| Objective:Ovarian epithelial cancer (OEC) accounts for 90% of all ovarian cancers and is the leading cause of death in gynecological malignancy. To date, due to lack of effective early detection techniques, postoperative recurrence and chemoresistance, the 5-year survival rate is still about 30%. Studies found that about 90% of human ovarian tumors originated from the ovarian surface epithelium (OSE), but the etiology and pathogenesis has remained unclear. Therefore, it is of great significance to study the pathogenesis of epithelial ovarian cancer, for the prevention and treatment of this disease.In recent years, epidemiologic studies indicate that follicle-stimulating hormone (FSH) plays an important role in ovarian carcinogenesis, which directly reflects that FSH can promote malignant transformation of OSE cells and proliferation of ovarian cancer cell in vitro. But the mechanism of FSH action on ovarian cancer, especially for proliferation and apoptosis still poorly understand. It is generally considered that the effects of FSH on cells are mediated by FSH receptor (FSHR), but little is known about the signaling transduction pathways and the specific mechanisms after FSH combined with FSHR. Most domestic scholars study this issue on tissue levels, it has become a hot spot to study the etiology and pathogenesis of epithelial ovarian cancer on the molecular mechanism and the signal transduction pathways. Promote or block these signals and their transduction processes will be a key strategy for prevention and treatment of ovarian cancer. So we set out to determine the effects of FSH on the proliferation and apoptosis of SKOV-3 and 3AO cells cultured in vitro as well as the cell cycles. Meanwhile, the signaling transduction pathways of which might had been led by FSH on the proliferation, apoptosis and SKOV-3 and 3AO cell cycles were studied. Our experiment was designed to provide a new viewpoint and a defined strategy for preventing and treating epithelial ovarian cancer.Methods:The human ovarian serous cancer cell line SKOV-3 and mucinous cancer cell line 3AO were cultured in RPMI 1640 culture solution containing 10% heat-inactivated FBS under a humidified 5%CO2 atmosphere, at 37℃, cells were applied to the experiment when they entered the logarithmic phase.1. We stimulated SKOV-3 and 3AO cell with FSH at different concentrations (0,10,20,40,80,160mIU/mL) in different time courses (Oh, 12h,24h,48h,72h), and used light microscope to observe the cell morphological changes.2. MTT assay:MTT assay was used to determine cell proliferation. The cultures were initiated in 96-well plates at a density of 1×105/mL.Cells were treated for different times(0h,12h,24h,48h,72h)with various concentrations (0,10,20,40,80,160mIU/mL) of FSH with five replicate wells for each concentration. We choose OmIU/mL as the control group. The relative number of adherent cells was then determined by the MTT method, in order to select the peak concentration and the time of FSH.3. PI labed FCM was used to detect the apoptotic rates and cell cycle phase of SKOV-3,3AO cells in different experimental groups. Cells were grouped as follows:(1) Control group, cells were only cultured in RPMI-1640 supplemented with 10%heat-inactivated FBS (2) LY294002 group, cells were treated with 10μmol/L LY294002 for 30min,40 mIU/mL FSH was added to the cells;(3) FSH group, cells were treated with 40 mIU/mL FSH for 48h (4) LY294002+FSH group, after cells were treated with 10μmol/L LY294002 for 30min.40 mIU/mL FSH was added to the the culture supernatants4. Western blot analysis:We measured the protein expression of FSH receptor (FSHR), serine-threonine protein kinase B (Akt), p-Akt (Thr308) and mutant type p53 protein in different experimental groups. Cells were grouped in accordance with methods 3. The proteins were extracted from SKOV-3 cells 48h later after treated and 3AO 24h later.5. All statistical analysis were performed with SPSS 13.0 statistical software package.Results:1. We treated SKOV-3 and 3AO cells with FSH in different concentration and observed the cell morphological changes under inverted phase contrast microscope. As the concentration increased and the time extended, the proliferation rate and intensity increased significantly. Treatment with the specific PI3K/Akt inhibitor LY294002 can inhibit the proliferation and induce apoptosis of SKOV-3 and 3AO cells. After cells were treated with LY294002, the shape of some cells began to change gradually. At first, the long shape turned round, but the cell membrane integrity. And then the cells started to shrink, round cells gradually increased. Then the volume of floated cells generally turned smaller than before, and the cytoplasm condensed, morphology became irregular, membrane got incomplete. In the end, the adhesion of these cells increasingly weakened, and floated.2. The MTT results showed that after we treated SKOV-3 cells in different time (12h,24h,48h and 72h) and with FSH in different concentration, (0,10,20,40,80,160mIU/mL), all the concentrations of FSH could markedly promote the multiplication of SKOV-3 and 3AO, There was significant difference in the proliferation of SKOV-3,3AO cells when compared with the control group. (P＜0.05).3. The apoptotic rates of SKOV-3 and 3AO cells were detected by PI labed FCM, the results showed:FSH restrained the apoptosis in SKOV-3 and 3AO cells, while the specific PI3K/Akt inhibitor LY294002 had a reversal effect, it induced apoptosis of SKOV-3 and 3AO cells.(1) After FCM analysis, we found that when the cells were treated by FSH at 48 hours, the apoptotic rates of SKOV-3 cells in FSH group (0.26%±0.01%) decreased compared with control group (0.33%±0.03%) (P＜0.05).LY294002 group (1.43%±0.04%) and LY294002+FSH group (1.41%±0.03%) increased markedly compared with control group and FSH group, (P＜0.05).Furthermore, no significant difference was observed in apoptotic rate of SKOV-3 cells between LY294002 group and LY294002+FSH group (P＞0.05).(2) The apoptotic rates of 3AO cells in FSH group (0.29%±0.05%) decreased compared with control group (0.39%±0.04%)(P＜0.05).LY294002 group (1.54%±0.03%) and LY294002+FSH group (1.52%±0.07%) increased markedly compared with control group and FSH group, (P＜0.05).Furthermore, no significant difference was observed in apoptotic rate of 3AO cells between LY294002 group and LY294002+FSH group (P＞0.05).4. The cell cycle phase of SKOV-3 and 3AO cells were detected by PI labed FCM showed:When cells were treated by FSH, the percentage of S stage increased, FSH could forward transition of SKOV-3 and 3AO in vitro from G1 to S phase, while the specific PI3K/Akt inhibitor LY294002 had a significant blockade at G0/G1 phase.(1) At G0/G1 phase, the number of SKOV-3 in FSH group (38.87%±0.76%) decreased greatly compared with those in control group (41.63%±0.41%) (P＜0.05), and the number of SKOV-3 in LY294002 group (45.23%±0.40%)and LY294002+FSH group (46.7%±0.53%) was significantly higher than that in FSH group (P＜0.05); At S phase, the number of SKOV-3 increased notably in FSH group (46.90%±0.36%) compared with those in control group (43.53%±0.25%) (P＜0.05), and the number of SKOV-3 in LY294002 group (41.20%±1.34%) and LY294002+FSH group (40.57%±0.86%) were lower than that in FSH group and Control group (P＜0.05); At G2/M phase, the number of SKOV-3 in FSH group (11.57%±0.41%), LY294002 group (13.63%±0.15%) and LY294002+FSH group (12.87%±0.31%) decreased significantly compared to those in control group (14.9%±0.10%) (P＜0.05); Furthermore, at any phase, no significant difference was found in the number of SKOV-3 between LY294002 group and LY294002+FSH group (P>0.50).(2) At G0/G1 phase, the number of 3AO in FSH group (45.50%±1.25%) decreased greatly compared with those in control group (50.67%±0.71%), (P＜0.05). and the number of 3A0 in LY294002 group (53.83%±1.00%) and LY294002+FSH group (52.47%±0.38%) was significantly higher than that in FSH group (P＜0.05). No significant difference was found in the number of 3AO between LY294002 group and LY294002+FSH group (P＞0.50); At S phase, the number of 3AO increased notably in FSH group (26.13%±0.40%) compared with those in control group (23.07%±0.45%) (P＜0.05), and the number of 3AO in LY294002 group (17.40%±0.65%) and LY294002+FSH group (20.17%±0.25%) were lower than that in FSH group and Control group (P＜0.05).There was significant difference between LY294002 group and LY294002+FSH group (P＞0.50); At G2/M phase, the number of 3AO in FSH group (28.43%±0.55%), LY294002 group (28.87%±0.31%) and LY294002+FSH group (27.57%±0.37%) decreased significantly compared to those in control group(26.30%±0.70%)(P＜0.05), Furthermore, no significant difference was found in the number of 3AO between LY294002 group and LY294002+FSH group (P＞0.50).4. Results of Western-blotting:(1) Western blot showed that the protein of FSH receptor (FSHR) were expressed in both SKOV-3 and 3AO cells, and no significant differences were observed in the protein expressions of FSHR among different groups (P>0.05). In addition, the level of FSHR protein was low in SKOV-3 cells compared with3AO(P＜0.05).(2) Western blot showed that there were no significant differences in protein expression of Akt among different groups in SKOV-3 and 3AO (P＞0.05).(3) While Western blot analysis documented the expression of p-Akt(Thr308) in SKOV-3 increased greatly in FSH group (0.9433±0.002) compared to those in Control group (0.8254±0.020) (P＜0.05),and in LY294002 group (0.3664±0.008) and LY294002+FSH group (0.6498±0.022) were significantly lower than that in FSH group (P＜0.05). And for 3AO significant differences were observed in the expressions of p-Akt (Thr308) among FSH group (0.3488±0.006), LY294002 group (0.1931± 0.006) and LY294002+FSH group (0.1669±0.007) when compared with Control group (0.2905±0.003) (P＜0.05). FSH group increased greatly compared with those in control group (P＜0.05), and in LY294002 group and LY294002+FSH group were significantly lower than that in FSH group (P＜0.05).(4) The expression of mtp53 protein increased greatly in FSH group compared with those in control group in SKOV-3 and 3AO cells (P＜0.05), and in LY294002 group and LY294002+FSH group were significantly lower than that in FSH group (P＜0.05).Conclusion:1. The proteins of FSH receptor (FSHR) were expressed in both SKOV-3 and 3AO cells, but the level of FSHR protein was low in SKOV-3 cells.2. FSH can induce the effect of proliferation and suppress the apoptosis in ovarian cancer cell line SKOV-3 and 3AO, while the specific PI3K/Akt inhibitor LY294002 has a reversal effect, it can inhibit the proliferation and induce apoptosis of SKOV-3 and 3AO cells.3. FSH can postively regulate the effect of cell cycle in SKOV-3 and 3AO. FSH can forward transition of SKOV-3 and 3AO from G1 to S phase in vitro, the PI3K/Akt inhibitor LY294002 arrest cell cycle of them at G0/G1 phase, and thus negatively regulate cell cycle progression of SKOV-3 and 3AO.4. At translational levels, FSH can enhanc the expression of p-Akt (Thr308) and mutant type p53 protein expression is also up-regulated in SKOV-3 and 3AO in vitro; the PI3K/Akt inhibitor LY294002 can antagonized the enhancement effects of FSH on Akt phosphorylation and mtp53 expression.5. The FSH positively regulates the PI3K/Akt signal transduction pathways via activting phosphorylation of Akt, further lead to mutant type p53 protein expression increase in SKOV-3 and 3AO cells. FSH stimulation of mutant type p53 protein expression is mediated through the PI3K/Akt pathway, and thus curbs the proliferation, induces apoptosis and postively regulates cell cycle of SKOV-3 and 3AO cells in vitro. 6. FSH may be related to the formation and development of ovarian epithelial cancer in vitro.
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