Medroxyprogesterone Acetate Induced Apoptosis In Epithelial Ovarian Cancer Skov-3 Cells In Vitro

Epithelial ovarian cancer (EOC) is the most common tumor of genital system, and also the fifth leading cause of cancer death in women as well as the first lethal gynecological malignancy. Ovarian cancer has a high incidence of metastasis that generally remains localized within the peritoneal cavity. In recent years, epidemiological studies and long-term clinical practice show that progesterone may antagonize the effect of estrogen to protect the ovary, preventing the occurrence of ovarian cancer. Some studies also suggested that progesterone can inhibit ovarian cancer cell proliferation and induce cancer cell apoptosis. But the specific mechanism remain unknow. Medroxyprogesterone Acetate (MPA) is one kind of progestin drugs, the most common uses of MPA are as the the progestin component of menopausal hormone replacement therapy and also as an oral or depot-injected contraceptive. Medroxyprogesterone is also used as a treatment for endometriosis, dysmenorrhea, and amenorrhea. Because MAP has anti-estrogen function and has capablity to promote the proliferative endometrium transform to secretory endometrium, it has been widely used as an adjuvant treatment of estrogen and progesterone receptor-positive andmetrial cancer.Being a important regulatory sites in cell survival pathway, PI3K/Akt pathway play a vital role in cell's proliferation and apoptosis. Numerous reports suggest a role of PI3K signaling in invasion and metastasis in various kinds of carcinomas. Akt has been also shown to be amplified or over-expressed in ovarian cancer, implying that it also plays a role in ovarian oncogenesis. Survival factors induce Bcl-2 pathway activition via several protein kinase signaling pathways including activation of PI3K/Akt pathway, which directly determined the cell proliferation or apoptosis. So, we assume that MPA induce EOC cells apoptosis via regulation PI3K/Akt pathway.There are many long-term clinical practices show that progestin used as a adjunctive therapy in the treatments of EOC, could be obtained a good curative effect. But because of the specific mechanism of progestin remain unknow, there is no consensus about whether progesterone can be applied as adjuvant treatment of ovarian cancer. The present study attempts to reveal the mechanisms of medroxyprogesterone acetate induced apoptosis in ovarian cancer cells, and provide a new theoretical basis for the treatment of ovarian cancer. Part I Medroxyprogesterone Acetate Induce Human Ovarian Cancer Cell Line SKOV-3 Apoptosis in VitroObjective 1. To observation of medroxyprogesterone acetate (MPA) depressed SKOV-3 cells proliferation in vitro.2. To observation of MPA depressed SKOV-3 cells proliferation in vitro, after cells were treated by PI3K inhibitor LY294002.Methods 1. Epithelial ovarian cancer SKOV-3 cells at exponential phase were subcultured at 7×103 cells/well into 96-well microplates and cultured overnight. Cells were treated with serial concentrations (0.1, 1,10,100μmol/l) of MAP for 12, 24 or 48h. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.2. SKOV-3 cells were treated with serial concentrations (1, 5, 12.5, 25μmol/L)LY294002 for 1h before treated with different concentrations of MAP. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.Result 1. Medroxyprogesterone acetat inhibited the growth of SKOV-3cell line effectively, in a time-and-dose dependent way (P<0.01).2. MPA inhibition of SKOV-3 cell proliferation effect could be blocked by LY294002Conclusion 1. MPA inhibited the growth of SKOV-3cell line effectively, in a time-and-dose dependent way (P<0.01). And the growth inhibition functions of MPA can be blocked by LY294002. Key words Epithelial ovarian cancer (EOC) ; medroxyprogestogen acetate (MPA);apoptosis Part II Influence of MPA on apoptosis in epithelial ovarian cancer cell line SKOV-3 via inhibition of PI3K/Akt signaling pathwayObjective 1. Investigate the influence of PI3K/Akt signaling pathway in SKOV-3 cell apoptosis which induced by MPA.2. Reveal the functions of non-classic progesterone receptors in hunman ovarian cancer cells apoptosis.Methods 1. SKOV-3 cells treat with serial concentrations MAP for 24h, lysed cells and collection of protein, Akt, p-Akt, p-Bad and Bcl-2 protein expression was detected by Western blotting.2. SKOV-3 cells treat with serial concentrations MAP for 24h, lysed cells and collection of protein, activity of Caspase-3 and Caspase-8 was measured by Caspase-3 and Caspase-8 assay kits.3. SKOV-3 cells were treated with different concentrations of MPA role of 24, 48 h; Annexin V / PI double staining flow cytometry to detect cell apoptosis.Result 1. The expression of p-Akt , p-Bad and Bcl-2 protein in the SKOV-3 cells were decreased in all medroxyprogestogen groups and significant decreased in high dose medroxyprogestogen group (P<0.05). The expression of p-Akt protein was decreased to 72% in the low dose group and 65% in high dose group. And the levels of p-Bad decreased to 52% in the low dose group and 33% in high dose group. The levels of Bcl-2 protein was descended to 90% in low dos group and 57% in high dose group. The expression of Akt protein was no significant difference in both medroxyprogestogen gurops and control group.4. After treated with serial concentrations of MPA for 24h, Caspaes-3 and -8 activity gradually increased in a dose-dependent manner. When cells were treated with low dose MPA the Caspase-3 increased 60.1% and Caspase-8 increased 47.1%; when cells were treated with high dose MPA the Caspase-3 increased 82.6% and Caspase-8 increased 94.6%5. After treated with serial concentrations of MPA for 12h, the apoptosis of SKOV-3 cells increased in a dose-dependent manner. When cells were treated with low dose MPA the apoptosis rate is 8.8%; when treated it with high dose the the apoptosis rate increased to 37%.Conclusion 1. MPA can inhibit the PI3K/Akt signal pathway, reducing the level of Bad phosphorylation, induced SKOV-3 cells apoptosis in vitro.2. The cells most sensitive to the drug, when the drug effects time was 24h and MPA concentration from 1μmol L to 10μmol / L, increase the drug concentration or drug effect can not be extended significantly improve the cell apoptosis.