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The medroxyprogesterone acetate vitro induced apoptosis of epithelial ovarian cancer SKOV-3

Author: LiYan
Tutor: ChengWenJun
School: Nanjing Medical University
Course: Obstetrics and Gynecology
Keywords: MPA Epithelial ovarian cancer PI3K/Akt signal transduction pathway Phosphorylation Apoptosis
CLC: R737.31
Type: Master's thesis
Year: 2010
Downloads: 29
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Abstract

Epithelial ovarian cancer is the most common tumors of the female reproductive system, and the mortality rate ranks first in gynecological malignancies. Pathological type of ovarian cancer is complex, and easy to transfer and widely disseminated biological behavior, prognosis is poor. In recent years, a large sample of epidemiological studies and long-term clinical practice indicate that progesterone can antagonize the effect of estrogen, ovarian protection, the prevention of the occurrence of ovarian cancer. At the same time, reported in the literature, progesterone can inhibit ovarian cancer cell proliferation and promote apoptosis, and its specific mechanism remains to be seen. Medroxyprogesterone acetate (Medroxyprogesterone Acetate, MPA) is a progestin drugs as menopausal women in progestin component of hormone replacement therapy can also be used as a contraceptive drugs, used to treat endometriosis syndrome, dysmenorrhea and amenorrhea. MPA has anti-estrogenic effect and role in endometrial promote endometrial proliferative phase to the secretory phase transition, and thus has been widely used in the adjuvant therapy of estrogen and progesterone receptor positive endometrial cancer. Phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway as an important cell survival signaling pathway regulatory sites play an important role in cell survival and apoptosis regulation process. Studies have shown that the pathogenesis of ovarian cancer may PI3K/Akt expression or function abnormalities, most ovarian cancer can Akt overexpression. In addition, the PI3K/Akt signaling pathway is one of the Bcl-2-mediated apoptosis pathway upstream regulatory procedures, the PI3K/Akt pathway activation status directly affect cell proliferation and apoptosis. Therefore, we assume that the MPA in addition by classic progesterone receptor pathway, whether by adjusting the PI3K/Akt signaling transduction pathway activation status affect proliferation and apoptosis of epithelial ovarian cancer cells. Although the results of the clinical trial treatment progesterone treatment of epithelial ovarian cancer as a secondary drug to get good results, but because of its mechanism of action is not clear, and therefore whether progesterone routine use as a supplementary drug in the clinical treatment of ovarian cancer has not yet reached a consensus. This study attempts to explore the MPA in vitro induced ovarian cancer cell apoptosis mechanisms may exist to provide a new theoretical basis for progesterone treatment of ovarian cancer. The first part of the MPA in vitro epithelial ovarian cancer SKOV-3 cell lines induced apoptosis purposes: 1. Observed MPA in vitro inhibition of progesterone nuclear receptor-negative epithelial ovarian cancer cell line SKOV-3 proliferation. MPA 2. Observed PI3K inhibitor LY294002 change SKOV-3 inhibited the proliferation of epithelial ovarian cancer cell lines. Method: 1. Epithelial ovarian cancer SKOV-3 cells were seeded in 96-well plates containing 10% fetal bovine serum RPMI-1640 culture medium filled with conventional culture to serum-free medium, for adherent cultured for 24h, respectively. added at a concentration of 0.1,1,10,100 μmol / L MPA, respectively role 12h, 24h, 48h, MTT (MTT) colorimetric assay relative vigor. Epithelial ovarian cancer SKOV-3 cell lines were seeded in containing routinely cultured in 96-well plates containing 10% fetal bovine serum RPMI-1640 culture medium to serum-free medium, for adherent cultured for 24h were added after 1,5,12.5,25 μmol / L LY294002 (PI3K inhibitor) for 1h, and then were added concentration 0.1,1,10,100 μmol / L MPA drug action 12h, 24h after 48 hours, tetramethyl even triazole salt (MTT) assay to detect the relative activity of the cells. Results: MPA inhibition time - dose-dependent manner in vitro proliferation of SKOV-3 cells, the longer the drug action, the greater the concentration of the drug, the more obvious toxic effects on the cells (P lt; 0.05). 2. MPA inhibit the proliferation of SKOV-3 cells in vitro can be blocked by LY294002. Conclusion: 1. MPA dose-dependent manner time - suppression SKOV-3 cell proliferation in vitro, this inhibition of proliferation can be blocked by the PI3K inhibitor LY294002. The second part of MPA by inhibiting the PI3K/Akt signaling transduction pathway induced apoptosis of epithelial ovarian cancer SKOV-3 purposes: 1. Research PI3K/Akt signal transduction pathway in MPA-induced progesterone nuclear receptor-negative SKOV-3 role to play in the process of apoptosis. 2. Further explore the possible role in the induction of epithelial ovarian cancer cells during apoptosis of non-classic progesterone receptor. Method: 1. Collect different concentrations of MPA role SKOV-3 cells 24 h after cleavage of the total protein in cell extracts Western Blot assay cells of Akt phosphorylated Akt (phosphorylated Akt, p-Akt), phosphorylation of Bad ( phosphorylated Bad, p-Bad) and Bcl-2 protein expression levels. 2. Collect different concentrations of MPA for 24h after SKOV-3 cells, cleavage of the total protein in cell extracts, using the aspartate-specific cysteine ??proteolytic enzymes (Cysteine ??aspartic acid specific protease, Caspase) vitality detection kit detection intracellular Caspase-3 and Caspase-8 enzyme activity. 3. SKOV-3 cells were treated with different concentrations of MPA after 12h, Annexin V / PI double staining flow cytometry to detect apoptosis. Results: With increasing doses of MPA uses SKOV-3 cells, p-Akt, p-Bad and Bcl-2 protein expression levels gradually reduce (P lt; 0.05). Compared with the control group, when MPA concentration 0.1μmol / L p-Akt expression reduced by approximately 28%, p-Bad expression decreased by about 48%, Bcl-2 expression is reduced by about 10%,; When MPA concentration rose to 100μmol / L, p-Akt expression levels decreased by approximately 35%, p-Bad expression decreased by about 76%, the Bcl-2 protein expression decreased by about 43%. Akt expression did not change significantly (P gt; 0.05) between the control group and the experimental group of different drug concentrations. 2. MPA role after 24h, Caspase-3 and Caspase-8 in the intracellular enzyme activity was significantly higher (P lt; 0.05). The MPA concentration 0.1μmol / L, Caspase-3 and Caspase-8 enzyme activity rose to 60.1% and 47.1%, Caspase-3 and Caspase-8 enzyme activity increased by 82.6%, respectively; MPA concentration rose to 100μmol / L and 94.6%. 3. MPA induced a dose-dependent manner with epithelial ovarian cancer SKOV-3 cell line apoptosis (P lt; 0.05). Incubated for 12h, the apoptosis of the control group was 1.8%, low dose MPA group (0.1μmol / L) apoptosis rate rose to 8.8%, MPA concentration of 100μmol / L, the apoptosis rate increased to 37.9% . Conclusion: MPA by non-progesterone nuclear receptor-dependent pathway inhibits PI3K/Akt signal transduction pathway activation, reducing free intracellular levels of Bcl-2, SKOV-3 cells induced apoptosis. 2. MPA concentration 1μmol / L ~ 10μmol / L, the drug action time for 12 ~ 24h, the cell is most sensitive to the drug, the inhibition of cell proliferation promote apoptosis strongest; further increase the concentration of the drug or extend the time of drug action not significantly increase the rate of apoptosis.

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