Introduction
Ovarian cancer (OC) is one of the commonest female malignancies and accounts for the leading death rates among the gynecologic cancers [
1,
2]. The main reasons of the poor prognosis of OCs are the delayed diagnosis due to the very subtle symptoms at the early stage of ovarian carcinogenesis [
3] and the easiness of spreading through blood dissemination [
4] and peritoneal transplantation [
5,
6]. Surgical treatment is the first choice to remove ovarian cancers if the tumours are well-differentiated, in relative small sizes and/or confined to the ovary [
7,
8]. However, the patients with advanced OCs have to be operated for debulking the disease and then treated by standard chemotherapy such as a dose-dense paclitaxel and carboplatin regimen [
9,
10]. Although the therapeutic outcome has been improved by more accurate staging of the disease and more aggressive surgical excision of tumor spots in the abdomen, the overall survival rates remain unoptimistic because of the frequent tumour recurrence and severe toxic effects of the anticancer agents [
11-
13]. For these reasons, it would be necessary to explore more efficient and lesser toxic agent(s) with clearer molecular targets for better adjuvant management of ovarian cancers.
Resveratrol (3,5,4′-trihydroxy-
trans-stilbene) has been regarded as a non-toxic polyphenolic compound that can be found in grapes, berries, peanuts and red wine [
14]. A body of evidence has demonstrated that resveratrol is able to inhibit the growth of many cancers such as bladder cancer, breast cancer and primary brain tumors [
15-
17]. Increasing data have shown that resveratrol can exert its biological effects on cancer cells by altering multiple molecular targets [
18,
19]. For example, it suppresses growth and induces apoptosis of human medulloblastoma cells accompanied with inhibition of STAT3 activation and transcription [
18]. More importantly, the anticancer doses (100 μM to 200 μM) of resveratrol have little harmful effect on glial cells and neurons in central nervous system and transitional epithelial cells of the urinary bladder [
15,
17,
19]. The inhibitory effects of resveratrol on ovarian cancer cells have been documented as well [
20,
21]. Although some studies have shown certain molecular alterations in resveratrol-treated ovarian cancer cells, such as down-regulation of Akt/GSK signaling [
22] and VEGF expression [
23], the critical event(s) among those alterations remains largely unknown. It is therefore necessary to address this point by comprehensively analyzing the statuses of ovarian cancer-related signaling pathways as well as their downstream genes.
Some signaling transduction pathways are found to be activated in the processes of ovarian carcinogenesis and play favorable roles in cell growth and survival [
24-
26]. For instance, hyperactive Jaks/STAT3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells [
27]. Similarly, Wnt/beta-catenin pathway also contributes to the proliferation of human ovarian cancer cell [
28] and inhibition of Notch signaling, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy [
25]. The data obtained from other cancer systems reveal that resveratrol can inhibit the signaling pathways mediated by STAT3, Wnt and Notch when exerting its cancer suppressive effects [
18,
29,
30]. The current study thus refers to the above findings as a cue and/or a cutting edge to identify the critical molecular event(s) caused by resveratrol in ovarian cancer cells.
Discussion
Ovarian cancer is one of the most lethal malignancies due to its strong spreading tendency via different dissemination routes including peritoneal implantation [
6,
39]. Because of the difficulty to remove cancer cells radically, adjuvant chemotherapy is employed to reduce the risk of tumor relapse [
40]. Nevertheless, the therapeutic outcome of OC patients is not optimistic due to the frequent drug resistance of cancer cells and severe toxic effects of anticancer drugs [
41,
42]. Apparently, more effort should be made to explore safer and more effective agent for ovarian cancer patients. It has been known that the anticancer doses of resveratrol are non-toxic to some kinds of normal cells/tissues [
15,
43], suggesting the potential values of this compound in the treatment of human cancers. Resveratrol also exerts inhibitory effects on ovarian cancer cells [
21,
22]. The current study further demonstrates that the resveratrol sensitivities of ovarian cancer cells are not identical, because 100 μM resveratrol is sufficient to cause G1 phase arrest and remarkable apoptosis in CAOV-3 while a dose of 120 μM is required to induce similar cellular events in OVCAR-3 cells. Although the underlying reason(s) leading to the differential resveratrol sensitivities remains to be disclosed, our results have potential translational values because 1) both OVCAR-3 and CAOV-3 cells are resistant to cis-platinum (cis-diamminedichloroplatinum), a commonly used agent in anti-OC therapy [
43,
44], 2) 120 μM resveratrol is harmless to normal neural and urothelial cells in vivo [
15,
45] and 3) long-term intra peritoneal administration of 150 mg (657 μM)/kg/per day resveratrol dose not affect the life quality of the rats including the reproductive ability [Li-Xia Zhong et al. unpublished data]. In this context, it would be considered that resveratrol may be a promising candidate to treated ovarian cancers, especially those with cis-platinum resistance.
A body of evidence suggests that many molecular alterations occur during ovarian carcinogenesis, of which the activated Notch, Wnt and STAT3 signaling pathways are supposed to play active roles in the carcinogenic process by up-regulating the expression of some tumor promoting genes such as c-Myc, survivin and Bcl-2 [
46,
47]. On the other hand, resveratrol possesses multifaceted targeting capacities [
48,
49] and the cancer-associated signaling pathways mediated by STAT3, Wnt2 and/or Notch1/2 have been known as its molecular targets [
35]. Although the inhibitory effects of resveratrol on ovarian cancer cells have been documented, the critical molecular event(s) caused by resveratrol remain largely unknown. In this study, the statuses of STAT3, Notch and Wnt2 signaling in OVCAR-3 and CAOV-3 cells and the influences of resveratrol in them are investigated. It is revealed that all of the three signaling pathways are activated in the two cell lines because of the co-existence of nuclear translocations of phosphorylated STAT3 (p-STAT3), HES1 and β-catenin. After resveratrol treatment, nuclear labeling of p-STAT3, β-catenin and HES1 become rare and weakened, indicating the concurrent inhibition of the biological activities of STAT3, Notch and Wnt2 signaling by this multi-targeting compound and its correlation with the suppressed growth of ovarian cancer cells.
c-Myc, Bcl-2 and survivin are well known cancer promoter genes and their transcription can be triggered by STAT3, Notch and Wnt signaling, respectively [
18,
46,
47]. In accompany with distinct growth arrest and apoptosis, the expression of these three genes is down-regulated in resveratrol-treated OVCAR-3 and CAOV-3 cells, which may be considered as the consequence of concurrent STAT3, Notch and Wnt inactivation. It has been recognized that reduction or absence of membrane E-cadherin distribution indicates the dedifferentiation states of epithelial cells and is one of the major reasons of β-catenin cytoplasmic accumulation and nuclear translocation [
50,
51]. In resveratrol treated OVCAR-3 cell population, the level of E-cadherin expression is increased and more abundant β-catenin membrane distribution can be observed, indicating the favorable effects of resveratrol on cell differentiation and inhibitory effect on Wnt signaling. Based on this notion, the lack of membranous β
-catenin labeling of resveratrol-treated CAOV-3 cells would be largely due to the silenced E-cadherin expression. Even though, CAOV-3 cells remain sensitive to resveratrol, suggesting that the presence or absence of E-cadherin expression and β
-catenin nuclear translocation may not be the critical factor in determining the responsiveness of OC cells to resveratrol.
Although resveratrol suppressed STAT3, Notch and Wnt activations have been evidenced in OVCAR-3 and CAOV-3 cells, it is still unclear which of them are/is closely linked with resveratrol-caused cell crisis. To address this issue, the two ovarian cancer cells are treated by selective inhibitors of Notch, Wnt and STAT3 respectively and their responses are compared with that of their resveratrol-treated and normally cultured counterparts. The results reveal that Notch inhibitor L-685,458 and Wnt inhibitor XAV-939 efficiently block nuclear translocations of HES1 and β-catenin, but neither of them leads to distinct growth inhibition of OVCAR-3 and CAOV-3 cells. On the other hand, STAT3 inhibitor AG490 suppresses proliferation and induces apoptosis in the extents as similar as that of resveratrol-treated cells. These findings thus suggest 1) the critical roles of STAT3 activation in the growth and survival of human ovarian cancer cells and 2) STAT3 signaling as the common oncotarget of resveratrol in cancer cells with different origins [
52]. Although simply block of Notch or Wnt signaling transduction has little inhibitory effect on the two OC cell lines so far checked, the implications of their inactivation can not been overlooked because of their compensatory roles in regulating the expression of ovarian cancer-related genes including the ones examined in this study.
Conclusion
Our current study demonstrate the efficiencies of resveratrol in inhibiting human ovarian cancer cells in terms of remarkable G1 phase accumulation, increased apoptosis fraction and concurrent suppression of Wnt, Notch and STAT3 signaling as well as their downstream cancer-related gene expression. Treatments with Wnt, Notch or STAT3 selective inhibitor reveal that only AG490, a JAK-specific inhibitor, inhibits OVCAR-3 and CAOV-3 cells in the extent as similar as that of resveratrol, suggesting the importance of STAT3 activation in the maintenance and survival of ovarian cancer cells. It is therefore possible that the activated STAT3 signaling is the critical molecular target of resveratrol and this polyphenol compound would be an alternative option in the management of ovarian cancers, especially the ones insensitive to conventional therapeutic drugs.
Acknowledgments
This work was supported by National Key Research Fund from National Department of Science and Technology (2008ZX09101-049), the grants from National Natural Science Foundation of China (No. 81450016, 81272786, 81071971, 81072063 and 30971038), Research Fund for PhD supervisors from National Education Department of China (20122105110005), Program Fund for Liaoning Excellent Talents in University (LJQ2012078) and Program for Changjiang Scholar and Innovative Research Team in University (PCSIRT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZLX carried out the analyses of cellular and molecular effects of resveratrol on ovarian cancer cells. LH participated in data analyses and morphological examination. MLW carried out cell culture and the statistical analysis. XYL participated in sample preparation for protein and RNA analyses. ZMJ carried out Western blotting and RT-PCR analyses. XYC participated in immunocytochemical staining. JL involved in the design of the study and performed the statistical analysis. YZ participated in the design of the study and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.