| Objectives To study the efficacy of salinomycin on human cervical carcinoma Hela cells in celluar proliferation and apoptosis and explored the possible molecular regulation mechanism.Methods(1)Put the cervical cancer Hela into 96- well plates, each hole 5000 cells, after 24 hours, were randomly selected from 12 hole, one group as the blank control group, more than nine groups, add salinomycin respectively 0, 0.375, 1.5, 6, 12, 24, 48, 96, 120 u g/m L, the 0 ug/m L group as the negative control group, to measure the effect of 24 h, 48 h, 72 h OD value by enzyme standard instrument, and map the inhibition rate to the role of salinomycin concentration,the diagram of the time;(2)According to the above results, select the salinomycin concentration which cell inhibition close IC50 salinomycin concentration, respectively for 6, 12, 24 ug/m L three groups, ditto method to set up the control group, 2 m L 5×105 cells per m L of cell inoculation on 6 orifice, training after 24 h, add to each well respectively at a concentration of 0, 6, 12, 24 mg/m L salinomycin, collecting cells after 24 h, centrifugal fixed, PI staining, to measure the cell cycle and apoptosis rate by flow cytometry instrument;(3) Ditto grouping, 10 ml of 5×106 cells per ml cells suspension were added to the the five petri dish respectively, cultivated after 24 h,added to each dish at a concentration of 0, 6, 12, 24 ug/ml salinomycin, extract the total cellular protein in each group after 24 h, to detect Bax, Bcl- 2, caspase 3 gene expression by Western Blot method.Results(1)The method of CCK 8 inspection found that the cell proliferation inhibition rate rose to 96.35% from 0.89%,after the affectting of 24-72 h by the salinomycin of 0.375-120 ug/ml. We found that the cell proliferation inhibition rate was positively correlated with salinomycin concentration and the action time,the difference was statistically significant(P<0.05);(2) By flow cytometry,we testing the cell apoptosis rate of the Hela cells which affected 24 hours by salinomycin.We found that the Hela cell apoptosis rate(11.15 + 0.70) % increased to(45.1 + 0.21) %. And the apoptosis rate increase linearly with the increase of concentration, statistically significant difference(P<0.05).(3)By flow cytometry,we testing the cell cycle of the Hela cells which affected 24 hours by salinomycin. We found that the cells in G0 / G1 phase increased from(59.56 + 0.93) % to(74.31 + 0.57) %,when the concentration of salinomycin move from 6 up to 24 u g/m L. And the higher the concentration of salinomycin,the arrest of the cells in G0 / G1 phase is more obvious, the differences was statistically significant(P<0.05). It has no obvious regular changes in the G2 phase and S phase;(4) By Western Blot method,we found that the Bcl-2 protein expression significantly less than the negative control group, and negatively related to the concentration, the difference was statistically significant(P<0.05), Bax protein and Caspase 3 protein expression are significantly higher than the negative control group, and were positively correlated with concentration, the differences was statistically significant(P<0.05).Conclusion(1) Salinomycin has a strong inhibitory effect in vitro proliferation of the human cervical cancer cells,and has good concentration dependence and time dependence;(2) Salinomycin can induce Hela cells apoptosis and the mechanism may be by enhancing the expression of bax, caspase 3 gene,decreasing the expression of bcl-2 gene. |
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