biomol 5

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Phytomedicine 22 (2015) 462–468

Contents lists available at ScienceDirect

Phytomedicine

journal homepage: www.elsevier.com/locate/phymed

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid

from Cajanus cajan in breast cancer cells

Yujie Fu a,b,1, Onat Kadioglu c,1, Benjamin Wiench c, Zuofu Wei a,b, Chang Gao d, Meng Luo a,b,Chengbo Gu a,b, Yuangang Zu a,b, Thomas Efferth c,∗

a Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, Chinab Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin, Chinac Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germanyd Peking University People’s Hospital, Beijing 100044, China

a r t i c l e i n f o

Article history:

Received 19 February 2015

Accepted 26 February 2015

Keywords:

Apoptosis

Breast cancer

Cell cycle

Microarray

Pharmacogenomics

Fabaceae

a b s t r a c t

Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown

to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and

apoptosis-related mechanisms have not been analyzed yet.

Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution,

mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation

assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA mi-

croarray hybridization and Ingenuity pathway analysis.

Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 μM.

The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray pro-

filing revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal

replication pathways.

Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading

to cell cycle arrest and apoptosis.

© 2015 Elsevier GmbH. All rights reserved.

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Abbreviations

BCIP/NBT 5-bromo-4-chloro-3′-indolyphosphate

p-toluidine salt/nitro-blue tetrazolium chloride

BRCA 1/2 breast cancer resistance genes 1/2

BSA bovine serum albumine

CSA cajanin stilbene acid

DAPI 4′,6-diamidine-2-phenylindole

DMEM dulbecco’s minimal essential medium

DMSO dimethyl sulfoxide

EGFR epidermal growth factor receptor

ER estrogen receptor

HER2 human epidermal growth factor receptor 2

MMP mitochondrial membrane potential

PBS phosphate buffered saline

PI propidium iodide

PMSF phenylmethanesulfonyl fluoride

PR progesterone receptor

∗ Corresponding author. Tel.: +49 6131 3925751; fax: +49 6131 23752.

E-mail address: [email protected] (T. Efferth).1

Both authors contributed equally to this work.

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http://dx.doi.org/10.1016/j.phymed.2015.02.005

0944-7113/© 2015 Elsevier GmbH. All rights reserved.

123 rhodamine 123

DS sodium dodecyl sulfate

BST tris-buffered saline/Tween 20

ntroduction

Breast cancer is one of the most common types of tumor (DeSantis

t al. 2014; Donepudi et al. 2014; Siegel et al. 2014; Zagouri et al.

014) and the American Cancer Society estimated around 230,000

ew cases of invasive breast cancer diagnosed in women and about

0,000 breast cancer deaths in USA in 2013 (DeSantis et al. 2011).

ome hormone receptors, i.e. estrogen and progesterone receptors

ER, PR) and growth factor receptors such as HER2 are referred

s important prognostic factors for breast cancer (Donepudi et al.,

014; Kawano et al. 2013; Kwast et al. 2014; Pourzand et al. 2011).

he majority of breast cancer cases are hormone-dependent and

R-positive. Anti-estrogenic therapies may be effective to improve

he prognosis of breast cancer patients, however, breast tumors

an develop resistance toward anti-hormonal drugs (Josefsson and

einster 2010; Musgrove and Sutherland 2009; Osborne and Schiff

011) and targeting one receptor is frequently inadequate due to

ultiple alternative routes to resist the detrimental effects of anti-

ancer drugs (Osborne and Schiff 2011; Zhang et al. 2014b). Thus,

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Y. Fu et al. / Phytomedicine 22 (2015) 462–468 463

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Fig. 1. Chemical structure of CSA.

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he search for active compounds against breast cancer represents still

critical issue. Various studies have evaluated natural compounds

n terms of their inhibitory effects on cancer-related signaling path-

ays (Cerella et al. 2015; Chinembiri et al. 2014; Jafari et al. 2014;

arrelli et al. 2014; Millimouno et al. 2014; Pourahmad et al. 2014;

ztiller-Sikorska et al. 2014; Turrini et al. 2014). Indeed, many anti-

ancer drugs are derived from natural compounds (Al-Tweigeri et al.

010; Driscoll and Marquez 1994; Fotia et al. 2012; Icli et al. 2011;

ee et al. 2014). Targeting breast cancer-related receptors (ER, PR,

GFR, etc.) and influencing cancer-related pathways related to apop-

osis, cell cycle and DNA damage possess a high potential against

reast cancer.

BRCA-1 and -2 are important proteins for breast cancer progres-

ion by regulating cell cycle and DNA damage. Mutations in the BRCA-1

nd BRCA-2 genes play a role for breast carcinogenesis (Gangi et al.

014; Xu et al. 2012). Functional loss by mutation leads to deficient

NA damage repair and cell cycle control (Narod and Foulkes 2004;

hahid et al. 2014; Venkitaraman 2002; Wiltshire et al. 2007; Zhou

nd Elledge 2000). Down-regulation of BRCA-1 and/or -2 may cause

ncreased DNA damage and apoptosis. In tumor cells, targeting those

roteins may be an effective strategy to direct the fate of cancer cells

hrough apoptosis.

Another critical protein in breast cancer is p21. This tumor sup-

ressor is either mutated or downregulated to favor excessive cell

roliferation and eventually metastasis. p21 also plays a role in DNA

amage-related pathways. The mutation status of p21 may deter-

ine breast cancer susceptibility (Akhter et al. 2014), whereas up-

egulation of p21 is linked with anticancer activity by apoptosis induc-

ion (Aziz et al. 2014). Thus, natural compounds causing up-regulation

f p21 and inducing apoptosis may be effective against breast cancer.

A novel compound from Pigeon Pea (Cajanus cajan (L.) Millsp.) is

ajanin stilbene acid (CSA) (Wu et al. 2009). CSA’s abundance is quite

ow and it is difficult to isolate. Thus, the bioactivity of CSA may have

een overseen in the past. However, the profound cytotoxicity of this

ompound indicates that this compound may be valuable for cancer

reatment. The cellular and molecular mechanisms of action for CSA

re still not well understood. In the present investigation, we analyzed

he activity of CSA in ER-positive MCF-7 cells in terms of cell cycle and

poptosis regulation. For this purpose, we applied two experimental

pproaches:

(1) We investigated well-known parameters such as cell cycle dis-

tribution, mitochondrial membrane potential, DNA fragmen-

tation, expression of Bax and Bcl-2 proteins, and activation of

caspases.

(2) By using gene expression profiling, we found that the BRCA-1-

related-DNA damage response pathway was directly affected

by CSA treatment and various genes playing role in this path-

way were down-regulated. Moreover, p21 was up-regulated,

implying the ability of CSA to affect both the DNA damage re-

sponse and cell proliferation in MCF-7 breast cancer cells.

aterials and methods

hemicals

Cajanin stilbene acid (CSA, purity � 98%) was isolated from Pigeon

ea (Cajanus cajan (L.) Millsp.) roots (Fig. 1). A 10 mg/ml stock solu-

ion of CSA was prepared in dimethyl sulfoxide (DMSO) and stored at

80 °C. Rhodamine 123 (R123) and propidium iodide (PI) were ob-

ained from Sigma-Aldrich Inc. (St. Louis, MO). Deionized water was

sed in all experiments. Hoechst 33258 was purchased Sigma-Aldrich

Taufkirchen, Germany).

ell culture

Human MCF-7 breast cancer cells were obtained from the In-

titute of Molecular Biology (University of Mainz, Germany). They

ere maintained under standard conditions (37 °C, 5% CO2) in DMEM

edium (Gibco BRL, Eggenstein, Germany) supplemented with 10%

etal calf serum and 1% penicillin/streptomycin (100 U/ml penicillin,

00 μg/ml streptomycin). Cells were passaged twice weekly. All ex-

eriments were performed with cells in the logarithmic growth phase.

ell cycle analysis

MCF-7 cells (1 × 106 cells/well) were seeded into 6-well plates.

fter 24 h, cells were treated with CSA at serial dilutions (0, 8.88,

1.83 or 14.79 μM) for 48 h. Then, cells were centrifuged, washed

ith PBS, stained with 50 μg/ml DAPI (Partec, Münster, Germany)

nd analyzed by flow cytometer (Partec, Münster, Germany). The cell

ycle phases were analyzed with FACScan and CellQuest software

Becton Dickinson, Mountain View, CA).

easurement of mitochondrial membrane potential

The mitochondrial membrane potential has been measured by

123 (Cao et al. 2007). MCF-7 cells (1 × 106 cells/well) were seeded

nto 6-well plates. After 24 h, cells were treated with CSA (8.88, 11.83

r 14.79 μM) for 48 h or left untreated. Then, cells were harvested

nd washed twice with PBS. Cell pellets were resuspended in 2 ml

resh medium containing 1.0 μM R123 and incubated at 37 °C in a

hermostatic bath for 30 min with gentle shaking. MCF-7 cells were

eparated by centrifugation, washed twice with PBS, stained with

μg/ml PI and analyzed by flow cytometry.

For microscopic analysis, cell monolayers were treated with the

ame protocol as described above and subjected to a confocal laser

canning microscope (C1-LU3EX, Nikon, Sendai, Japan).

NA fragmentation assay

Apoptotic DNA fragments appear as DNA ladder consisting of

ultimers of 180–200 bp (Tilly and Hsueh 1993). MCF-7 cells

1 × 106 cells/ml) were seeded in 6-well plates and exposed to CSA

8.88, 11.83 or 14.79 μM) for 48 h or left untreated. Then, cells were

ollected by centrifugation. DNA was isolated with a commercial iso-

ation kit (Watson Biotechnologies Inc, Shanghai, China) according to

he manufacturer’s instructions. The DNA was separated in 1% agarose

el and visualized by ultraviolet illumination (Image Master VDS-CL,

okyo, Japan) after staining with ethidium bromide.

orphological observation of nuclear change

MCF-7 cells (1 × 106 cells/ml) were seeded in 6-well plates and

reated with CSA (8.88, 11.83 or 14.79 μM) for 48 h at 37 °C or left

ntreated. Cells were collected, washed, fixed in 4% paraformalde-

yde for 30 min and stained with 5 μg/ml Hoechst 33258 for 5 min at

oom temperature. The apoptotic cells were visualized using inverted

uorescence microscope (Nikon TE2000, Tokyo, Japan).

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464 Y. Fu et al. / Phytomedicine 22 (2015) 462–468

Fig. 2. (A) Cell cycle distribution of MCF-7 cells after treatment with different concen-

trations of CSA for 48 h. (1) 0 μM; (2) 8.88 μM; (3) 11.83 μM; (4) 14.79 μM. (B) Cell

cycle distribution of MCF-7 cells after treatment with 14.79 μM for different times. (1)

0 h; (2) 6 h; (3) 12 h; (4) 24 h. (C) Effect of CSA on cell cycle in MCF-7 tumor xenografts.

(1) Negative control, (2) positive control (20 mg/kg cyclophosphamide), (3) low dose

CSA (15 mg/kg), (4) high dose CSA (30 mg/kg). The tumor cells were arrested at S and

G2M phase. The results were comparable to the data in vitro.

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Western blotting

MCF-7 cells were treated with CSA (8.88, 11.83 or 14.79 μM) for

48 h or left untreated. For protein isolation, medium was removed,

cells were washed twice with ice-cold PBS, then lysed using cell lysis

buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM

sodium pyrophosphate, 1 mM EDTA, 1% Na3CO4, 0.5 μg/ml leupeptin,

1 mM phenylmethanesulfonyl fluoride (PMSF)]. The lysates were col-

lected by scraping from the plates and then centrifuged at 10,000 rpm

at 4 °C for 5 min.

Total protein samples (20 μg) were loaded on 12% of SDS-

polyacrylamide gels for electrophoresis, and then transferred onto

PVDF transfer membranes (Millipore, Billerica, USA) at 0.8 mA/cm2

for 2 h. Membranes were blocked at room temperature for 2 h with

blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes

were then incubated overnight at 4 °C with primary antibodies (anti-

caspase-3, anti-Bax, anti-Bcl-2, anti-β-actin) at a dilution of 1:250

(Biosynthesis Biotechnology Company, Beijing, China) in blocking so-

lution. After thrice washings in TBST for each 5 s, membranes were

incubated for 1 h at room temperature with alkaline phosphatase

peroxidase-conjugated anti-mouse secondary antibody (1:500 dilu-

tion) in blocking solution. Detection was performed by the BCIP/NBT

Alkaline Phosphatase Color Development kit (Beyotime Institute of

Biotechnology) according to the manufacturer’s instructions. Bands

were then recorded by a digital camera (Canon, EOS 350D, Tokyo,

Japan).

mRNA microarray analysis

Total RNA from MCF-7 cells after 72 h of treatment with CSA at IC50

concentration (54.77 μM) or DMSO solvent control was extracted us-

ing RNeasyR©

mini kit (Qiagen Inc., Valencia, CA, USA) according to

the manufacturer’s instructions. RNA quality was verified by elec-

trophoresis using the Nano Chip assay on an Agilent 2100 Bioana-

lyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples

with RNA index values greater than 8.5 were selected for expression

profiling. RNA concentrations were determined using the NanoDrop

spectrophotometer (Nano-Drop Technologies, Wilmington, DE). Total

RNA was labeled and converted to cDNA (Eberwine et al. 1992). Then,

fluorescent cRNA (Cyanine 3-CTP) was synthesized and purified using

QIAgen RNeasyR©

kit. After fragmentation of the cRNA, samples were

hybridized on Whole Human Genome RNA chips (8 × 60 K Agilent) by

following the One-Color Microarray-Based Gene Expression Analysis

Protocol (Agilent Technologies GmbH) for 17 h at 65 °C. Microarray

slides were washed and scanned with Agilent Microarray Scanning

system. Images were analyzed and data were extracted, background

subtracted and normalized using the standard procedures of Agilent

Feature Extraction Software. The expression data obtained was fil-

tered with Chipster data analysis platform. These steps include filter-

ing of genes by two times standard deviation of deregulated genes

and subsequent assessment of significance using empirical Bayes t-

test (p < 0.05). Pathway analysis was done by using the Ingenuity

Pathways Analysis software (version 5.5) from Ingenuity Systems

Table 1

Primer nucleotide sequences, concentrations and annealing

Gene Sequence

BRCA-1 Fw: 5′-TCAATGGAAGAAACCACCAAGGT-3′

Rev: 5′-CATTCCAGTTGATCTGTGGGC-3′

BRCA-2 Fw: 5′-GTTTGTGAAGGGTCGTCAGA-3′

Rev: 5′-AGAACTAAGGGTGGGTGGTG-3′

p21 Fw: 5′-GCGATGGAACTTCGACTTTGT-3′

Rev: 5′-GGGCTTCCTCTTGGAGAAGAT-3′

RPS13 Fw: 5′-GGTTGAAGTTGACATCTGACGA-3′

Rev: 5′-CTTGTGCAACACATGTGAAT-3′

Redwood City, CA, USA) and –log (p-value) was used to estimate

he significances of pathways and biological functions.

eal time RT-PCR

In order to validate the microarray data, BRCA-1, BRCA-2 and p21

enes were selected and the total RNA isolated for the microarray ex-

eriment was used for real time RT-PCR. RPS-13 was used as reference

ene for standardization. All measurements were done in duplicates

nd the average fold change values were provided. Table 1 depicts the

rimer nucleotide sequences, primer concentrations and annealing

emperatures. Real time RT-PCR reactions and the fold change calcula-

ions were conducted as described previously (Panossian et al. 2013).

esults

ell cycle analysis

Exposure of MCF-7 cells to CSA (8.88–14.79 μM) for 48 h in vitro

aused a dose-dependent G2M arrest (19.64–27.48% compared to

2.88% in untreated cells) and S arrest (17.46–32.16% compared to

3.40% in untreated cells) (Fig. 2A). CSA (5μg/ml) arrested MCF-7 cells

t the S and G2M phase also in a time-dependent manner (Fig. 2B).

temperatures.

Concentration (nM) Annealing temperature (°C)

250 59

250 59

250 59

250 59

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Y. Fu et al. / Phytomedicine 22 (2015) 462–468 465

Bax

Bcl2

beta-

actin

beta-

actin

active

caspase 3

pro-

caspase 3

% Bax

% Bcl2

% pro-caspase 3

% active caspase 3

(A)

(C) (D) (F)

(E)

(B)

Fig. 3. (A) Morphological analysis of nuclear fragmentation and apoptosis of MCF-7 cells treated with 14.79 μM CSA for 48 h by fluorescence microscopy. (1) Untreated cells; (2)

cells treated with CSA. The experiment was repeated three times and representative photographs are shown. (B) Assessment of apoptosis in MCF-7 cells by the DNA fragmentation

assay. M, DNA size marker; lane 1, untreated cells; lanes 2–4, treatment with 8.88, 11.83 or 14.79 μM CSA. (C) CSA-mediated upregulation of Bax and downregulation of Bcl-2 as

determined by Western blotting. MCF-7 cells were treated with CSA (8.88, 11.83 or 14.79 μM) for 48 h. The test was repeated three times and representative blots are shown. (∗p

value < 0.05, ∗∗p value < 0.01). (D) Mitochondrial membrane potential of MCF-7 cells treated with CSA or left untreated as assayed by flow cytometry (1) 0 μM, (2) 8.88 μM, (3)

11.83 μM, (4) 14.79 μM. (E) Mitochondrial membrane potential of MCF-7 cells treated with CSA or untreated assayed by confocal laser scanning microscopy (1) 0 μM, (2) 8.88 μM,

(3) 11.83 μM, (4) 14.79 μM. (F) Effect of CSA on caspase-3 activity as assayed by Western blotting. MCF-7 cells were treated with CSA (8.88, 11.83 and 14.79 μM) for 48 h. The test

was repeated three times and representative blots are shown. (∗p-value < 0.05, ∗∗p-value < 0.01).

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hen, we used single-cell suspensions obtained from MCF-7 xenograft

umors after excision from nude mice. Again, a dose-dependent in-

rease in S and G2M phase cells was observed after treatment with 15

r 30 mg/kg CSA (Fig. 2C, histograms 3 and 4) compared to untreated

ontrols (Fig. 2C, histogram 1). Cyclophosphamide is a standard drug

or breast cancer therapy and was used as control compound. Cy-

lophosphamide did not affect the G2M phase, but the S phase

Fig. 2C, histogram 2).

ssessment of apoptosis

Hoechst 33258 staining showed considerable morphological

hanges in nuclear chromatin of CSA-treated MCF-7 cells. Untreated

ontrol cells did not show chromatin condensation and their nuclei

ere stained in less bright and homogeneous blue color (Fig. 3A, pho-

ograph 1). In contrast, CSA (14.79 μM for 48 h), caused very intense

taining of condensed and fragmented chromatin and the formation

f typical apoptotic bodies. Only a few nuclei displayed normal mor-

hology (Fig. 3A, photograph 2).

NA laddering

Apoptosis-related DNA laddering was visible after treatment of

CF-7 cells with increasing CSA concentrations for 48 h (Fig. 3B, lanes

–4). Untreated control cells did not induce apoptosis (lane 1). DNA-

addering was also observed in MCF-7 xenograft tumors treated with

SA or cyclophosphamide (data not shown).

arkers of the mitochondrial apoptosis

Western blot analysis revealed that CSA-treated MCF-7 cells

own-regulated Bcl-2 expression, but up-regulated Bax expression

Fig. 3C). CSA-induced apoptosis was associated with mitochondrial

epolarization. In MCF-7 cells, CSA at doses of 8.88–14.79 μM led to

ose-dependently increased percentages of mitochondrial depolar-

zation (��m) from 97.03 to 61.26% (Fig. 3D). Mitochondrial mem-

rane potentials were measured by laser scanning microscopy and

omparable results were obtained (Fig. 3E). Furthermore, CSA led to a

ose-dependent increase of caspase-3 activity as observed by West-

rn blotting (Fig. 3F).

ifferential gene regulation by CSA

Upon CSA treatment at the IC50 concentration, 363 genes were

ifferentially regulated after 24 h and 659 genes after 72 h, as ana-

yzed by microarray-based mRNA hybridizations. We subjected these

enes to Ingenuity Pathway Analysis. Many cell cycle and apoptosis

elated pathways were observed to be affected upon CSA treatment

s shown in Fig. 4A. BRCA1 in DNA damage response (Fig. 4B) and

ell cycle control of chromosomal replication (Fig. 4C) were the most

ffected pathways with –log (p-value) of 12.5 and 11.3 respectively.

RCA-1 and BRCA-2 were down-regulated by 2.990- and 3.364-fold,

espectively, whereas p21 was up-regulated by 5.223-fold upon CSA

reatment. Microarray data and the deregulation of those genes were

alidated by real time RT-PCR as can be seen in Table 2. The correlation

oefficient between mRNA expression values determined by microar-

ay hybridization and real-time RT-PCR was 0.99 (Pearson Correlation

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466 Y. Fu et al. / Phytomedicine 22 (2015) 462–468

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-valu

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(A)

(B)

(C)

Fig. 4. (A) Identification of canonical signaling pathways regulated upon CSA treatment in MCF-7 cells. Transcriptome-wide gene expression of cells treated with the IC50

concentration of CSA was compared to gene expression in untreated cells. The evaluation of differentially expressed genes was performed using the Ingenuity Pathway Analysis

software version 5.5. Each bar represents the ratio of the number of genes in a particular pathway, whose expression is correlated with cellular response toward CSA (IC50).

(B) The BRCA1-related DNA damage response pathway as the most affected pathway upon CSA treatment. Genes labelled green were down-regulated and genes labelled red were

up-regulated. (C) The cell cycle control of chromosomal replication pathway as the second most affected pathway upon CSA treatment. Genes labelled green were down-regulated.

(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2

Validation of microarray-based mRNA expression by quanti-

tative real-time RT-PCR.

Gene Method Fold change

BRCA-1 Microarray −2.99

RT-PCR −4.93

BRCA-2 Microarray −3.36

RT-PCR −4.91

p21 Microarray 5.22

RT-PCR 1.66

mRNA expression values from microarray hybridization and

real-time RT-PCR were significantly correlated (R = 0.99;

p = 0.013; Pearson correlation test).

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Test). These results clearly indicate that CSA influences DNA damage

and cell cycle related pathways and the expression of three important

genes playing role in DNA damage response pathway and cell cycle

control.

Discussion

In the present study, we investigated the anti-cancer activity of

CSA, a compound isolated by us from Pigeon Pea (Cajanus cajan)

(Wu et al. 2009) in terms of apoptosis and cell cycle regulation. The

cellular and molecular mechanisms of CSA’s mode of action are still

not well understood and we hypothesized that CSA may act on cell

cycle and apoptosis related pathways. CSA induced arrest in the G2M

phase of the cell cycle in a time- and concentration-dependent man-

ner. If unreleased, G2M arrest can ultimately lead to apoptosis. By

DNA laddering assay and fluorescence microscopy, we found that CSA

ndeed induced apoptosis. Apoptosis induction was associated with

cl-2 down-regulation, Bax up-regulation, caspase-3 activation and

epolarization of the mitochondrial membrane potential, suggesting

hat CSA activated the mitochondrial pathway of apoptosis in MCF-7

ells. Various studies have shown that targeting those pathways and

nducing cell cycle arrest and apoptosis serve as a valuable strategy

or cancer drug discovery process (Evan and Vousden 2001; Kim et al.

014; Wang et al. 2014; You and Park 2014; Zhang et al. 2015; Zhang

t al. 2014a; Zheng et al., 2014).

Gene expression profiling studies yield valuable information to

nderstand molecular mechanisms of different cancer types (Drukker

t al. 2014; Fina et al. 2015; Fu et al. 2014; Yuan et al. 2014; Zubor et al.

015). The mode of action of a compound and its potential as an anti-

ancer agent can be evaluated via gene expression profiling studies

Iorio et al. 2009; Nunez et al. 2008; Righeschi et al. 2012; Schmeits

t al. 2014; Zhou et al. 2005). Therefore, we applied mRNA microarray

nalyses to unravel modes of action of CSA. The deregulated genes af-

er CSA treatment were subjected to Ingenuity Pathway analyses to

dentify affected signaling routes. Intriguingly, among the top signal-

ng pathways were G2M arrest pathways. This is a strong hint that cell

ycle arrest in G2M and induction of apoptosis are important modes

f action of CSA toward cancer cells. BRCA-1 and BRCA-2 were down-

egulated upon CSA treatment, indicating that DNA damage and repair

athways were affected. Proteins (p21, BRCA-1 and BRCA-2) playing

ole in DNA damage response pathway (Pawlik and Keyomarsi 2004)

ere deregulated upon CSA treatment. Up-regulation of p21, down-

egulation of BRCA-1 and BRCA-2 imply that uncontrolled proliferation

as to some extent normalized and DNA damage was accumulated

eading to apoptosis. As tumor suppressor p21 plays a critical role in

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Y. Fu et al. / Phytomedicine 22 (2015) 462–468 467

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ell cycle regulation, excessive cell proliferation and metastasis can

e halted via p21 up-regulation (Garcia-Tunon et al. 2006; Tanaka and

ino 2014). Our results on CSA can be reconciled with more general

ndings in cancer biology that tumors activate DNA damage response

athways such as BRCA-1/2 upon exposure to DNA-damaging agents

Cheung-Ong et al. 2014). It is worth speculating that CSA may be

ven more cytotoxic, if combined with other DNA-damaging drugs

uch doxorubicin and cisplatin.

We conclude that CSA may act on breast cancer cells by target-

ng multiple tumorigenic pathways leading to cell cycle arrest and

poptosis. Our data indicate that CSA possesses therapeutic poten-

ial against breast cancer. Further preclinical and clinical studies are

arranted to clarify the therapeutic potential of CSA.

onflict of interest

The authors declare that they have no conflict of interest.

cknowledgments

We gratefully acknowledge the financial support from Special

und of Forestry Industrial Research for Public Welfare of China

201004040), Importation of International Advanced Forestry Science

nd Technology, National Forestry Bureau (2012-4-06), Heilongjiang

rovince Science Foundation for Excellent Youths (JC200704) and

roject for Distinguished Teacher Abroad, Chinese Ministry of Edu-

ation (MS2010DBLY031).

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