Molecular Mechanism Of Resveratrol Against Ovarian Cancer Research

Background and objectives: Ovarian cancer(OC) is one of the commonest female malignancies,and there is an estimated 200,000 new cases of ovarian cancer with more than 120,000 deaths each year across the world, so the mortality accounts for the leading death rates among the gynecologic cancers.The ovary is located in the depths of pelvic cavity, so that the early symptoms are not obvious, and about 75% of patients are diagnosed as advanced disease with metastasis. At present, for the patients with the tumors in advanced stages,combination of operation with chemotherpay regimen is standard treatment.Although the therapeutic outcome has been somewhat 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 recurrence and severe toxic effects of anti-cancer drugs.Combination chemotheray drugs based on platinum not only kill tumor cells, but also damage the normal tissues and cells surrounding tumor.For the patients, the side effect of the chemotherapy drugs is unbearable,so it is difficult to continue the next stage. Therefore,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 polygonum cuspidatum, grapes, mulberries, peanuts and so on. Its biological effect mainly has cardiovascular protection,anti-mutation,anti-inflammatory,anti-oxidation,anti-aging,anti-tumor,induced apoptosis, protecting the central nervous system,etc. A body of evidence has demonstrated that resveratrol is able to inhibit the growth of many cancers such as bladder cancer, breast cancer,medulloblastoma,etc.The more important thing is that resveratrol has no obvious toxic and side effect on the body and normal tissues.So resveratrol has a high medicinal value on anti-tumor. Recent data show that resveratrol can exert its biological effects on cancer cells by altering multiple molecular targets. For example, it suppresses growth and induces apoptosis of human medulloblastoma cells accompanied with S phase retardation,and the low expression of c-Myc、cyclinB and cyclinD1.We also found that resveratrol could up-regulated CYP1A1 but down-regulated CYP1B1 expression and change the expression of some cancer-associated genes by altering the activities of Wnt、Notch and STAT3 signal pathways.The inhibitory effects of resveratrol on ovarian cancer cells have been documented as well. Although some studies have shown certain molecular alterations in resveratrol-treated ovarian cancer cells, the critical event(s) among those alterations remains largely unknown. It is therefore necessary to address this point by comprehensive analyses of 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. For instance, hyperactive Jaks/STAT3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells. Similarly, Wnt pathway also contributes to the proliferation of human ovarian cancer cell and inhibition of Notch signaling, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy. 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. The above findings thus provide a cue and/or a cutting edge to identify the critical molecular event(s) caused by resveratrol in ovarian cancer cells.For the above reasons, our work was carried out using two ovarian cancer cell lines, explored the molecular mechanism of resveratrol against ovarian caner,in order to provid theories for clinical therapy. The research contents include:(1) To study the effects of resveratrol on human ovarian cancer cells, analyze the sensitivity、dose- and time-correlation and the nature of killing cells with two human ovarian cancer cell lines treated by resveratrol.(2) To analyze the statuses of Notch-, Wnt-and STAT3-mediated signaling and downstream genes in human ovarian cancer cells treated by resveratrol, CAOV-3 and OVCAR-3 cells.(3) To explore the biological consequence(s) of selective inhibition of individual signaling to the two cell lines, investigating the key molecular targets of ovarian cancer treated by resveratrol.. Materials and methods: Human ovarian cancer CAOV-3 and OVCAR-3 cells were cultured respectively in DMEM containing 12% fetal bovine serum under 37 oC and 5% CO2 condition and in RPMI1640. Then the two ovarian cancer lines were treated by 100μM and 120μM resveratrol. Their responses to the treatment were explored by H&E staining, MTT assay, TUNEL staining,trypan blue staining and flow cytometry. The statuses of Wnt, Notch and STAT3 signaling and downstream genes in them,such as Survivin,c-Myc,Bcl-2,were analyzed by multiple experimental approaches, immunocytochemical(ICC) staining, RT-PCR and Western-blotting. Selective inhibitors of Notch(L-685,458),Wnt(XAV939) or STAT3(AG490) signaling were employed to treat CAOV-3 and OVCAR-3 cells.To elucidate the significance of individual signaling pathways for ovarian cancers,their responses were treated by H&E staining, MTT assay, trypan blue staining and immunocytochemical(ICC) staining, Results: 1.Resveratrol caused growth arrest and apoptosisMTT assay and viable cell counting were performed revealed that the growth of CAOV-3 and OVCAR-3 cells was suppressed by resveratrol treatment in dose- and time-related fashions. H/E staining and TUNEL assay showed frequent apoptotic death in resveratrol-treated cell populations. Flow cytometry demonstrated that 120μM resveratrol treatment caused distinct G1-phase arrest of the two OC cell lines. 2.The functions of resveratrol on Notch,Wnt and STAT3 signaling pathways of the two cervical cancer cell lines(1) The influence of resveratrol on Notch signal pathway in ovarian cancer cell lines.Immunocytochemical/ICC staining showed that Notch1 、Notch2 and Hes1 were expressed in CAOV-3 and OVCAR-3 cells and mainly distributed in cytoplasm.When the two cell lines were treated with 120μM resveratrol, the expression level of Notch2 and Hes1 were significantly reduced.While the expression of Notch1 was enhanced in the former, reduced in the latter.RT-PCR and western-blotting showed the same results as Immunocytochemical/ICC staining.(2) The influence of resveratrol on Wnt signal pathway in ovarian cancer cell lines.Immunocytochemical/ICC staining showed that Wnt2 and β-catenin were expressed in CAOV-3 and OVCAR-3 cells,and Wnt2 mainly distributed in cytoplasm, β-catenin were located in cytoplasm and nuclei in CAOV-3 cell, but only in cytoplasm in OVCAR-3 cell. When the two cell lines were treated with 120μM resveratrol, the expression level of Wnt2 significantly reduced in CAOV-3 cell,and increased in OVCAR-3 cell. The expression of β-catenin reduced and located on the cell memberane.The expression of E-cadherin were incresed on the cell membrane in OVCAR-3. Western-blotting and RT-PCR showed the same results as Immunocytochemical/ICC staining.(3) The influence of resveratrol on STAT3 signal pathway in ovarian cancer cell lines.Immunocytochemical/ICC staining showed that STAT3 and p-STAT3 was expressed in the normally cultured OVCAR-3 and CAOV-3 cells with distinct nuclear translocation. When the two cell lines were treated with 120 μM resveratrol, the expression level of STAT3 and p-STAT3 were reduced both in the cytoplasm and nuclei. Western-blotting and RT-PCR showed the same results as ICC staining.(4) The influence of resveratrol on downstream genes of Notch,Wnt and STAT3 signal pathways in ovarian cancer cell lines,Survivin,c-Myc and Bcl-2.Immunocytochemical/ICC staining,western-blotting and RT-PCR showed that Survivin, c-Myc and Bcl-2 decreased in both CAOV-3 and OVCAR-3 cells compared with their normally cultured counterparts. Western-blotting and RT-PCR showed the same results as ICC staining. 3.Variable sensitivities of CAOV-3 and OVCAR-3 cells to three pathway inhibitors(1) Notch inhibitor L-685,458 had no effect on proliferation and survival of CAOV-3 and OVCAR-3 cells.MTT assay and H/E staining show that when 8μM Notch inhibitor L-685,458-treated OVCAR-3 and CAOV-3 cells,there is no distinct morphologic change and growth inhibition in comparison with their normally cultured counterparts.(2) Wnt inhibitor XAV939 had no effect on proliferation and survival of CAOV-3 and OVCAR-3 cells.MTT assay and H/E staining show that when 10μM Wnt inhibitor XAV-939-treated OVCAR-3 and CAOV-3 cells, there is no distinct morphologic change and growth inhibition in comparison with their normally cultured counterparts.(3) The expression of p-STAT3 was inhibited, ans apoptosis-induced by the STAT3 inhibitor AG490 in CAOV-3 and OVCAR-3 cells,Immunocytochemical/ICC staining showed that STAT3 phosphorylation was inhibited in two human ovarian cancer cell lines upon 80μM AG490 treatment for 48 hours, accompanied with similar growth suppression rates(74%) as that of resveratrol-treated populations(68%) in OVCAR-3 cells and 82% versus 77% in CAOV-3 cells. Conclusions: 1. Resveratrol caused growth arrest and apoptosis in ovarian cancer cells. Compared with CAOV-3 cell, it is more sensitive for OVCAR-3 to resveratrol.So there is different sensitivity in both two huaman ovarian cancer cell lines treatmented by resveratrol. 2. Notch, Wnt and STAT3 signaling pathways were activated in the ovarian carcinogenic processes by up-regulating the expression of some tumor promoting genes such as Survivin, c-Myc and Bcl-2.Resveratrol inhibits the signaling pathways and their downstream genes.Therefore, anti-cancer function of resveratrol may has the multiple targets. 3. Resveratrol altered β-catenin intracellular distribution patterns,inducing the cell differentiation.This may be connected with up-regulaion of E-cadherin on membrane,expecially OVCAR-3 cell. 4. Notch inhibitor L-685,458 and Wnt inhibitor XAV-939-treated CAOV-3 and OVCAR-3 cells showed that there is no distinct morphologic change and growth inhibition.For proliferation and survival of ovarian cancer cell, Notch and Wnt signal pathways is not critical. 5. The expression of p-STAT3 in CAOV-3 and OVCAR-3 cells were reduced by STAT3 inhibition AG490,and the activity of STAT3 signal pathway inhibited. It promots that STAT3 signaling pathway may be the most important among the signaling pathway in ovarian cancer cells treated by resveratrol. For the proliferation and growth of ovarian cancer,the activation of STAT3 signal pathway is critical.