Proliferation Inhibition, Apoptosis And The Machenism In Human Ovarian Cancer Skov3 Cells Induced By Cpd 5

Objective: This study was designed to investigate the proliferation inhibition, apoptotic induction and to explore the mechanism in human ovarian cancer SKOV3 cells treated by Cpd 5 to provide experimental data for clinical application of Cpd 5 as an novel anticancer drug for human ovarian cancer. Methods: SKOV3 cells were cultured regularly and divided into three groups: negative control; experimental groups treated by Cpd 5 at various concentrations; and the positive control treated by 5μg/ml DDP. The morphological changes of the cells after treatments were observed by microscope. Trypan blue staining and MTT (methylthiazolyltetrazolium) assay were used to detect cell growth inhibition induced by the chemical. AnnexinV-EGFP/ PI staining was employed for quantifying apoptotic cells by fluorescence microscope and flow cytometry. The apoptotic cells were identified by TUNEL (TdT-mediated dUTP nick-end labeling) and Hoechst-33258 staining. Afterwards the AI (apoptotic index) was calculated. Expression of Cdc25A in SKOV3 cells was investigated by Immunofluorescence staning. Estrogen receptorα(ERα),Caspase-3 and Cdc25A in the cells were immunocytochemically detected. Results: Cell morphological changes could not be seen in the negative control and low concentration groups(5,10μmol/L) treated by Cpd 5. The apparence of apoptotic morphology emerged at the concentrations of 30μmol/L for 24h treatment and 40, 50and 60μmol/L after 12h treatment respectively, including cell shrinkage, membrane blebbing, nuclear condensation and increasement of the transmittance, and there were also different amounts of cells floating in the media. The ratios of the cells growth inhibition calculated by MTT were 2.77%, 5.19%, 10.61%, 41.15%, 71.37%, 82.90% and 89.81%, in the groups treated by Cpd 5 at the concentrations of 5, 10, 20, 30, 40, 50 and 60μmol/L for 48h, respectively. The proliferation inhibition was investigated after treatment at different concentrations for 12, 24, 48 and 72h, for further identifying the inhibitory effects. The cells were inhibited by Cpd 5 in a dose-time- dependent manner. The inhibition ratio in higher-dose groups(30, 40, 50 and 60μmol/L) were higher than that in positive control(DDP) signifficantly(p<0.01). The cellular vitality descended with increase of hours and the concentrations of Cpd 5, which was similar to that by MTT assay. Analyzed by flow cytometry, the apoptotic cells accounted for 9.25%, 20.07% and 56.16%, after treatment by 30μmol/L Cpd 5 for12, 24 and 48hours, respectively. The ratio of apoptotic cells in 50μmol/L group was significantly higher than that in 30μmol/L group. The morphological changes emerged from the cells treated by Cpd 5 at the concentrations of 40, 50 and 60μmol/L for 12h, under a microscope. By AnnexinV/PI staining and fluorescence microscopy, the ratios of the apoptotic cells increased significantly in the groups induced by Cpd 5 at different concentrations, in comparison with the control. Stained by Hoechst-33258, the apoptotic cells increased in the groups treated by 20μmol/L Cpd 5 for 24h and 48h. Furthermore, the ratios of apoptotic cells increased significantly in the groups treated by 30, 40, 50 and 60μmol/L Cpd 5 for different time-points. The apoptotic cells observed by TUNEL assay revealed that there were no positive cells in the negative control. While after the treatment by Cpd 5, some cells appeared to be shrinked, pyknosis, chromatin margination or agglutination and scattered apoptotic body. The nuclei in positive cells were stained with brown. ERαwas located in the nuclei of SKOV3 cells and the expression of ERαwas weak or unconspicuous in negtive control. After treatment by Cpd 5, the expression of ERαappeared to be apparente and reinforced accompanied with increase of hours and Cpd 5 concentrations. The expression of Caspase-3 was low or not seen in negtive control,while over-expressed both in cytoplasm and nucleus, especially the latter, in the groups treated by Cpd 5, And its expression also showed a time-dose-dependence on Cpd 5. Another protein Cdc25A Immunocytochemically emerged in the nuclei of the cells and the positive cells reduced with the concentrations increased of the drug. Conclusion: Based on the data above, it was concluded that Cpd 5 inhibits proliferation and induces apoptosis in SKOV3 cells in a dose-time-dependent manner. The mechanisms of the apoptosis induced by Cpd 5 seems to be associated with the activation of Caspase-3 and inactivation of Cdc25A. The other passway, ERα, in SKOV3 cells may be down-regulated by Cpd 5.