR pathway in the ovary of laying hens
To further explore the mechanisms involved in the genistein-induced changes in the
ovarian carcinogenesis we studied the effects of genistein on the mammalian target of
rapamycin (mTOR) survival signaling which has also been shown to be associated with
oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that
phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low
or high genistein significantly decreased the levels of phosphorylated proteins of mTOR
p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data
demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in
ovarian cancer cells
Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens
It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is
one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to
Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As
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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
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66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
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67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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Article File
Figure 1
Figure 2
Figures 3-6
illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the
genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary
by activating the Nrf2-induced cellular stress responses
DISCUSSION
Although surgical and chemotherapeutic interventions have improved the overall survival
rates effective treatment of ovarian cancer is limited due to the major challenges such as
clinicopathological and genetic heterogeneity lack of early detection strategies for the disease
tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore
chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents
provides a rational approach to reduce the incidence and mortality rates of ovarian cancer
Epidemiological studies have shown that the dietary intake of soybean is associated with reduced
risks of various types of cancer including ovarian cancer (46) Genistein the most abundant
isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer
(17-23) Although numerous epidemiological and in vitro studies have demonstrated that
genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a
lack of well-characterized studies that address the efficacy and mechanisms of action of genistein
in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present
study therefore we utilized the laying hen model to prospectively test the chemopreventive
effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the
molecular mechanisms underlying the actions of genistein on the initiation and progression of
ovarian cancer
Laying hens have been shown to develop spontaneous ovarian cancer at a high rate
providing an appropriate natural experimental model of human ovarian cancer (25) The fact that
laying hens and humans share some similarities in reproductive physiology and hens have also
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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
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47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
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proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
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63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
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2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Figure 4 Cancer Research
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301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-
induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological
classification of ovarian tumors based on the tumor stage and grade have indicated that similar to
humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are
observed in hens (26) Recent studies have reported that several biomarkers of human ovarian
cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth
factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear
Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been
identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in
the laying hen model recapitulates the etiology and disease progression in human hens have
been previously used in studies testing the effects of chemopreventive agents such as oral
contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)
To the best of our knowledge the present study is the first study to investigate the
chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot
study we conducted a three-armed randomized controlled trial to assess the effects of genistein
intervention on the incidence of spontaneous ovarian cancer in laying hens Our data
demonstrated that genistein significantly and dose-dependently reduced the incidence rate of
ovarian cancer consistent with previously published epidemiological and in vitro findings In
addition we observed an increased survival rate in genistein-treated animals although the effect
was not significant Histological analysis of the ovarian tumors revealed that two subtypes of
ovarian cancer serous and mucinous carcinomas were observed in these hens However the
prevalence of different subtypes did not significantly vary between control and genistein-treated
hens Genistein intake also significantly decreased the number and size of ovarian tumors in
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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
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1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
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44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
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Article File
Figure 1
Figure 2
Figures 3-6
hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis
of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing
genistein whereas there was no change in the serum daidzein levels of these animals confirming
that the tumor inhibitory effect has been linked specifically to genistein
It is well documented that tumor initiation and progression in the ovary has been
associated with chronic inflammation which is activated by oxidative stress (52) Therefore we
first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the
control and genistein-fed animals Our results showed that genistein supplementation resulted in
a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein
exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways
Based on this finding we hypothesized that genistein could ameliorate oxidative stress and
inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways
which are involved in the pathogenesis of ovarian cancer
NF-κB signaling which is a critical molecular link between inflammation and cancer is
known to regulate key processes in several malignancies including ovarian cancer (53-55)
Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian
cancer patients and is associated with growth and progression of ovarian tumors (56-58)
Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-
κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation
adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment
including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the
suppression of inflammatory processes Our data showed that genistein supplementation
significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at
the protein level resulting in the induction of apoptosis
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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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2016951088-95
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1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
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44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
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45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
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56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
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2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
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Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
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Article File
Figure 1
Figure 2
Figures 3-6
It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in
ovarian cancer (59) It is a complex signaling network transducing signals from various growth
factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G
protein-coupled receptors into intracellular messages by generating phospholipids which
activate downstream effectors including AKT and mTOR via phosphorylation (42) Once
activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6
kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)
resulting in increased translation of target genes involved in cell cycle cell survival metabolism
motility angiogenesis chemoresistance and genomic instability (60) In our study we observed
that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K
and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting
that genistein may have the potential to enhance the efficacy of therapeutic agents that target
PI3KAKTmTOR signaling cascade in ovarian cancer cells
Nrf2 signaling pathway has been shown as one of the major defense mechanisms to
protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the
cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-
associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent
proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of
Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation
allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements
(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant
activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are
associated with poor prognosis in patients with ovarian cancer (65) Recent studies have
demonstrated that not only healthy cells but also various cancer cells including ovarian tumors
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer
cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
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autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway
Neurosci Lett 2009 461131-135
42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
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culture models Antioxid Redox Signal 2005 780-91
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KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
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Article File
Figure 1
Figure 2
Figures 3-6
can protect themselves against oxidative stress by activating the transcription factor Nrf2 the
master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)
Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer
prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)
Our results showed that genistein intervention significantly and dose-dependently increased the
expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the
involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer
In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate
that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by
modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support
and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a
biologically relevant in vivo model providing a strong rationale for clinical studies to assess the
protective effects of genistein which may ultimately lead to better clinical outcomes and
improved overall survival rates for patients diagnosed with ovarian cancer
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40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
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42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
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43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
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44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
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30 Rodriguez-Burford C Barnes MN Berry W Partridge EE Grizzle WE
Immunohistochemical expression of molecular markers in an avian model a potential model for
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81373-379
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32 Rasouli E Jahanian R Improved performance and immunological responses as the result of
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34Mocka EH Stern RA Fletcher OJ Anderson KE Petitte JN Mozdziak PE Chemoprevention
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isoflavones ameliorate the adverse effects of chemotherapy in children Nutr Cancer
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Arlington VA Association of Official Analytical Chemists 1990
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human serum by HPLCUV LC-GC North America 2004 22 362ndash365
38 Sahin K Orhan C Tuzcu M Sahin N Hayirli A Bilgili S Kucuk O Lycopene activates
antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
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39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
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cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
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42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
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45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
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48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
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hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
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54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
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SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
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57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
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58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
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59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
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2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
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Article File
Figure 1
Figure 2
Figures 3-6
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10 Myung SK Ju W Choi HJ Kim SC Soy intake and risk of endocrine-related
gynaecological cancer a meta-analysis British Journal of Obstetrics and Gynaecology 2009
1161697-1705
11 Rossi M Negri E Lagiou P Talamini R Dal Maso L Montella M Franceschi S La
Vecchia C Flavonoids and ovarian cancer risk A case-control study in Italy International
Journal of Cancer 2008 123895-898
12 Chang ET Lee VS Canchola AJ Clarke CA Purdie DM Reynolds P Anton-Culver
H Bernstein L Deapen D Peel D Pinder R Ross RK Stram DO West DW Wright W
Ziogas A Horn-Ross PL Diet and risk of ovarian cancer in the California Teachers Study
cohort American Journal of Epidemiology 2007 165802-813
13 Bandera EV King M Chandran U Paddock LE Rodriguez-Rodriguez L Olson SH
Phytoestrogen consumption from foods and supplements and epithelial ovarian cancer risk
apopulation-based case control study BMC Womens Health 2011 1140
14 Takimoto CH Glover K Huang X Hayes SA Gallot L Quinn M Jovanovic BD Shapiro A
Hernandez L Goetz A Llorens V Lieberman R Crowell JA Poisson BA Bergan RC Phase I
pharmacokinetic and pharmacodynamic analysis of unconjugated soy isoflavones administered
to individuals with cancer Cancer Epidemiol Biomarkers Prev 2003 12 1213-1221
15 Yildiz F Phytoestrogens in functional foods CRC Press Boca Raton FL 2005
16 Pike AC Brzozowski AM Hubbard RE Bonn T Thorsell AG Engstrom O
Ljunggren
J Gustafsson JA Carlquist M Structure of the ligand-binding domain of oestrogen receptor
beta
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
in the presence of a partial agonist and a full antagonist EMBO Journal 1999 184608-4618
17 Chen X Anderson JJ Isoflavones inhibit proliferation of ovarian cancer cells in vitro via an
estrogen receptor-dependent pathway Nutr Cancer 2001 41165e71
18 Novak-Hofer I Cohrs S Grunberg J Friedli A Schlatter MC Pfeifer M Altevogt P
Schubiger PA Antibodies directed against L1-CAM synergize with Genistein in inhibiting
growth and survival pathways in SKOV3ip human ovarian cancer cells Cancer Lett 2008
261193-204
19 Choi EJ Kim T Lee MS Pro-apoptotic effect and cytotoxicity of genistein and genistin in
human ovarian cancer SK- OV-3 cells Life Science 2007 801403-1408
20 Ouyang G Yao L Ruan K Song G Mao Y Bao S Genistein induces G2M cell cycle
arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint
pathways Cell Biology International 2009 331237-1244
21 Gossner G Choi M Tan L Fogoros S Griffith KA Kuenker M Liu JR Genistein
induced apoptosis and autophagocytosis in ovarian cancer cells Gynecologic Oncology
2007 10523-30
22 Luo H Jiang BH King SM Chen YC Inhibition of cell growth and VEGF expression in
ovarian cancer cells by flavonoids Nutrition and Cancer 2008 60800-809
23 Rucinska A Kirko S Gabryelak T Effect of the phytoestrogen genistein-8-C-glucoside on
Chinese hamster ovary cells in vitro Cell Biology International 2007 311371-1378
24 Stakleff KD Von Gruenigen VE Rodent models for ovarian cancer research Int J Gynecol
Cancer 2003 13405-12
25 Fredrickson TN Ovarian tumors of the hen Environ Health Perspect 1987 7335-51
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
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26 Barua A Bitterman P Abramowicz JS Dirks AL Bahr JM Hales DB Bradaric MJ
Edassery SL Rotmensch J Luborsky JL Histopathology of ovarian tumors in laying hens a
preclinical model of human ovarian cancer Int J Gynecol Cancer 2009 19531-539
27 International Chicken Genome Sequencing C Sequence and comparative analysis of the
chicken genome provide unique perspectives on vertebrate evolution Nature 2004 432695-716
28 Jackson E Anderson K Ashwell C Petitte J Mozdziak PE CA125 expression in
spontaneous ovarian adenocarcinomas from laying hens Gynecol Oncol 2007 104192-198
29 Hakim AA Barry CP Barnes HJ Anderson KE Petitte J Whitaker R Lancaster JM
Wenham RM Carver DK Turbov J Berchuck A Kopelovich L Rodriguez GC Ovarian
adenocarcinomas in the laying hen and women share similar alterations in p53 ras and HER-
2neu Cancer Prev Res 2009 2114-121
30 Rodriguez-Burford C Barnes MN Berry W Partridge EE Grizzle WE
Immunohistochemical expression of molecular markers in an avian model a potential model for
preclinical evaluation of agents for ovarian cancer chemoprevention Gynecol Oncol 2001
81373-379
31 Akdemir F Sahin K Genistein supplementation to the quail effects on egg production and
32 Rasouli E Jahanian R Improved performance and immunological responses as the result of
dietary genistein supplementation of broiler chicks Animal 201591473-1480
33 Harris EA Fletcher OJ Anderson KE Petitte JN Kopelovich L Mozdziak PE Epithelial
cell tumors of the hen reproductive tract Avian Dis 20145895-101
34Mocka EH Stern RA Fletcher OJ Anderson KE Petitte JN Mozdziak PE Chemoprevention
of spontaneous ovarian cancer in the domestic hen Poult Sci 2017 961901-1909
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35 Tacyildiz N Ozyoruk D Yavuz G Unal E Dincaslan H Dogu F Sahin K Kucuk O Soy
isoflavones ameliorate the adverse effects of chemotherapy in children Nutr Cancer
2010621001-1005
36 AOC Association of Official Analytical Chemists Official Methods of Analysis 15th ed
Arlington VA Association of Official Analytical Chemists 1990
37 Karatepe M Simultaneous determination of ascorbic acid and free malondialdehyde in
human serum by HPLCUV LC-GC North America 2004 22 362ndash365
38 Sahin K Orhan C Tuzcu M Sahin N Hayirli A Bilgili S Kucuk O Lycopene activates
antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer
cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
41 Byun YJ Kim SK Kim YM Chae GT Jeong SW Lee SB Hydrogen peroxide induces
autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway
Neurosci Lett 2009 461131-135
42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
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44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
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55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
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Figure 1 Cancer Research
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Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
Subscriptions
Reprints and
pubsaacrorgDepartment at
To order reprints of this article or to subscribe to the journal contact the AACR Publications
Permissions
Rightslink site Click on Request Permissions which will take you to the Copyright Clearance Centers (CCC)
89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
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Article File
Figure 1
Figure 2
Figures 3-6
in the presence of a partial agonist and a full antagonist EMBO Journal 1999 184608-4618
17 Chen X Anderson JJ Isoflavones inhibit proliferation of ovarian cancer cells in vitro via an
estrogen receptor-dependent pathway Nutr Cancer 2001 41165e71
18 Novak-Hofer I Cohrs S Grunberg J Friedli A Schlatter MC Pfeifer M Altevogt P
Schubiger PA Antibodies directed against L1-CAM synergize with Genistein in inhibiting
growth and survival pathways in SKOV3ip human ovarian cancer cells Cancer Lett 2008
261193-204
19 Choi EJ Kim T Lee MS Pro-apoptotic effect and cytotoxicity of genistein and genistin in
human ovarian cancer SK- OV-3 cells Life Science 2007 801403-1408
20 Ouyang G Yao L Ruan K Song G Mao Y Bao S Genistein induces G2M cell cycle
arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint
pathways Cell Biology International 2009 331237-1244
21 Gossner G Choi M Tan L Fogoros S Griffith KA Kuenker M Liu JR Genistein
induced apoptosis and autophagocytosis in ovarian cancer cells Gynecologic Oncology
2007 10523-30
22 Luo H Jiang BH King SM Chen YC Inhibition of cell growth and VEGF expression in
ovarian cancer cells by flavonoids Nutrition and Cancer 2008 60800-809
23 Rucinska A Kirko S Gabryelak T Effect of the phytoestrogen genistein-8-C-glucoside on
Chinese hamster ovary cells in vitro Cell Biology International 2007 311371-1378
24 Stakleff KD Von Gruenigen VE Rodent models for ovarian cancer research Int J Gynecol
Cancer 2003 13405-12
25 Fredrickson TN Ovarian tumors of the hen Environ Health Perspect 1987 7335-51
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
26 Barua A Bitterman P Abramowicz JS Dirks AL Bahr JM Hales DB Bradaric MJ
Edassery SL Rotmensch J Luborsky JL Histopathology of ovarian tumors in laying hens a
preclinical model of human ovarian cancer Int J Gynecol Cancer 2009 19531-539
27 International Chicken Genome Sequencing C Sequence and comparative analysis of the
chicken genome provide unique perspectives on vertebrate evolution Nature 2004 432695-716
28 Jackson E Anderson K Ashwell C Petitte J Mozdziak PE CA125 expression in
spontaneous ovarian adenocarcinomas from laying hens Gynecol Oncol 2007 104192-198
29 Hakim AA Barry CP Barnes HJ Anderson KE Petitte J Whitaker R Lancaster JM
Wenham RM Carver DK Turbov J Berchuck A Kopelovich L Rodriguez GC Ovarian
adenocarcinomas in the laying hen and women share similar alterations in p53 ras and HER-
2neu Cancer Prev Res 2009 2114-121
30 Rodriguez-Burford C Barnes MN Berry W Partridge EE Grizzle WE
Immunohistochemical expression of molecular markers in an avian model a potential model for
preclinical evaluation of agents for ovarian cancer chemoprevention Gynecol Oncol 2001
81373-379
31 Akdemir F Sahin K Genistein supplementation to the quail effects on egg production and
32 Rasouli E Jahanian R Improved performance and immunological responses as the result of
dietary genistein supplementation of broiler chicks Animal 201591473-1480
33 Harris EA Fletcher OJ Anderson KE Petitte JN Kopelovich L Mozdziak PE Epithelial
cell tumors of the hen reproductive tract Avian Dis 20145895-101
34Mocka EH Stern RA Fletcher OJ Anderson KE Petitte JN Mozdziak PE Chemoprevention
of spontaneous ovarian cancer in the domestic hen Poult Sci 2017 961901-1909
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
35 Tacyildiz N Ozyoruk D Yavuz G Unal E Dincaslan H Dogu F Sahin K Kucuk O Soy
isoflavones ameliorate the adverse effects of chemotherapy in children Nutr Cancer
2010621001-1005
36 AOC Association of Official Analytical Chemists Official Methods of Analysis 15th ed
Arlington VA Association of Official Analytical Chemists 1990
37 Karatepe M Simultaneous determination of ascorbic acid and free malondialdehyde in
human serum by HPLCUV LC-GC North America 2004 22 362ndash365
38 Sahin K Orhan C Tuzcu M Sahin N Hayirli A Bilgili S Kucuk O Lycopene activates
antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer
cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
41 Byun YJ Kim SK Kim YM Chae GT Jeong SW Lee SB Hydrogen peroxide induces
autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway
Neurosci Lett 2009 461131-135
42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
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Article File
Figure 1
Figure 2
Figures 3-6
26 Barua A Bitterman P Abramowicz JS Dirks AL Bahr JM Hales DB Bradaric MJ
Edassery SL Rotmensch J Luborsky JL Histopathology of ovarian tumors in laying hens a
preclinical model of human ovarian cancer Int J Gynecol Cancer 2009 19531-539
27 International Chicken Genome Sequencing C Sequence and comparative analysis of the
chicken genome provide unique perspectives on vertebrate evolution Nature 2004 432695-716
28 Jackson E Anderson K Ashwell C Petitte J Mozdziak PE CA125 expression in
spontaneous ovarian adenocarcinomas from laying hens Gynecol Oncol 2007 104192-198
29 Hakim AA Barry CP Barnes HJ Anderson KE Petitte J Whitaker R Lancaster JM
Wenham RM Carver DK Turbov J Berchuck A Kopelovich L Rodriguez GC Ovarian
adenocarcinomas in the laying hen and women share similar alterations in p53 ras and HER-
2neu Cancer Prev Res 2009 2114-121
30 Rodriguez-Burford C Barnes MN Berry W Partridge EE Grizzle WE
Immunohistochemical expression of molecular markers in an avian model a potential model for
preclinical evaluation of agents for ovarian cancer chemoprevention Gynecol Oncol 2001
81373-379
31 Akdemir F Sahin K Genistein supplementation to the quail effects on egg production and
32 Rasouli E Jahanian R Improved performance and immunological responses as the result of
dietary genistein supplementation of broiler chicks Animal 201591473-1480
33 Harris EA Fletcher OJ Anderson KE Petitte JN Kopelovich L Mozdziak PE Epithelial
cell tumors of the hen reproductive tract Avian Dis 20145895-101
34Mocka EH Stern RA Fletcher OJ Anderson KE Petitte JN Mozdziak PE Chemoprevention
of spontaneous ovarian cancer in the domestic hen Poult Sci 2017 961901-1909
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
35 Tacyildiz N Ozyoruk D Yavuz G Unal E Dincaslan H Dogu F Sahin K Kucuk O Soy
isoflavones ameliorate the adverse effects of chemotherapy in children Nutr Cancer
2010621001-1005
36 AOC Association of Official Analytical Chemists Official Methods of Analysis 15th ed
Arlington VA Association of Official Analytical Chemists 1990
37 Karatepe M Simultaneous determination of ascorbic acid and free malondialdehyde in
human serum by HPLCUV LC-GC North America 2004 22 362ndash365
38 Sahin K Orhan C Tuzcu M Sahin N Hayirli A Bilgili S Kucuk O Lycopene activates
antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer
cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
41 Byun YJ Kim SK Kim YM Chae GT Jeong SW Lee SB Hydrogen peroxide induces
autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway
Neurosci Lett 2009 461131-135
42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
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FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
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Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
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Manuscript
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Article File
Figure 1
Figure 2
Figures 3-6
35 Tacyildiz N Ozyoruk D Yavuz G Unal E Dincaslan H Dogu F Sahin K Kucuk O Soy
isoflavones ameliorate the adverse effects of chemotherapy in children Nutr Cancer
2010621001-1005
36 AOC Association of Official Analytical Chemists Official Methods of Analysis 15th ed
Arlington VA Association of Official Analytical Chemists 1990
37 Karatepe M Simultaneous determination of ascorbic acid and free malondialdehyde in
human serum by HPLCUV LC-GC North America 2004 22 362ndash365
38 Sahin K Orhan C Tuzcu M Sahin N Hayirli A Bilgili S Kucuk O Lycopene activates
antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers Poult Sci
2016951088-95
39 Chan DW Liu VW Tsao GS Yao KM Furukawa T Chan KK Ngan HY Loss of MKP3
mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer
cells Carcinogenesis 2008 291742-1750
40 Gloire G Legrand-Poels S Piette J NF-kappaB activation by reactive oxygen species
fifteen years later Biochem Pharmacol 2006 721493-1505
41 Byun YJ Kim SK Kim YM Chae GT Jeong SW Lee SB Hydrogen peroxide induces
autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway
Neurosci Lett 2009 461131-135
42 Hay N and Sonenberg N Upstream and downstream of mTOR Genes Dev 2004 181926-
1945
43 Itoh K Chiba T Takahashi S Ishii T Igarashi K Katoh Y Oyake T Hayashi N Satoh K
Hatayama I Yamamoto M Nabeshima Y An Nrf2small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements Biochem
Biophys Res Commun 1997 236313-322
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
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Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
Subscriptions
Reprints and
pubsaacrorgDepartment at
To order reprints of this article or to subscribe to the journal contact the AACR Publications
Permissions
Rightslink site Click on Request Permissions which will take you to the Copyright Clearance Centers (CCC)
89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
44 Ryter SW Choi AM Heme oxygenase-1 redox regulation of a stress protein in lung and cell
culture models Antioxid Redox Signal 2005 780-91
45 Cho KR Shih IeM Ovarian cancer Annu Rev Pathol 2009 4287-313
46 Spagnuolo C Russo GL Orhan IE Habtemariam S Daglia M Sureda A Nabavi SF Devi
KP Loizzo MR Tundis R Nabavi SM Genistein and cancer current status challenges and
future directions Adv Nutr 2015 6408-419
47 Murdoch WJ Van Kirk EA Alexander BM DNA damages in ovarian surface epithelial cells
of ovulatory hens Exp Biol Med 2005 230429-433
48 Zhuge Y Lagman JA Ansenberger K Mahon CJ Daikoku T Dey SK Bahr JM Hales DB
CYP1B1 expression in ovarian cancer in the laying hen Gallus domesticus Gynecol Oncol 2009
112171-178
49 Urick ME Giles JR Johnson PA VEGF expression and the effect of NSAIDs on ascites cell
proliferation in the hen model of ovarian cancer Gynecol Oncol 2008 110418-424
50 Trevintildeo LS1 Buckles EL Johnson PA Oral contraceptives decrease the prevalence of
ovarian cancer in the hen Cancer Prev Res 2012 5343-349
51 Urick ME Giles JR Johnson PA Dietary aspirin decreases the stage of ovarian cancer in the
hen Gynecol Oncol 2009 112166-170
52 Cramer DW Welch WR Determinants of ovarian cancer risk II Inferences regarding
pathogenesis J Natl Cancer Inst 1983 71717-721
53 Coussens LM Werb Z Inflammation and cancer Nature 2002 420860-867
54 Hernandez L Hsu SC Davidson B Birrer MJ Kohn EC Annunziata CM Activation of NF-
kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian
cancer Cancer Res 2010 704005-4014
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
Subscriptions
Reprints and
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To order reprints of this article or to subscribe to the journal contact the AACR Publications
Permissions
Rightslink site Click on Request Permissions which will take you to the Copyright Clearance Centers (CCC)
89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
55 Karin M The IkappaB kinase - a bridge between inflammation and cancer Cell Res 2008
18334-342
56 Annunziata CM Stavnes HT Kleinberg L Berner A Hernandez LF Birrer MJ Steinberg
SM Davidson B Kohn EC Nuclear factor kappaB transcription factors are coexpressed and
convey a poor outcome in ovarian cancer Cancer 2010 1163276-3284
57 Darb-Esfahani S Sinn BV Weichert W Budczies J Lehmann A Noske A Buckendahl AC
Muller BM Sehouli J Koensgen D Gyorffy B Dietel M Denkert C Expression of classical
NF-kappaB pathway effectors in human ovarian carcinoma Histopathology 2010 56727-739
58 Guo LM Pu Y Han Z Liu T Li YX Liu M Li X Tang H MicroRNA-9 inhibits ovarian
cancer cell growth through regulation of NF-kappaB1 FEBS J 2009 2765537-5546
59 Huang J Zhang L Greshock J Colligon TA Wang Y Ward R Katsaros D Lassus H
Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL
Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian
cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618
60 Laplante M Sabatini DM mTOR signaling in growth control and disease Cell 2012
149274-293
61 Kansanen E Jyrkkanen HK Levonen AL Activation of stress signaling pathways by
electrophilic oxidized and nitrated lipids Free Radic Biol Med 2012 52973-982
62 Taguchi K Motohashi H Yamamoto M Molecular mechanisms of the Keap1ndashNrf2 pathway
in stress response and cancer evolution Genes Cells 2011 16123-140
63 Niture SK Khatri R Jaiswal AK Regulation of Nrf2-an update Free Radic Biol Med 2014
6636-44
64 Malhotra D Portales-Casamar E Singh A Srivastava S Arenillas D Happel C Shyr C
Wakabayashi N Kensler TW Wasserman WW Biswal S Global mapping of binding sites for
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
Subscriptions
Reprints and
pubsaacrorgDepartment at
To order reprints of this article or to subscribe to the journal contact the AACR Publications
Permissions
Rightslink site Click on Request Permissions which will take you to the Copyright Clearance Centers (CCC)
89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network
analysis Nucleic Acids Res 2010 385718-5734
65 Liao H Zhou Q Zhang Z Wang Q Sun Y Yi X Feng Y NRF2 is overexpressed in ovarian
epithelial carcinoma and is regulated by gonadotrophin and sex-steroid hormones Oncol Rep
2012 271918-1924
66 Konstantinopoulos PA Spentzos D Fountzilas E Francoeur N Sanisetty S Grammatikos
AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian
cancer Cancer Res 2011 715081-5089
67 Lau A Villeneuve NF Sun Z Wong PK Zhang DD Dual roles of Nrf2 in cancer
Pharmacol Res 2008 58262-270
68 Kou X Kirberger M Yang Y Chen N Natural products for cancer prevention associated
with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28
69 Ma X Zhang J Liu S Huang Y Chen B Wang D Nrf2 knockdown by shRNA inhibits
tumor growth and increases efficacy of chemotherapy in cervical cancer Cancer Chemother
Pharmacol 2012 69485-494
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
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Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Table 1 Histopathology classifications of the reproductive tumors of hens
Adenocarcinoma Characteristics
Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most
common classification
Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular
pattern present but not distinct
Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least
common classification
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
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Reprints and
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89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens
Item Dietary Genistein levels mghen per day --P--
X2 301 5248 10626
Tumor ()
Incidence
Reduction
30100
30
-
19100
19
-367
10100
10
-667
0002
X2=12701
Serous carcinoma ()
Incidence
Reduction
19100
19
-
11100
11
-421
6100
6
-684
0017
X2=8144
Mucinous carcinoma ()
Incidence
Reduction
11100
11
-
8100
8
-273
4100
4
-636
0175
X2=3485
Adenocarcinoma incidence ()
Grade 1 530
167
719
368
510
500
0084
X2=4943
Grade 2 1830
600
819
421
310
300
0196
X2=3258
Grade 3 730
233
419
211
210
200
0968
X2=0064
Number of tumors1 37100
19100 10100 00001
Sizehen mm1
281plusmn050a
109plusmn027b 047plusmn016
b 00001
Sizehen with tumors only
mm1
686plusmn085 526plusmn081 470plusmn067 0249
Size range of tumors mm 1ndash22 1ndash12 2ndash8 -
Survival () 83100
83
88100
88
91100
91
0228
X2=2953
1Data are presented as the means and standard errors a-b Means in the same line without a common
superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
200
250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journalSign up to receive free email-alerts
Subscriptions
Reprints and
pubsaacrorgDepartment at
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Permissions
Rightslink site Click on Request Permissions which will take you to the Copyright Clearance Centers (CCC)
89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
FIGURE LEGENDS
Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the
ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed
Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens
Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining
original magnification x40
Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and
daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without
a common superscript differ significantly (P lt 005)
Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)
expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative
blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel
C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a
representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are
percent of the control a-c Means in the same line without a common superscript differ
significantly ( P lt 005)
Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian
tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
200
300
400
500
c
b
a
A
Genistein levels mghen per day
Gen
iste
in n
mo
ll
301 5248 10626
0
20
40
60
80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
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b
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-actin
p-mTOR
Genistein levels mghen per day
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pe
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ntr
ol
301 5248 10626
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-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
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a
b
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-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
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c
b
a
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-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the
same line without a common superscript differ significantly (P lt 005)
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
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c
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a
A
Genistein levels mghen per day
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iste
in n
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Genistein levels mghen per day
Dai
dze
in n
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301 5248 10626
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b
c
C
Genistein levels mghen per day
Ova
ry M
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g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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a
b
c
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-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
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ntr
ol
301 5248 10626
0
50
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a
b
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-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
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a
b
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-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
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c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Figure 1 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
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c
b
a
A
Genistein levels mghen per day
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iste
in n
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Genistein levels mghen per day
Dai
dze
in n
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301 5248 10626
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1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
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n
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lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
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a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
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a
b
c
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-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Figure 2 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
100
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c
b
a
A
Genistein levels mghen per day
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iste
in n
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20
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B
Genistein levels mghen per day
Dai
dze
in n
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301 5248 10626
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3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
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n
mo
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g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
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a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
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-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
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250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
301 5248 10626
0
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c
b
a
A
Genistein levels mghen per day
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iste
in n
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301 5248 10626
0
20
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80
B
Genistein levels mghen per day
Dai
dze
in n
mo
ll
301 5248 10626
0
1
2
3
4a
b
c
C
Genistein levels mghen per day
Ova
ry M
DA
n
mo
lm
g p
rote
in
Figure 3 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
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a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
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-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
Figure 4 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
100
150
a
b
c
A
-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
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250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
200
250
c
b
a
B
-actin
HO-1
Genistein levels mghen per day
HO
-1 p
erc
en
t o
f co
ntr
ol
Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
Material
Supplementary
89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
Manuscript
Authoredited Author manuscripts have been peer reviewed and accepted for publication but have not yet been
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89httpcancerpreventionresearchaacrjournalsorgcontentearly201901161940-6207CAPR-17-02To request permission to re-use all or part of this article use this link
Cancer Research on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Article File
Figure 1
Figure 2
Figures 3-6
301 5248 10626
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a
b
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-actin
p-mTOR
Genistein levels mghen per day
p-m
TO
R
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
B
-actin
p-p70S6K1
Genistein levels mghen per day
p-p
70
S6
K1
p
erc
en
t o
f co
ntr
ol
301 5248 10626
0
50
100
150
a
b
c
C
-actin
p-4E-BP1
Genistein levels mghen per day
p-4
E-B
P1
p
erc
en
t o
f co
ntr
ol
Figure 5 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
301 5248 10626
0
50
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150
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250
c
b
a
A
-actin
Nrf2
Genistein levels mghen per day
Nrf
2
pe
rce
nt
of
co
ntr
ol
301 5248 10626
0
50
100
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Figure 6 Cancer Research
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
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301 5248 10626
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HO
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Figure 6 Cancer Research
on June 9 2020 copy 2019 American Association forcancerpreventionresearchaacrjournalsorg Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
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Supplementary
89DC1
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289
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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian
Updated version
1011581940-6207CAPR-17-0289doi
Access the most recent version of this article at
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89DC1
httpcancerpreventionresearchaacrjournalsorgcontentsuppl201901161940-6207CAPR-17-02Access the most recent supplemental material at
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289