ESC-3 induces apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling in vitro and in vivo

  • Authors:
    • Qi-Rui Fu
    • Wei Song
    • Yi-Tao Deng
    • Hua-Liang Li
    • Xiao-Mei Mao
    • Chen-Lu Lin
    • Ya-Hui Zheng
    • Shu-Ming Chen
    • Qiong-Hua Chen
    • Qing-Xi Chen
  • View Affiliations

  • Published online on: November 18, 2016     https://doi.org/10.3892/ijo.2016.3773
  • Pages: 241-251
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Abstract

Apoptosis, programmed cell death under physiological or pathological conditions, plays a critical role in the tissue homeostasis of eukaryotes. It is desirable to prevent the occurrence and metastasis of cancer through inducing apoptosis. Our previous study demonstrated that apoptosis could be induced by extract from crocodile in human cholangiocarcinoma. ESC-3, a novel cytotoxic compound isolated from the extract induced apoptosis in Mz-ChA-1 cells via the mitochondria-dependent pathway in a dose-dependent manner. In this study, ESC-3 significantly inhibited the proliferation of A2780 cells and arrested the cells at G2/M phase. After exposure to ESC-3, A2780 cells displayed typical morphological changes and the ability of colony-forming was remarkably inhibited. ESC-3 could significantly upregulate the expression of Bax proteins while Bcl-2 protein remained unchanged, resulting in the elevation of Bax/Bcl-2 ratio, which usually could induce apoptosis. The critical protein of Wnt signaling (β-catenin) was significantly downregulated, whereas Hes1, the downstream protein of Notch signaling, was remarkably attenuated through upregulating the expression of P53. In addition, xenograft models demonstrated that ESC-3 effectively suppressed the growth of OvCa tumors (T/C=42%). Western blot analysis of PCNA and VEGF confirmed that ESC-3 could inhibit the growth and metastasis of OvCa tumors. In conclusion, apoptosis could be induced by ESC-3 through Wnt/β-catenin and Notch signaling in vitro and in vivo, and might have therapeutic potential for the treatment of human OvCa.

Introduction

In all gynecologic cancers, ovarian cancer (OvCa) is one of the most lethal, mostly diagnosed at advanced stages for lack of the effective prior-diagnostic methods (1). It ranked the fifth most common cause of cancer-related death among women in the United States (2). After tumor cytoreductive surgery or administration of platinum-based chemotherapy, almost all the patients developed recurrent and disseminated malignancies with multiple drug resistance (3,4). Approximately 30% of epithelial ovarian cancer patients died in less than five years even with the progress in therapeutic methods (5). Therefore, it is urgent and essential to develop a novel non-toxic drug for improving the existing therapy.

Bile is composed by large amounts of bile acids such as chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), cholic acid (CA), and deoxycholic acid (DCA) (6). Snake bile has been found to possess anti-inflammatory, anti-convulsion and analgesic physiological functions (7). In a previous study, we found that extracts from Crocodylus siamensis bile could induce apoptosis effectively in human cholangiocarcinoma cells lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and liver cancer cell (SMMC-7721) (8). After purifying from the extracts, we obtained a more effective inducer ESC-3 (9) and studied the localization of prohibitin during apoptosis of human cholangiocarcinoma Mz-ChA-1 cells (10). However, it is still unclear whether ESC-3 could suppress the growth of ovarian tumor and the xenograft tumorigenesis in vivo.

In this study, we firstly demonstrated the antitumor effects of novel inducer ESC-3 on human ovarian carcinomas cell lines (A2780 cells, SKOV-3 cells and OVCAR-3 cells) in vitro and elucidated its inducing apoptosis mechanism. We also employed A2780 xenograft models to confirm the effectiveness and the potential as a candidate for ovarian cancer therapy.

Materials and methods

Cell preparation

SKOV-3 and IOSE-80 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml). The human OvCa A2780 cells were cultured in DMEM, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml). OVCAR-3 cells were cultured in complete medium supplemented with 10 µg/ml insulin. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cell proliferation assay

Cell viability was determined using the CCK-8 assay. A2780 cells were treated with ESC-3 at different concentrations (0, 5, 10, 20, 40 and 80 µg/ml) for 24, 48 and 72 h, respectively. Cell viability was determined using CCK-8 according to the manufacturer's instructions. Briefly, 4×103 cells per well were seeded in a 96-well plate and incubated at 37°C for 24 h. Subsequently, cells were treated with different concentrations of ESC-3 for 24, 48 and 72 h respectively. Then 10 µl WST-8 dye was added to each well, cells were incubated at 37°C for 1 h and the absorbance was finally determined at 450 nm using a microplate reader.

Morphological changes

The cells from control group and the group treated with ESC-3 (40 µg/ml) for 48 h were seeded onto coverslips and grown for 24 h. After washing with PBS three times, the cells were stained with Giemsa staining solution/Hoechst 33258/AO&EB and observed under standard inverted phase-contrast microscope or a fluorescence microscope.

Colony-forming assay

Cells were plated into a 6-well culture plate (1,200 cells/well) and allowed to adhere for 12 h before treatment. The next day, cells were treated with ESC-3 and equal volumes of DMSO. After 48 h, ESC-3-containing media was removed, and cells were allowed to form colonies in serum-free media for 14 days, and then the colonies were fixed with a solution of acetic acid and methanol (1:3) for 15 min, stained with Giemsa for 15 min and counted manually.

Flow cytometry assay

Cells were treated with ESC-3 (5, 10, 20, 40 and 80 µg/ml) for 48 h and then collected and washed twice with PBS. After fixing in ice-cold 70% ethanol for 12 h, the samples were washed twice with PBS and then incubated with 10 mg/ml RNase and 1 mg/ml PI (propidium iodide) for 30 min in the dark. Finally, the samples were evaluated by Flow Cytometry 500, and the data were analyzed using Cell Fit software. The Annexin V-fluorescein isothiocyanate (V-FITC)/PI double staining assay was conducted to quantify cell apoptotic proportion according to the manufacturer's instructions. Briefly, after exposure to 40 µg/ml ESC-3 the A2780 cells were harvested and stained with Annexin V-FITC and PI for 20 min at room temperature. Following washing with PBS, we used Flow Cytometry 500 to detect the fluorescence of the cells.

Western blot analysis

To explore the mechanism of apoptosis induced by ESC-3, proteins were extracted with RIPA buffer (10 mM Tris, 150 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.1% deoxycholate, 1 mM PMSF, 2 mM sodium fluoride, and 1 mM sodium orthovanadate), then centrifuged at 13,000 rpm for 30 min at 4°C. Briefly, equivalent amounts of proteins were analyzed by 10–15% SDS-PAGE, then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were then incubated with specific primary antibodies. Finally, proteins were visualized with peroxidase-coupled secondary antibody, using the ECL system (Pierce Co., USA) for detection.

Xenograft models

Female Balb/c nude mice (16±2 g) were purchased from SLRC Laboratory Animal Co., Ltd. Shanghai, China. The animals were kept on 12-h-light/12-h-dark cycle under the condition of a constant temperature of 21–22°C and 60–65% humidity. Additionally, they were maintained on standard pellet diet and water ad libitum throughout the experiments. The experimental procedures were performed in accordance with the guidelines for the humane treatment of animals set by the Laboratory Animal Center. Briefly, ~5×106 A2780 cells were subcutaneously injected into nude mice to establish human ovarian cancer xenograft. When the tumor reached a volume of 100 mm3, the mice were randomized to control and treatment groups, then the control groups received corn oil (every three days, i.g. administration) and the other groups 50 mg/kg ESC-3 (every other three days, i.g. administration) for 30 days. Tumor volume (V) was calculated as V = (length × width2)/2. The tumor volume at day n was expressed as relative tumor volume (RTV) according to the following formula: RTV = TVn/TV0, where TVn is the tumor volume at day n and TV0 is the tumor volume at day 0. Therapeutic effects of treatment were expressed in terms of T/C (%) using the calculation formula T/C (%) = mean RTV of the treated group/mean RTV of the control group × 100%. Tumors and internal organs (heart, liver, spleen, lung and liver) were fixed in formalin and processed for hematoxylin-eosin staining. The samples were processed by the following published standard methods. In brief, the sections (4–5-µm) mounted on glass slides were deparaffinized, rehydrated through grated alcohols to distilled water, stained with hematoxylin and eosin, and then observed under a light microscope (Olympus BH-2).

Results

ESC-3 inhibits cell proliferation and colony-forming ability in human OvCa cell lines

To evaluate effects of ESC-3 on proliferation of A2780 cells using CCK-8 assays, A2780 cells were treated with ESC-3 at different concentrations (5, 10, 20, 40 and 80 µg/ml) for 24, 48 and 72 h, respectively. As shown in Fig. 1A, after treated with different concentration of ESC-3, cell proliferation became slower compared to that of untreated cells (P<0.01). With ESC-3 concentrations of 5, 10, 20, 40 and 80 µg/ml for 48 h, the inhibition rates were 18, 22, 33, 57 and 76%, respectively. Besides, ESC-3 significantly suppressed the proliferation of SKOV-3 cells and inhibited colony-formation ability as shown in Fig. 3A and G. After exposure to 60 µg/ml ESC-3, the changes in cell morphology occurred with typical trait of apoptosis: cell shrinkage, chromatin condensation, apoptotic body formation, dense nuclei (Fig. 3C). However, ESC-3 did not induce apoptosis in human ovarian carcinomas OVCAR-3 effectively (Fig. 3H). Our data indicated that ESC-3 could inhibit the proliferation of A2780 cells and SKOV-3 cells in a dose- and time-dependent manner.

To investigate whether morphological changes happened after ESC-3-treatment, we used an optical inverted microscope to visualized morphological features. As shown in Fig. 1B, after treated with 40 µg/ml ESC-3, A2780 cells were smaller in size and close to rotundity compared to the control group. With Hoechst 33258 staining, the treated cells emitted a higher fluorescence intensity and were smaller than those of the control group in size. After AO/EB staining, the treated cells displayed orange and red fluorescence, while the untreated cells emitted a low green fluorescence in a homogeneous manner. To determine the ability of colony-forming, 2,000 A2780 cells were seeded into and treated with 40 µg/ml ESC-3. As shown in Fig. 1C and D, the ESC-3-treated cells showed a significant decrease in colony number compared to the untreated A2780 cells. There results suggested that ESC-3-treated A2780 cells displayed typical morphological features of apoptosis and a significant reduction in the colony-forming ability (P<0.01).

ESC-3 causes cell cycle arrest and induces apoptotic cell death in A2780 and SKOV3 cell lines

To confirm whether the inhibition of cellular proliferation was associated with the cell cycle distribution, we performed a cell cycle analysis after exposure to different concentration of ESC-3 (5, 10, 20, 40 and 80 µg/ml). As shown in Fig. 2A and B, after treated with ESC-3 for 48 h, the cell cycle distribution of A2780 cells was altered in a dose-dependent manner. The proportion of cells at the G2/M increased from 22.1 to 55.3% (P<0.01), while the percentage of cells in G0/G1 phase was 63.8% in the control group and decreased to 30.3% after treatment with 40 µg/ml ESC-3 (P<0.01). On the other hand, the proportion of ESC-3-treated at S phase displayed non-significance compared to untreated cells. ESC-3 caused cell cycle arrest and induced apoptotic cell death in SKOV-3, which could confirm the consistency of the in vitro study in A2780 cells as shown in Fig. 3E. Our data indicated that ESC-3 arrested A2780 cells and SKOV-3 cells at G2/M phase and suppressed cell proliferation. The protein level of CDK1 and cyclin B1 were decreased after exposure to ESC-3 compared with the untreated group in A2780 cells (Fig. 2C).

We preformed flow cytometric analysis using dual staining with Annexin V and propidium iodide to distinguish between early apoptotic and late apoptotic cells. As shown in Fig. 2D and E, the apoptotic proportion of cells with 40 µg/ml was 13.5% compared to untreated cells with 2.5% apoptotic proportion (P<0.05). Therefore, we demonstrated that apoptosis could be induced by ESC-3.

ESC-3 induces A2780 apoptotic cell death through Wnt/β-catenin and Notch pathway

To investigate the apoptosis mechanism induced by ESC-3, the expression of apoptosis-related (Bax, Bcl-2 and P53) and pathway-related (Wnt2, β-catenin, Notch1, Notch2 and Hes1) proteins were measured by western blotting and quantified using ImageJ software. As shown in Fig. 4A and B, the proteins level of Bax were significantly increased (P<0.01) after exposure to ESC-3 for 24 h, while the change in expression of Bcl-2 proteins remained non-significant; therefore, the ratio of Bax to Bcl-2 increased (P<0.01) significantly compared to the untreated group. Moreover, the protein levels of P53 were remarkably increased in a dose-dependent manner. As shown in Fig. 4E and F, the expression of Wnt2 proteins were decreased to 65% protein levels of the untreated group, and the expression of β-catenin at the protein levels decreased (P<0.01) significantly compared the proteins obtained from the untreated cells. Besides, the expression of Notch1 and Notch2 proteins, the receptor located at cell membrane initiating the Notch pathway, decreased significantly (P<0.01) in a dose-dependent manner; the expression levels of the Hes1 proteins, the downstream proteins of Notch pathway, decreased (P<0.01) obviously (Fig. 3C and D). Our data suggested that the Wnt/β-catenin and Notch pathway might play a significant role in induction of cell apoptosis by ESC-3.

ESC-3 inhibits the growth of A2780 xenograft tumor in Balb/c nude mice without noticeable toxicity

To determine the antitumor effects of ESC-3, 1×106 A2780 cells were injected subcutaneously into the right flank of Balb/c nude mice to build the tumor xenograft models as shown in Fig. 5A. During the study, the body weight and tumor volume of nude mice was tracked every three days to detect the non-toxic and effectivity of ESC-3 in vivo. As shown in Fig. 5D and E, the body weight of nude mice treated with ESC-3 displayed non-significant changes compared to the control group, while the volume demonstrated apparent difference between the treated and the control group (at the 15th day P<0.05 and at the 21st day P<0.01). After administration (i.g) with ESC-3 for 24 days, the mice were sacrificed (Fig. 5B) and the tumors was excised (Fig. 5C) and weighed (Fig. 5F), the tumors from ESC-3-treated mice were smaller and lighter than those of the control group (P<0.01). As shown in Fig. 5G, hematoxylin-eosin staining of ESC-3-treated pathological paraffin sections displayed typical apoptotic features: condensed chromatin and pyknotic nuclei. To confirm the non-toxicity of ESC-3 further, the viscus of Balb/c nude mice were excised, weight and stained with hematoxylin-eosin, it showed that the relative visceral coefficient have no remarkable difference between the control group and ESC-3-treated group (Fig. 5H), and hematoxylin-eosin staining of ESC-3-treated viscus pathological paraffin sections displayed no significant changes in organizational structure (Fig. 5I). Our data demonstrated that ESC-3 effectively suppressed the growth of A2780 xenograft tumors (T/C=42%) without affecting the weight and viscus of nude mice as shown in Tables I and II. Therefore, the ESC-3 is efficient and non-toxic to ovarian cancer.

Table I

Evaluation system of ESC-3 on ovarian xenograft models.

Table I

Evaluation system of ESC-3 on ovarian xenograft models.

Mean volume
(t=0)
Mean volume
(t=24)
RTVT/C
Control group95.53 mm31,323.32 mm313.85
Treated group91.61 mm3545.04 mm35.9542%

[i] RTV, relative tumor volume; RTV = TVn/TV0, where TVn is the tumor volume at day n and TV0 is the tumor volume at day 0. Therapeutic effects were expressed in terms of T/C (%) using the calculation formula T/C (%) = mean RTV of the treated group/mean RTV of the control group × 100% (n=8).

Table II

Weight of nude mouse body, tumor and viscera at the 24th day.

Table II

Weight of nude mouse body, tumor and viscera at the 24th day.

No.HeartLiverSpleenLungKidneyBodyTumor
  10.09141.50330.23460.12270.321221.54.1511
  20.11151.33870.15550.12730.392922.72.2685
  30.11581.42380.11710.13100.303117.21.6610
  40.09831.39530.13160.14450.338119.53.0444
  50.11561.35700.24250.19930.329821.82.8495
  60.10551.16780.11080.13700.301121.13.8889
  70.10611.33550.12330.13210.305519.52.9054
  80.09651.13360.10640.14970.296819.33.6030
Mean0.10511.33190.15270.14300.323620.33.0465
  90.12461.35000.14020.16500.345722.30.4304
100.10901.46540.10720.12660.344621.11.3244
110.11251.34350.13220.14990.333220.90.6967
120.12031.51700.13610.18410.350321.81.2925
130.09131.22720.15140.11610.325218.10.4968
140.11281.40280.17100.16510.358720.31.6308
150.11861.40500.10230.15200.312416.81.3545
160.11471.26060.08990.14540.353519.50.5738
Mean0.11301.37340.12880.15050.340520.1   0.9750b

a P<0.05,

b P<0.01 compared to the control group (n=8). Control group numbered 1–8 (every three days, i.g. 100 µl corn oil). Treated numbered 9–16 (every three days, i.g. 100 µl 80 mg/kg ESC-3).

Tumor inhibition is induced by ESC-3 through Wnt/β-catenin and Notch pathway

After tumors were treated (i.g) with 80 mg/kg ESC-3 every three days for 24 days, the expression of proteins obtained from the tumors were measured by western blot analyses to determine the consistency of the results in vitro and in vivo. We examined the expression levels of apoptosis-related and pathway-related proteins, including Bax, Bcl-2, P53, Wnt2, β-catenin, Notch1, Notch2, Hes1, VEGF and PCNA. As shown in Fig. 6A and E, the result of western blot analyses revealed that the expression of Bax was significantly increased ~2.9-fold (P<0.01) after administration (i.g) with ESC-3, while proteins of Bcl-2 remained about the same compared to the control group; therefore, the ratio of Bax to Bcl-2 also increased (P<0.01). Moreover, we observed that the levels of P53 proteins obtained from ESC-3-treated increased ~3.3-fold (P<0.01), which displayed the consistency also observed in vitro and in vivo. Furthermore, we determined the proteins in Wnt/β-catenin and Notch pathway, as shown in Fig. 6B and F, the expression of Notch1 and Notch2 proteins decreased ~1.21-fold (P<0.05) and 0.06-fold respectively, the proteins level of Hes1, the downstream effector proteins of Notch pathway, were decreased (P<0.01) significantly after administration (i.g) of ESC-3 for 24 days. ESC-3 has a significant effect on the expression levels of Wnt2 and β-catenin proteins based on the ESC-3-treated tumors (Fig. 6C and G). Furthermore, the expression of VEGF and PCNA were significantly decreased at the protein level compared with the control group (Fig. 6D and H).

Discussion

Apoptosis, programmed cell death under physiological or pathological conditions, plays a critical role in the tissue homeostasis of eukaryotes (11). It is desirable to prevent the occurrence and metastasis of cancer through inducing apoptosis (12). The cancer cells have the ability to sustain chronic proliferation through the cell cycle, which is the most fundamental feature (13). Our data were suggested that ESC-3 significantly suppressed the proliferation of A2780 cells and inhibited colony-formation ability. After exposure to different concentrations of ESC-3, the A2780 cells were arrested at G2/M phase through downregulation of CDK1 and cyclin B1, two critical G2/M transition regulators (14). The primary indicators of apoptosis under physiological and pathological conditions, are the morphological changes (11,15,16). After exposure to 40 µg/ml ESC-3, the changes in cell morphology occurred with typical trait of apoptosis: cell shrinkage, chromatin condensation, apoptotic body formation, and dense nuclei. Our results were in agreement with the study by Horowitz et al (17), concluding that chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) possess the ability to induce apoptotic phenomenon in ovarian cancers. Then, SKOV-3 and OVCAR-3 cell lines were analyzed in vitro. The results suggested that ESC-3-treated SKOV-3 cells displayed typical morphological features of apoptosis and a significant reduction in the colony-forming ability. Besides, ESC-3 caused cell cycle arrest and induced apoptotic cell death in SKOV-3, which confirmed the consistency with the in vitro study in A2780 cells. However, ESC-3 did not induce apoptosis in human ovarian carcinomas OVCAR-3 effectively, this difference in suppression of ESC-3 on A2780, SKOV-3 and OVCAr-3 cell proliferation confirmed the different phenotypes of these three ovarian cancer cell lines, which emphasizes the need to recognize the heterogeneity of cancer cell populations (1821). Furthermore, we used normal human ovarian epithelial cells (IOSE-80) to perform the CCK-8 assay. We found that, compared to the control, ESC-3 dose-dependently inhibited A2780 cells and SKOV-3 cells, but not IOSE-80 in cell proliferation as shown in Figs. 1A and 3A and B.

The Bcl-2 family plays a vital role in regulating of apoptosis, including Bax and Bcl-2, of which the former induces apoptosis and the latter prevents apoptosis (22). In the present study, our data demonstrated that ESC-3 could significantly upregulate the expression of Bax proteins while the protein levels of Bcl-2 remained steady, resulting in the elevation of Bax/Bcl-2 ratio which usually induces apoptosis (23). In the previous study, bile extract from crocodile could induce apoptosis in human cholangiocarcinoma through the mitochondrial pathway (24). Furthermore, ESC-3 isolated from Crocodylus siamensis bile through a Sephadex LH-20 column (Pharmacia, Sweden) and an RP-18 reversed-phase column (25×0.3 cm), induced apoptosis in Mz-ChA-1 cells in a dose-dependent manner via the mitochondria-dependent pathway (9). In this study, we demonstrated the mechanism of apoptosis induced by ESC-3 in human OvCa carcinomas. The development and progression of several malignancies have association with the Notch signal pathway (25,26). There are four Notch receptors the Notch1, 2, 3 and 4 and five ligands (Delta-like1, 3 and 4) in Notch signaling mediating via cell-to-cell contact (27). A basic platform consisting of the ternary complex (Notch-CSL-MAM) could recruit coactivators including p300 to increasing the expression levels of Notch pathway downstream proteins (2830), however, this process could be blocked with the abnormal elevation of P53, a tumor suppressor in human cancers (31). Our results suggested that ESC-3 could significantly (P<0.01) upregulate the expression of P53, while hes1 remarkably decreased at the proteins levels. As previous research reported that Hes1, the downstream protein of Notch pathway, could be suppressed by the abnormal elevation of P53 through the combination with Notch-CSL-MAM complex, which might disturb the tendency of dose-response at the protein level (31). Our data do not indicate whether the association between p53 and the NTCs is mediated by direct association with MAM or through additional proteins. However, it was reported that P53 can be combination with MAM directly to block the recruitment of a coactivator. The associations between Wnt signaling and ovarian cancer confirmed that Wnt signaling played a critical role in the embryonic development of ovary and homeostasis including proliferation, differentiation, and migration (32,33). Our data suggested that ESC-3 could downregulate the expression of Wnt2 at the protein levels, whereas, the protein levels of β-catenin, the key effector in Wnt signaling, were decreased (P<0.01) significantly. In conclusion, ESC-3 induced apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling as shown in Fig. 7.

The xenograft models were employed to confirm the consistency with the in vitro assays and to determine the non-toxicity and effectiveness of ESC-3. Our data in vivo demonstrated that 80 mg/kg dose of ESC-3 every three days was highly effective and did not have toxic side effects. ESC-3 also effectively inhibited the expression of the proliferation marker PCNA (34), indicating its important role in the growth of ovarian tumors. Furthermore, the expression of vascular endothelial growth factor (VEGF), playing a critical role in angiogenesis (proliferation, migration and survival of endothelial cells) in cancer (35,36), was significantly decreased measured by western blotting. The molecular mechanism demonstrated the consistency between result in vitro and in vivo after treated with ESC-3. Therefore, our data suggested that ESC-3 was a safe, natural and effective compound in ovarian cancer therapy.

In conclusion, ESC-3 is a novel active compound that could arrest the A2780 cells and SKOV-3 cells at the G2/M phase and cyclin B1 proteins and induce apoptosis in a dose-dependent manner via the Wnt/β-catenin and Notch pathway, moreover, xenograft models displayed the consistency as showed in the results in vitro. Therefore, ESC-3 could be a potential therapeutic in ovarian carcinomas.

Acknowledgments

This study was supported by the Natural Science Foundation of China (grant nos. 81571418 and 81402309), the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (grant no. J1310027) and by the Natural Sciences Foundation of Fujian Province, China (2016J05105).

Abbreviations:

SCB

siamese crocodile bile

OvCa

ovarian cancer

FBS

fetal bovine serum

DMEM

Dulbecco's modified Eagle's essential medium

CCK-8

cell counting kit-8

DMSO

dimethyl sulfoxide

FITC

fluorescein isothiocyanate

PI

propidium iodide

PVDF

polyvinylidene difluoride

PMSF

phenylmethanesulfonyl fluoride

VEGF

vascular endothelial growth factor

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January-2017
Volume 50 Issue 1

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Spandidos Publications style
Fu Q, Song W, Deng Y, Li H, Mao X, Lin C, Zheng Y, Chen S, Chen Q, Chen Q, Chen Q, et al: ESC-3 induces apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling in vitro and in vivo. Int J Oncol 50: 241-251, 2017
APA
Fu, Q., Song, W., Deng, Y., Li, H., Mao, X., Lin, C. ... Chen, Q. (2017). ESC-3 induces apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling in vitro and in vivo. International Journal of Oncology, 50, 241-251. https://doi.org/10.3892/ijo.2016.3773
MLA
Fu, Q., Song, W., Deng, Y., Li, H., Mao, X., Lin, C., Zheng, Y., Chen, S., Chen, Q., Chen, Q."ESC-3 induces apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling in vitro and in vivo". International Journal of Oncology 50.1 (2017): 241-251.
Chicago
Fu, Q., Song, W., Deng, Y., Li, H., Mao, X., Lin, C., Zheng, Y., Chen, S., Chen, Q., Chen, Q."ESC-3 induces apoptosis of human ovarian carcinomas through Wnt/β-catenin and Notch signaling in vitro and in vivo". International Journal of Oncology 50, no. 1 (2017): 241-251. https://doi.org/10.3892/ijo.2016.3773