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European Journal of Pharmacology
Manuscript Draft
Manuscript Number: EJP-51850
Title: Curcumin Modulates the AKT and PTEN Expression to Suppress Glioma
Cell Proliferation
Article Type: Research Paper
Section/Category: Neuropharmacology and analgesia
Keywords: Glioblastoma; Curcumin; PTEN; AKT; Mechanism
Abstract: Glioma is the most common neoplasm in the brain. Curcumin,as a
known polyphenolic compound extracted from turmeric,is clinically used
in the chemoprevention and treatment of some cancers. However, the degree
to which curcumin is involved in mediating the treatment of glioma and
the possible mechanisms of such an effect remains unknown. we detected
cell viability of glioma cells under curcumin by MTT assay and examined
the invasion and migration ability. The proliferation and apoptotic
proteins were detected. We made glioma-xenograft model and detected the
proliferation under curcumin treatment.We found that curcumin inhibited
the proliferation of U251 and U87 cells of glioma. Curcumin up-regulated
the invasion and migration of U251 and U87 cells. We detected that
curcumin decreased p-AKT and p-mTOR protein expression of U251 and U87
cells. Curcumin promotes apoptosis of U251 and U87 cells. We used some
databases to analyze the effect of PTEN gene expression on the survival
rate of glioma patients and to analyze the interaction between proteins.
Further, we found that curcumin promoted the PTEN and p53 expression, as
the tumor suppressor genes. In addition, we administered curcumin to U87-
xenograft and found that curcumin decreased the tumor volume, caused
necrosis of tumor tissue, and significantly enhanced the PTEN and P53
expression in vivo. These results indicate that curcumin may inhibit
proliferation by increasing the p-AKT/p-mTOR pathway, and promotes
apoptosis by enhancing the PTEN and p53 expression. Our study can provide
new insights into the molecular mechanisms by which curcumin can inhibit
glioma and its targeted interventions.
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Curcumin Modulates the AKT and PTEN Expression to Suppress Glioma
Cell Proliferation
Zexia Wang1,2
, Fei Liu1,2
, Liangzhu Yu1, Hongli Xia
3, Mincai Li
1,2#, Zhenwu Hu
1# 1 School of Basic Medical Sciences, Hubei University of Science and Technology, Xianning, 437100, P.R.China,
2 School of Pharmacy, Hubei University of Science and Technology, Xianning, 437100, P.R. China,
3 The Central Hospital of Xianning, Hubei University of Science and Technology, Xianning, 437100, P.R.China,
#Correspondence to School of Basic Sciences, Hubei University of Science and Technology, Xianning, 437100,
P.R.China
The email address for all author
Zexia Wang [email protected]
Fei Liu [email protected]
Liangzhu Yu [email protected]
Hongli Xia [email protected]
Mincai Li [email protected]
Zhenwu Hu [email protected]
ManuscriptClick here to view linked References
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Abstract
Background Glioma is the most common neoplasm in the brain. Curcumin,as a known
polyphenolic compound extracted from turmeric,is clinically used in the chemoprevention
and treatment of some cancers. However, the degree to which curcumin is involved in
mediating the treatment of glioma and the possible mechanisms of such an effect remains
unknown.
Methods we detected cell viability of glioma cells under curcumin by MTT assay and
examined the invasion and migration ability. The proliferation and apoptotic proteins were
detected. We made glioma-xenograft model and detected the proliferation under curcumin
treatment.
Results We found that curcumin inhibited the proliferation of U251 and U87 cells of glioma.
Curcumin up-regulated the invasion and migration of U251 and U87 cells. We detected that
curcumin decreased p-AKT and p-mTOR protein expression of U251 and U87 cells.
Curcumin promotes apoptosis of U251 and U87 cells. We used some databases to analyze
the effect of PTEN gene expression on the survival rate of glioma patients and to analyze
the interaction between proteins. Further, we found that curcumin promoted the PTEN and
p53 expression, as the tumor suppressor genes. In addition, we administered curcumin to
U87-xenograft and found that curcumin decreased the tumor volume, caused necrosis of
tumor tissue, and significantly enhanced the PTEN and P53 expression in vivo.
Conclusions These results indicate that curcumin may inhibit proliferation by increasing the
p-AKT/p-mTOR pathway, and promotes apoptosis by enhancing the PTEN and p53
expression. Our study can provide new insights into the molecular mechanisms by which
curcumin can inhibit glioma and its targeted interventions.
Key Words: Glioblastoma; Curcumin; PTEN; AKT; Mechanism
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Introduction
Glioma is a common primary neoplasm, including astrocytoma, oligoastrocytomas,
oligodendroglioma and glioblastomas[1]. Glioblastoma (GB) is one of the most aggressive
solid tumors patients with these tumors have a very poor prognosis. The poorly differentiated
polymorphic glioma cells have diverse morphologies, nuclear atypia, and undergo active
nuclear division. Their histological features show the different morphology such as necrosis,
vascular proliferation and thrombosis[2] The current treatment methods include the tumor
resection, chemotherapy, radiotherapy, anti-vascular targeted drugs. Because current
therapeutic strategies are ineffective it is important to find new treatments or better drugs.
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), isolated
from the spice turmeric of Curcuma,a natural polyphenolic compound [3,4]. This hydrophobic
compound has been used to treat a variety of human diseases, such as inflammatory,
neoplastic and some neurodegenerative diseases[5,6]. Previous studies showed that
curcumin inhibited cytotoxicity by decreasing oxidative damage caused by reactive oxygen
species[7,8,9]. Some studies have shown that curcumin has therapeutic properties and
preventive effects on many types of neoplasms. These reports demonstrated that curcumin
regulates several signaling pathways, including STAT3 and NF-κB transcription factor in the
proliferation, angiogenesis and metastasis[7,10]. However, the role of curcumin in gliomas is
unclear and further clinical studies are required.
Our study was designed to discover the potential ability of curcumin to suppress glioma
in vitro and in vivo. Our results suggest that curcumin inhibits the proliferation of glioma, and
that this effect is associated with the inhibition of AKT/mTOR signaling pathway and PTEN
activation.
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Materials and Methods
Materials
The curcumin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).
Anti-AKT, anti-mTOR, anti- caspase-3,anti-beclin,anti-PTEN,anti-p53 and anti-AT1R
antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All
secondary antibodies were from Proteintech Group (USA). Trizol reagent and the enhanced
chemiluminescence kit were from Beijing Applygen Technologies (Beijing, China). All
procedures using animals were conducted as described previously[11] and were in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH publication 85-23, revised 1996) and approved by the institutional animal care
and use committee at the Hubei University of Science and Technology, China.
Glioma cell culture.
The glioma cell lines, U251and U87, were cultured in Dulbecco's modified Eagle's medium
(DMEM) both supplemented with 10% fetal bovine serum (FBS) as described previously[12].
The cells were incubated under humidified conditions at 37˚C and 5% CO2. The medium
was changed every 3 days. The cells were passaged at 95% confluency for the following
experiments.
Cell proliferation assay.
The cell proliferation assay was examined as described previously[13,14]. U251and U87cells
were seeded into 96-well tissue culture plates and were incubated with 0, 10, 20 and 40 µM
curcumin. After incubation for 24-, 48-, and 72- h at 37˚C, the viability was detected with a 3
‑(4,5‑dimethyl- ylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay. The glioma
cells are cultured in media containing 0.5 mg/ml MTT for 2h. Dimethyl sulfoxide was added
to dissolve the formazan products and the absorbance was measured
spectrophotometrically at a 550 nm wavelength. Three replicates were performed.
Wound healing assays
The wound healing assays were performed by the cell migration as described prevously[15].
Cells were seeded into 6-well plates and cultured until the confluence reached 90%. A sterile
10-μl pipette tip generated a scratch through each well. The wound closure was observed
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after 0- and 24-h and photographed using a (insert make and model) microscope. The
number of U251 and U87 cells that migrated was calculated in 5 high-magnification fields
(200×) that were randomly selected. Three replicates were performed, and the data were
averaged for statistical analysis.
Transwell migration and invade assays
The migration assay was performed using a transwell system (Corning Costar, USA) as
described previously[12]. A total of 3×104 cells in 100 μL of serum-free media were added to
the top chamber 8-μm Costar polycarbonate transwells, and 500 μL of DMEM,
supplemented with 10% FBS and curcumin, were added to the bottom chamber. Culture
supernatant with DMSO served as the control group. After being incubated for 4 h, the cells
on the upper membrane surface were removed and the cells that migrated were fixed with 5%
(wt/vol) glutaraldehyde and stained with 0.1% (wt/vol) crystal-violet solution. Filter inserts
were inverted and the migratory cell number was determined by visualizing the
crystal-violet-stained cells directly on undersides of the inserts using a (insert make and
model here) microscope. Three replicates were performed, and the data were averaged for
statistical analysis.
Adhesion assay
Adhesion assay was performed as previously described[12] The 96-well plates were
preincubated with 20 μg/mL fibronectin (FN) fragment for 4 h, and 200 μL of U251 and U87
cells, with or without curcumin, were incubated for 60 min. The U251 and U87cells were
added at a density of 5 × 105 cells/well for 45 min at 37°C. The non-adhesive U251 and U87
cells were washed 3 times. The number of U251 and U87 cells that adhered to fibronectin
was calculated.
Western blots
The Western blot assay was performed as described previously[13,14]. Following dosing of
0-,10- and 20-µM curcumin treatment for 24 h, the protein was extracted with lysis buffer and
obtained after centrifugation. Equal amounts of protein extracts were separated by 10%
SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were
incubated with primary antibodies at 4˚C overnight. Bands were visualized using horseradish
peroxidase-conjugated anti-rabbit IgG and an enhanced chemiluminescence reagent,
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according to the manufacturer’s instructions. GAPDH was used as an internal control.
Proteins were quantified by densitometric analysis.
Cell apoptosis assay.
The apoptosis assay was performed as described previously[14]. Following dosing of 0-,10-,
and 20-µM curcumin for 24 h, the cells were washed three times. An Annexin V-fluorescein
isothiocyanate (FITC)-propidium iodide (PI) apoptosis kit was used to detect apoptosis. Cells
were washed three times and suspended at a density of 3x106 cells/ml in 1X Annexin
V-binding buffer. Annexin V-FITC and PI buffer were added to the cells which were incubated
for 15 min at room temperature. Cells treated with DMSO were used as control.
Database analysis
The relationship between PTEN expression and survival in glioma was analyzed by
PrognoScan database (http://www.prognoscan.org/)[16]. The threshold was set as cox
p-value<0.05. The correlation of PTEN expression and survival of glioma was analyzed by
Kaplan-Meier plotter database (http://linkedomics.org/ analysis/)[17]. The hazard ratio with 95%
confidence intervals and log-rank p-value were analyzed.
Antitumor activity assay in vivo
Antitumor activity was performed as previously described[9]. Female nude mice were
housed with pathogen-free conditions. All experimental procedures conformed to the animal
experiment guidelines of the Animal Care and Welfare Committee of Hubei University of
Science and Technology. U87 xenografts were performed by subcutaneous injections of
2.0×106 U87 cells in nude mice[9]. When the tumor volume reached approximate 100 mm3,
the mice were randomly divided into two groups (n = 5) and intraperitoneal. injections of
curcumin (60 mg/kg) or saline was administered daily for 14 days. The tumor size and
animal body weights were measured every 2 days. Tumor volume was calculated as
described[9]. The mice were sacrificed and the tumors were removed and weighed at the end
of the experiment.
Statistical analysis
All results were presented as mean ±SEM of at least three independent experiments.
Groups were analyzed using one-way ANOVA with Bonferroni’s post-hoc tests. Statistical
significance was set as P < 0.05.
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Results
1 Curcumin inhibits glioblastoma proliferation in a time- and dose-dependent manner.
Using an MTT assay, we measured the proliferation of U251 and U87 cells that were
treated with 0-, 10, 20-, and 40- µM curcumin for 24-,48-, and 72-h. As shown in Fig.1A, the
glioblastoma cells become small and appeared unhealthy due to the increasing
concentrations of curcumin. Treatments with 10- and 20-µM curcumin for 24 h significantly
inhibited cell proliferation, compared to the control group in both the U251 (Fig. 1B) and U87
(Fig. 1C) cells. Our results indicated that curcumin downregulates glioblastoma proliferation
in both U251 and U87 cells.
2 Curcumin inhibits the migration and invasion of glioblastoma in vitro
To detect the effects of curcumin on metastasis, the wound healing assay was used. The
morphological changes in migrating cells are presented in Fig. 2A. Following treatment with
0-, 10-, and 20-µM of curcumin for 24 h, the migration of U251 and U87 cells was inhibited
(Fig. 2B).
Next, we examined the invasion ability using the transwell assay. The invaded cells were
stained as shown in Fig. 2C. As displayed in Fig. 2D, treatments with10- and 20-µM of
curcumin significantly inhibited the invasion capacities of both U251 and U87 cells. These
data indicate that curcumin inhibits the migration of GB cells in a concentration-dependent
manner.
Next, we measured the adhesion ability of U251 and U87 cells. The adherent cells are
shown in Fig.3E. As displayed in the Fig.3F, treatments with10- and 20-µM of curcumin
inhibited the adhesion ability of both U251 and U87 cells.
3 Curcumin inhibits PI3K/mTOR protein activation
Since curcumin can inhibit the proliferation of glioblastoma, we determined whether
curcumin regulates the expression of AKT/mTOR in the cellular survival pathways of GB. To
this end we examined the protein expression of AKT/mTOR. As shown in Fig. 3A, no
differences between the non-phosphorylated (total) protein of AKT and mTOR in U251 cells
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in response to curcumin were observed. As phosphorylation is the activator, we also
measured the phosphorylation levels of P13K/mTOR signaling. As shown in Fig.3B, the
curcumin treatment group significantly decreased the phosphorylation of AKT(p-AKT) and
mTOR(p-mTOR) in U251 cells. In addition, the PI3K/AKT inhibitor LY294002 markedly
decreased the p-AKT and p- mTOR protein expression.
4 Curcumin enhances the apoptosis of glioblastoma cells.
We also determined whether curcumin-induced apoptosis in glioblastoma. The apoptosis of
U251 and U87cells was measured using the annexin V and PI staining (Fig. 4A) and was
quantified by the fluorescence intensity (Fig. 4B). The results suggested that treatment
with10- and 20-µM curcumin significantly enhanced the apoptosis in both U251 and U87
cells.
Next, we determined the protein expression of the apoptotic proteins, caspase-3 and
beclin-1. Western bolt analysis showed that the caspase 3 and beclin-1 proteins were
markedly enhanced in U251 and U87 cells (Fig. 4C). Our results suggested that curcumin
enhanced apoptosis in glioblastoma cells.
5 Curcumin restores PTEN expression in glioblastoma cells.
PTEN is widely distributed in glioma patients and this fact prompted us to analyze whether
the PTEN affected the survival of glioma patients. We used the PrognoScan database to
predict the overall survival (OS) of patients with brain tumors. The result of the analysis
showed that the glioma database (GSE4412) was statistically significant for OS
(p=0.000706) (Fig. 5A). The analysis result was confirmed in the linkedomics database
(sample 396). There was a significant difference in OS between the tumor patients and the
normal group (p= 0.001613) for the survival of glioma patients (Fig. 5B). These results
indicated that the low PTEN expression in the glioma patients confers a poor prognosis.
Next, we determined the effect of curcumin on the PTEN protein expression. As shown in
Fig. 5C, treatments with 10- and 20-µM of curcumin enhanced the PTEN expression in both
U251 and U87 cells.
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PTEN is a tumor suppressor and also interacts with other important proteins. Since curcumin
can enhance apoptosis in glioblastoma cells, we tested whether curcumin could attribute to
the restoration of the P53 expression in glioblastoma. As shown in the Fig.5C, the P53
protein expression increased significantly with curcumin treatment in U87 cells.
6 Curcumin inhibited tumor growth and increased the PTEN expression in
xenografts
To detect the inhibitory effect of curcumin on tumorigenesis in vivo, nude mice were used
to establish a xenograft tumor model. As shown in Fig. 6A, curcumin treatment significantly
inhibited tumor growth. In addition, tumor weight was significantly lower in the
curcumin-treated group, compared the control group (Fig. 6B). Hematoxylin and eosin (HE)
staining was used to show the morphological changes of tumor tissue in the xenograft tumor
model. We found that the tumor tissue in the curcumin-treated group contained focal
necrosis and had a reduction in angiogenesis (Fig. 6C). Further, the PTEN and P53
expression were significantly higher in the curcumin-treated group, compared to the control
group (Fig. 6D). Therefore, these data indicate that curcumin has a significant inhibitory
effect on glioma growth in vivo, and the mechanism appears to be the promotion of PTEN
and P53 expression.
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Discussion
We investigated the biological effects of curcumin in glioma cells and evaluated its
potential efficacy in vitro (italicize) and in vivo (italicize), which are caused by inhibitory
effects on tumors. We found that curcumin has a dose-dependent effect on proliferation and
migration of glioma cells and inhibits significantly the proliferation, migration, and
invasiveness of glioma cells. The inhibition was associated with the proliferating activity of
the AKT/mTOR signaling pathway under curcumin treatment. We also found that
curcumin-induced apoptosis and significantly regulated the expression of PTEN and P53 in
glioma cells. We also found that curcumin significantly inhibited tumor volume and caused
tumor necrosis, while restoring PTEN expression.
Our study indicates that curcumin inhibits the proliferation of glioblastoma cells in a
dose-dependent manner. We also found that curcumin decreased the migration and
invasion of glioma cells in vitro (italicize). The effect of antitumor of curcumin is consistent
with previous reports[18,19].
We also analyzed the possibility that the inhibition of tumor proliferation may be the
result of action of curcumin on the AKT/mTOR pathway. It is known that AKT/mTOR
activation can promote cell proliferation and has been confirmed to contribute to the
biological behaviors for many tumors[20,21]. The growth of tumors is associated with the
activation of the AKT/mTOR pathway[19,22]. We found that AKT/mTOR expression is
up-regulated to promote the proliferation of glioma cell. Given this observation, we
measured the proliferation, migration and invasion of U251 and U87 glioma cells, and found
that curcumin significantly inhibited these biological process in glioma cells.. In aggregate,
these data indicate that curcumin inhibits the proliferation, migration and invasion of glioma
cells in vitro (italicize) and was associated with the inhibition of AKT/mTOR activation.
Many chemotherapy drugs have the ability to induce cell apoptosis[23]. Apoptosis might
be mediated by intrinsic or extrinsic signaling pathways[24] The apoptosis of glioblastoma
was enhanced by curcumin treatment in the present study. Furthermore, caspase 3 and
beclin-1 are key regulators for intrinsic apoptotic signaling pathways[25,26] and induce the
mitochondrial release of caspase-activated cytochrome C[27]. We found that
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curcumin-induced apoptosis and was associated with decreased caspase 3 and beclin-1.
These findings suggest that curcumin can induce intrinsic apoptotic signaling involved in the
activation of apoptosis[28].
The PTEN gene is known as a negative regulator of AKT/mTOR signaling for
tumorigenesis[29,30]. PTEN inhibition is associated with invasive tumor growth, enhanced
metastasis, and poor prognosis for GB patients[31]. Our study showed that curcumin inhibited
the effects of phosphorylation of AKT and enhanced PTEN. Curcumin promoted the
expression of PTEN, in vitro. These findings are consistent with the inhibition of tumor
growth following r curcumin treatment in vitro (italicize) and in vivo[9,32].
The tumor suppressor protein, P53, may inhibit the growth of cancer cells by regulating
various molecular mechanisms, including apoptosis and DNA repair[33]. P53 decreased
proliferation, migration and invasion in many tumor cells[20]. Our study demonstrates that
curcumin enhances P53 expression in vitro and in vivo.
In summary, there are many cellular mechanisms by which curcumin can exert its
beneficial effects against GB. First, curcumin reduces the AKT/mTOR pathway and inhibits
GB cell proliferation and migration. Secondly, curcumin induces apoptosis. Curcumin
increases PTEN expression and reverses P53 expression. Finally, curcumin can
significantly inhibit tumor growth, induce tumor apoptosis, and up-regulates PTEN
expression in vivo.
Conclusion
Our findings show that curcumin treatment partially inhibits the proliferative activity of
glioma cells and suppresses the invasive behavior by down-regulating AKT/mTOR activity,
thereby inducing apoptosis of glioma cells and increasing PTEN and P53 expression.
Furthermore, this positive response to curcumin is dose-dependent and was confirmed in
U87-xenografts in vivo. These results suggest that further explorations in evaluating the
potential use of curcumin for the treatment of GB and other malignant tumors are warranted.
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Acknowledgments Not applicable.
Authors’ contributions LM and WZ designed the study. LM, WZ and LF performed the
experiment. XH analyzed the data. YL HZ contributed to the writing the manuscript. All
authors have read and approved the final manuscript.
Competing interests The authors declare that they have no competing interests.
Availability of data and materials All data generated or analyzed during this study are
included in this published article.
Funding This work was supported by the grants from the Fund of the Science and
Technology Department of Hubei Province (2018CFB737) and the Found of Hubei
University of Science and Technology (2018xzy02,2019xz01). No specific funding was
received for this study.
Declaration
Ethics approval and consent to participate All animals’ procedures were conducted in
accordance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH publication 85-23, revised 1996) and approved by the institutional animal care
and use committee at the Hubei University of Science and Technology, China. No ethical
approval for the use of the human glioma cell lines was required.
Page 17
Figure Legends
Figure 1 The effects of curcumin on the proliferation and viability of U251 and U87 cell lines.
The anti-proliferative effects of curcumin (0-, 10-, 20- and 40-µM) on U251 and U87 cell lines
were assessed by MTT assay after 48 h of treatment( U251 in A). The values are given as
mean ± S.E. of three independent experiments for U251(B) and U87(C) cell. *P<0.05 and
**P<0.01, compared to control group.
Figure 2 Curcumin inhibits the migration, invasion, and adherence of U251 and U87 cells in
vitro. After U251 and U87 cells were treated with different concentrations of curcumin, the
migration ability was determined by the wound healing assay (A). The invasion of the glioma
was measured by the transwell assay (C) and cell adherence was evaluated by the ECM
assay (E). The migration (B), invasion (D) and adherence (F) values are given as mean ±
S.E. for three independent experiments for U251 and U87 cells. *P<0.05 and **P<0.01,
compared to control group.
Figure 3. Curcumin suppresses AKT/mTOR activation in U251 cells. After U251 cells were
treated with 20 µM of curcumin or 25 µM of LY294002, the levels of AKT, p-AKT, mTOR,
p-mTOR and GAPDH were determined by the Western blot analysis A). The quantitative
analysis of AKT, p-AKT, mTOR and p-mTOR was conducted (B). Values are given as mean
± S.E. for three independent experiments. *P<0.05 and **P<0.01, compared to control
group.
Figure 4. Curcumin enhances apoptosis in glioma cells. After U251 cells were treated with
10- and 20-µM of curcumin, apoptotic cells were visualized by the Annexin V and PI staining
(A).The quantitative analysis of apoptotic cells was performed (B). The apoptosis proteins,
caspase-3 and beclin-1, were measured by the Western blot analysis (C). Values are
given as mean ± S.E. for three independent experiments. *P<0.05 and **P<0.01, compared
to control group.
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Figure 5. The functional analysis for the PTEN gene and the PTEN protein expression after
curcumin treatment. The overall survival of the PTEN gene in the glioma patients was
plotted from PrognoScan database(p-value=0.000706)(A) and was plotted from
Kaplan-Meier Plotter database(p-value=0.001613) (B). The PTEN and P53 protein were
detected by the Western blotting and were analyzed (C). Values are given as mean ± S.E.
for three independent experiments. *P<0.05, compared to control group.
Figure 6 Curcumin effectively inhibited the tumor growth and enhanced the PTEN and P53
expression in the xenograft tumor model of glioma. The tumor volume was detected io
different days(A). The tumor size and wet tumor weight (B) were measured at the time of
dissection. The histological features of the tumors were examined by HE (C). The PTEN and
P53 protein were detected by the Western blots and were quantified (D). Values are given
as mean ± S.E. for three independent experiments. *P<0.05, compared to control group.