The Roles Of Follicle Stimulating Hormone On Biological Behaviours Of Ovarian Cancer Cells And Its Molecular Mechanism

At present,ovarian malignancies is one of the three gynecological malignancies. Ovarian cancer is the most common histopathological type accounting for 85%-90% of all ovarian malignancies.Despite continuous improvement in surgical modalities, chemotherapeutics and radiotherapetics over the past several years,the 5-year survival rate for ovarian cancer with the most poor prognosis of all gynecological malignancies is about 30%-40%,which has not improved significantly.Clinically,Ovarian cancer is often found in postmenopausal women with high serum follicle stimulating hormone(FSH) and luteinizing hormone(LH) level.The risk for women who have many pregancies,oral contraceptives,hormone replacement therapy is low while their serum FSH and LH concentration is also low.In the past 20 years,previous studies have proven that FSH plays an importmant role in cancinogenesis.But little is known about the relationship between FSH and ovarian carcinogenesis,neither the roles of FSH on biological behaviours of ovarian cancer cells.The results of our experiments suggest that FSH may be related to proliferation, apoptosis,migration,invasion and the expression of vascular endothelial growth factor(VEGF) in ovarian cancer cell line SKOV-3 and ES-2.Through the research on the molecular mechanism between FSH and VEGF,our clinical data and animal trial, we may provide a new stratery for further studying the carcinogenesis of ovarian cancer.These experiments were devided into 4 parts,as follows:①The effects of FSH on proliferation,apoptosis,migration,invasion and VEGF;②The roles of FSH on VEGF in ovarian cancer cells;③Expression and AKT and pAKT in ovarian cancer;④The growth of ovarian cancer in vivo induced by FSH riment.SectionⅠ.The roles of FSH on biological behaviours of ovarian cancer cells Objectives To investigate whether FSH can induce the effects of proliferation,apoptosis,migration,invasion and the expression of VEGF in ovarian cancer cell line SKOV-3 and ES-2 in vitro.Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The proliferative effects of the cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) colorimetry.The apoptosis and cell cycle were showed by the flow cytometry.The matrix metalloproteinases-2(MMP-2) protein levels in supernatants were determined by zymography.The VEGF protein levels in supernatants were determined by enzyme-linked immunosorbent assay(ELISA).The protein levels of MMP-2 and VEGF in SKOV-3 and ES-2 cytoplasma were tested by Western blot methods.The expressions of MMP-2 and VEGF mRNA in SKOV-3 and ES-2 were determined by reverse-transcription polymerase chain reaction(RT-PCR).Migration and invasion assay can investigate the migratory and invasive activities.Results FSH can induce the proliferative effects in ovarian cancer cell line SKOV-3 and ES-2.FSH can suppress the apoptosis and induce the effect of cell cycle in SKOV-3 and ES-2.Expressions of MMP-2 and VEGF mRNA and protein increased with the concentration increasement in SKOV-3 and ES-2.The migratory and invasive activities were also up-regulated by FSH.Conclusions FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.SectionⅡ.The molecular mechanism between FSH and VEGFObjectives To investigate the molecular mechanism between FSH and VEGF in ovarian cancer cell.Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The cells were then harvested and the proteins or mRNA of pAKT/AKT or pERK/ERK or survivin or hypoxia inducible factor-1α(HIF-1α) were extracted for Western blot or RT-PCR or real-time PCR analysis.The nine 19-nucleotide(nt) DNA sequences targeting survivin,HIF-1αand AKT and one non-target siRNA were designed,and cloned into pRNA plasmid vectors.The recombinant plasmids were transfected into SKOV-3 or ES-2 cells.The expression of AKT or surviving or HIF-1αwas knocked down by RNA interference(RNAi) and accessed by Western blot.Moreover,to investigate the molecular mechanism of FSH, the specific inhibitor of PI3K/AKT pathway LY294002 or the specific inhibitor of MAPK pathway U0126 was also used to inhibit the phosphorylation of AKT or ERK1/2 and to study the relations in all these molecules.Results①FSH can stimulate survivin and HIF-1αexpression in SKOV-3 cells;②RNAi of SURVIVIN or HIF-1αboth blocked the expression of VEGF mRNA and protein,but only RNAi of survivin suppressed the increasement of VEGF mRNA and protein induced by FSH.FSH may increase the expression of VEGF through surviving;③FSH can induce the AKT phosphorylation in SKOV-3 and ES-2 cells, whereas it had no obvious influence on the ERK phosphorylation;④The specific PI3K inhibitor LY294002 or RNAi of AKT antagonized the enhancement effects of FSH on the AKT phosphorylation and survivin expression,whereas the specific ERK inhibitor U0126 only descreased the ERK phosphorylation and had no effect on the survivin expression.Conclusions FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.SectionⅢ.Expression of AKT and pAKT in ovarian cancerObjectives To examine the expressions of pAKT/AKT in ovarian cancer tissues and then explore the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.Methods The expression of pAKT/AKT was examined using the immunohistochemical method in the eighty-two samples of paraffin-embedded ovarian tumor tissue,including 21 ovarian benign serous cystadenomas,18 ovarian borderline serous cystadenomas,26 ovarian serous cystadenocarcinomas,and 17 ovarian clear cell carcinomas.The x2 analysis and Kaplan-Meier product-limit method was used to analyze the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.Results Compared with ovarian serous cystadenoma samples,ovarian serous cystadenocarcinoma samples had much higher positive rates for AKT and pAKT protein expression(P＜0.01).Expression of AKT and pAKT was higher in grade 2/3 than that in grade1 in the ovarian serous cystadenocarcinoma specimens(P＜0.01). We also observed strong correlations between the expression of AKT and pAKT and the 2-year or and 5-year survival rates in the ovarian serous cystadenocarcinoma cases(P＜0.01).Our results also show that the 5-year survival rate was only about 14.3%(3 of 21) in patients with ovarian serous cystadenocarcinoma who had AKT expression,whereas it was 80%(4 of 5) in those who did not have AKT expression; the 5-year survival rate was only 15%(3 of 20) in patients with ovarian serous cystadenocarcinoma who had pAKT expression,whereas it was about 66.6%(4 of 6) in those wh6 did not have pAKT expression.Conclusions These results suggest that the expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.SectionⅣ.The growth of ovarian cancer in vivo induced by FSHObjectives To investigate the function of FSH on the growth of ovarian cancer in vivo.Methods SKOV-3 or ES-2 cells were cultured and heterotransplanted into nude mice. The nude mice were treated with FSH(3 IU·d^-1)) or FSH(10 IU·d^-1) or physiological saline.The weights of the nude mice and the valumes of the transplantable tumors were measured every three or four days.At last the nude mice were executed and the serum FSH concentrations and the weights of the transplantable tumors were determined.Results The serum FSH concentrations of the nude mice treated with FSH were higher than those without FSH treatment(P＜0.05).The weights of the nude mice in the experimental and control group have not statistically significant difference.The valumes and weights of the transplantable tumors treated with FSH(3 IU·d^-1)) were both greater than those in the control group(P＜0.05).The weights of the transplantable tumors treated with FSH(10 IU·d^-1) were greater than those in the control group(P＜0.05),but the valumes of the transplantable tumors treated with FSH(10 IU·d^-1) were statistically nonsignificant.Conclusions FSH may be related to the formation and development of ovarian cancer in vivo.In summury,the results demonstrates that FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.The expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.FSH may be related to the formation and development of ovarian cancer in vivo.