Medroxyprogestogen enhances apoptosis of SKOV-3 cells via inhibition of the PI3K/Akt signaling pathway

Abstract

We sought to assess the effect of progestin on the apoptosis of epithelial ovarian cancer cell line SKOV-3 and via regulation of phosphorylation signaling in. Epithelial ovarian cancer cell line SKOV-3 was treated with medroxyprogestogen, phosphatidylinositol 3-kinase inhibitor LY294002 and vehicle control. Akt, phospho-Akt, Bcl-2 and phospho-Bad proteins were examined by immunoblotting assays. Medroxyprogestogen-induced apoptosis was assessed by MTT assays and Annexin V apoptosis assay. We found no significant difference in Akt and Bad expression in both the medroxyprogestogen groups and the control group. The levels of phospho-Akt, Bcl-2 and phospho-Bad were decreased in all the medroxyprogestogen groups and significantly decreased in the high dose mitogen-activated protein (MAP) group (10 µmol/L). Viability of SKOV-3 was reduced and apparent apoptosis of SKOV-3 cells was observed with increased doses of MAP. The findings suggest that medroxyprogestogen can induce SKOV-3 cell apoptosis by inhibiting Akt phosphorylation.

Keywords: Akt; apoptosis; medroxyprogestogen; ovarian cancer; phosphorylation.

Fig. 1. MAP suppresses SKOV-3 cell proliferation in vitro.

Epithelial ovarian carcinoma cells (SKOV-3) were seeded into 96-well plates at 7,000 cells//well. Cell were incubated with serial concentrations (0.1, 1, 10, 100 µmol/L) MAP for 12, 24 and 48 hours. Cytostatic effects were measured using crystal violet staining and expressed as mean survival (as compared with controls) ±SD at least three independent experiments. The results suggest that MAP suppresses SKOV-3 cell viability in a dose- and time-dependent manner. *P < 0.05, **P < 0.01 (two-way ANOVA) in comparison with controls.

Fig. 2. MAP induces apoptotic death of SKOV-3 cells

Epithelial ovarian carcinoma cells (SKOV-3) were incubated with serial concentrations (0.1, 1, 10, 100 µmmol/L) MAP for 24 and 48 hours. Annexin V/propidium iodide apoptosis assay was used to measure apoptosis of SKOV-3. The results show that compared with the control group, the apoptosis in all MAP treated groups increased. Cells were treated with low dose MAP for 48 hours and the apoptosis rate increased to 24%. When cells were treated with high dose MAP, the apoptosis rate increased to 31% at 24 hours and 43% at 48 hours. *P < 0.05, **P < 0.01.

Fig. 3. MAP induced-apoptosis is dependent on caspase-3 and caspase-8 activation.

SKOV-3 cells were pretreated with different concentrations of MAP (0, 0.1, 1, 10, and 100 µmol/L) for 24 hours. The cells were harvested and lysed. The lysate containing 100 µMg of protein was mixed with caspase-3/caspase-8 specific synthetic fluorescent substrate AC-DEVD-pNA/ AC-IEVD-pNA. After incubation at 37°C for 4 hours or more, the yellowish color from the pNA released from the substrate by the action of active caspase-3 or caspase -8 were determined at 405 nm. Columns represent the mean (±SD) values obtained from three independent experiments. *P < 0.05, **P < 0.01.

Fig. 4. The expressions of Akt, p-Akt and Bad and p-Bad, and Bcl-2 proteins in SKOV-3.

SKOV-3 cells were treated with serial concentrations of (0, 0.1, 1, 10, and 100 µmol/L) MAP for 24 hours. The protein levels of Akt and p-Akt (A), Bad and p-Bad (B), and Bcl-2 (C) were detected by Western blotting assays. Adobe Photoshop was used to measure the mean gray value of each band. The result suggested that exogenous MAP decreases p-Akt, Bcl-2 and p-Bad expressions in a dose-dependent manner, but causes no apparent changes in the expressions of Akt and Bad.

Fig. 5. MAP and LY294002 induce epithelial ovarian carcinoma cell apoptosis in vitro.

A: Epithelial ovarian carcinoma cells (SKOV-3) were seeded into 96-well plates at the concentration of 7,000 cells/well. Cell were treated with serial concentrations of LY294002 (1.0, 5, 12.5, and 25 µmol/L) for 1 hour. The cells were then incubated with serial concentrations (0.1, 1, 10, and 100 µmol/L) MAP for 24 hours. B: The cells were pretreated with LY294002 at the indicated doses. The cells were then incubated with 10 µmol/L MAP for 12, 24 and 48 hours. Cytostatic effects were measured using crystal violet staining and expressed as mean survival (as compared with controls) ±SD. Two-way analysis of variance was used to compare the measurement data between groups (^#P < 0.05), while t test for comparison within groups (*P < 0.05).