Dual Regulation Of Gonadotropin Releasing Hormone Analogues On Ovarian Cancer And Ovarian Function

Ovarian cancer is among the three most common gynecologic malignancies and is the leading cause of death from gynecologic cancer. Although operation, radiotherapy and chemotherapy are ever improving, the 5 year survival rate is still 30-40%. And the age at diagnosis is younger and younger. With the improvement of the consciousness of healthy and examination measures, more and more young patients of early stages were detected and cured. How can we provide more effective and safer treatments to raise the survival rate and life quality of ovarian cancer patients? How can we protect patients'reproductive function? These are the novel hotspots of ovarian cancer researches.According to the "gonadotropin theory", gonadotropin can promote the growth of ovarian cancer. Gonadotropin releasing hormone (GnRH) can inhibit the releasing of gonadotrophin and inhibit the growth and proliferation of ovarian cancer theoretically. GnRH, a decapeptide released by hypothalamus can regulate the synthesis and secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) to keep the normal function of the reproductive system.The half time of GnRH is quite short and we used GnRH analogues (agonist and antagonist) for research and application. Researches indicated that GnRH analogues can inhibit the growth of ovarian cancer cells in vitro and these were the effects outside H-P-O axis which mediated by GnRH receptors. There are two forms of GnRHR in mammals, GnRHR1 and GnRHR2. On the other hand, researches indicated that when children were exposed to chemotherapy the incidence of ovarian failure was 10%. This result indicated that we can create the artificial preadolescent state to protect ovary from chemotherapy. GnRH can inhibit the pituitary gland to release FSH and LH, make the ovaries occupy the impuberal level and protect the ovaries from the injury of chemotherapy drugs. Anti-Mullerian hormone (AMH) is a glycoprotein hormone which is secreted by granulosa cells of ovaries. AMH can inhibit the initial and circulative recruitment of follicles and regulate the procedure of follicular development. AMH is the earliest marker of ovary function. Our research tried to certify that GnRH analogues can inhibit ovarian cancer proliferation and protect ovarian function in vitro and in vivo, to compare the differences between agonist and antagonist, to provide the evidence for clinical application of GnRH analogues to cure ovarian cancer and protect ovarian function simultaneously. We chose goserelin and cetrorelix as agonist and antagonist in the following experiments.Our study consisted of 4 parts, as follows:1. The effects of GnRH analogues on the growth and proliferation of ovarian cancer cells.2. The effects of GnRH analogues on secretion of AMH, estradiol (E2) and progesterone (P) in human granulosa cells 3. The dual regulation of GnRH analogues on transplanted tumors and ovarian function on nude mice.4. The expression of GnRHR on the epithelial ovarian cancer tissues and their correlation with clinical stages and pathological grades.Sectionâ… The effects of GnRH analogues on the growth and proliferation of ovarian cancer cells in vitroObjective To investigate the effects of GnRH analogues on the growth and proliferation of ovarian cancer cells.Methods The expression of GnRHR1 and GnRHR2 in ES-2 and SKOV3-ip ovarian cancer cell lines were detected by RT-PCR. The ES-2 and SKOV3-ip ovarian cancer cell lines were treated by different concentrations (0,10-9,10-8,10-7,10-6,10-5 mol/L) of GnRH agonist goserelin or antagonist cetrorelix for 24 h. Cell proliferation was evaluated by SRB. The colony formation of ES-2 and SKOV3-ip cells were observed after treated by 10-5 mol/L goserelin or cetrorelix for 14 days. ES-2 and SKOV3-ip cell lines were treated by 10-5 mol/L goserelin or cetrorelix for 24 h, and the influence of cell cycle were detected by flow cytometer, and the expression of cyclin B1 and cyclin D1 were measured by western blot. We also compared the differences of pAKT expression at different time intervals (0,15,30,60,120,240 min) treated by 10-5 mol/L goserelin or cetrorelix in ES-2 and SKOV3-ip cells.Results GnRHR was expressed in ES-2 and SKOV3-ip cell lines. The results of SRB showed that GnRH analogues can inhibit the proliferation of ES-2 and SKOV3-ip cells on different levels, the higher the concentration is, the more obvious the effect is. In ES-2 cell lines,10-5 mol/L cetrorelix had more obvious inhibitory effect than goserelin (P<0.05).10-5 mol/L goserelin or cetrorelix can inhibit the colony formation. In SKOV3-ip cells, the inhibition effect was more obvious in cetrorelix treated groups (P<0.05). Flow cytometry revealed that goserelin or cetrorelix can increase the ratio of cells in Phase S, but it was not of statistical importance (P>0.05). Both goserelin and cetrorelix could reduce the expression of cyclin B1 and raise the expression of cyclin D1 (P<0.05) in ES-2 and SKOV3-ip cells. And western blot showed that 10-5 mol/L goserelin or cetrorelix could inhibit the expression of pAKT.Conclusion GnRHR1 and GnRHR2 are expressed in ES-2 and SKOV3-ip cell lines. Both GnRH agonist and antagonist can inhibit the growth and proliferation of ES-2 and SKOV3-ip cells.Sectionâ…¡The effects of GnRH analogues on the secretion of AMH,E2 and P in human granulosa cellsObjective To investigate the effects of GnRH analogues on the secretion of AMH,E2 and P in human granulosa cells.Methods The human granulosa cells (hGC) in primary cell culture were verified by immunocytochemistry with the markers of Calretinin, Inhibin a, EMA, Vimentin, CD99, Desmin, CD34 and Oil red and H-E dyeing. And the expression of GnRHRl and GnRHR2 on granulosa cells were measured by immunocytochemistry. The secretion of AMH in hGC in different groups (control, DDP, goserelin, goserelin +DDP, cetrorelix and cetrorelix+DDP) were detected by ELISA. And the secretion of E2 and P were detected by chemiluminescence.Results GnRHRl and GnRHR2 are expressed in hGC. Goserelin can stimulate the secretion of AMH, cetrorelix can stimulate the secretion of AMH obviously (P<0.05). But there was no influence on the secretion of E2 and P (P>0.05).Conclusion GnRHR1 and GnRHR2 are expressed in hGC. GnRH analogues can raise the secretion of AMH on hGC.Section III The dual regulation of GnRH analogues on transplanted tumors and ovarian function in nude miceObjective To investigate the dual regulation of GnRH analogues on transplanted tumors and ovarian function in nude mice.Methods ES-2 cells were cultured and heterotransplanted into 36 nude mice. The nude mice were divided into 6 groups:control (normal saline), DDP, goserelin, goserelin+DDP, cetrorelix and cetrorelix+DDP. The weight of nude mice, the volumes of transplanted tumors, the expression of Ki-67 in transplanted tumors, the estrus, serum AMH, FSH, E2and P levels, different levels of ovarian follicles and ovary volumes in each group were compared. The pathological morphology of ovaries and transplanted tumors of nude mice were observed by H-E staining. Results There was no difference on the weight of nude mice in 6 groups. Compared with control group, the tumor volumes in DDP, goserelin, goserelin+DDP, cetrorelix or cetrorelix+DDP group were much smaller from the 12th day (P<0.05). The expression of Ki-67 in DDP, goserelin and goserelin+DDP group was much lower than that in the control group (P<0.05). The frequencies of estrus in goserelin group, goserelin+DDP group or cetrorelix group was obviously decreased compared with the control group (P<0.05), the continuing time of estrus in goserelin+DDP group was prolonged (P<0.05). The expression of AMH in goserelin group was higher than that in the control group and there was no difference of the expression of FSH, E2 or P in different groups (P>0.05). The ratio of atretic follicle in goserelin group was much lower than that in DDP group (P<0.05). The ratio of primary and preantral follicles in goserelin group, goserelin+DDP group, cetrorelix group or cetrorelix+DDP group was higher than those in the DDP group (P<0.05). By pathomorphology observation, interstitial fibrosis and follicular atresia was observed in the ovaries in DDP group and hepatic metastasis occurred in control group with poor differentiation.Conclusion Goserelin or cetrorelix can inhibit the growth and proliferation of transplanted tumors in nude mice, meanwhile increase the secretion of AMH, decrease the frequencies of estrus and prolong the lasting time of estrus, raise the ratio of primary and preantral follicles so that the ovarian function of nude mice were protected.Section IV The expression of GnRHR on the epithelial ovarian cancer tissues and their correlation with clinical stages and pathological gradesObjective To investigate the expression of GnRHR(GnRHR1 and GnRHR2) and their correlation with clinical stages and pathological grades in the epithelial ovarian cancer tissues.Methods The expression of GnRHR 1 and GnRHR2 were detected in 54 epithelial ovarian cancer tissues by the immunohistochemistry. The correlations of GnRHR expression with clinical stages, pathological grades and metastasis were analysed.Results The expression of GnRHR1 or GnRHR2 was higher in stageâ… -â…¡than in stageâ…¢-â…£(P=0.01, P<0.01, respectively). The expression of GnRHR2 was higher in well differentiated than in moderate or poor differentiated tumors (P<0.01). There was no difference of GnRHR1 expression in different differentiation groups and there was no difference of GnRHR1 and GnRHR2 expression between primary and metastasic diseases (P>0.05). There was no correlation between GnRHR1 and GnRHR2 expression.Conclusion GnRHRl and GnRHR2 were extensively expressed in epithelial ovarian cancer tissues. The expression of GnRHR1 and GnRHR2 were higher in stagesâ… -â…¡. The expression of GnRHR2 was higher in well differentiation tissues. GnRHR1 and GnRHR2 were closely relevant to ovarian cancer proliferation and differentiation.In conclusion, GnRH analogues, both goserelin and cetrorelix can inhibit the growth and proliferation of ovarian cancer cells, and inhibit the growth of transplanted tumors in nude mice. GnRH analogues can stimulate the secretion of AMH of hGC, inhibit the recruitment of follicles and increase the ratio of primary and preantral follicles. The frequencies of the estrus of the nude mice were decreased and the estrus cycles were prolonged and the reserve function of the ovaries was protected. We observed these two effects in the same nude mice simultaneously. This study provides the evidences of the clinical application of GnRH analogues in the treatment of ovarian cancer and protection of reproductive function in young patients with early stage diseases.