The Effect Of Cinobufacin Injection On The Proliferation, Apoptosis And Cell Cycle Of Human Hepatoma HepG-2 Cells

Objective:To investigate the effect of cinobufacin injection on the proliferation,apoptosis and cell cycle of human hepatoma HepG-2 cells, and discuss the possible mechanism.Method:After the intervention of Cinobufacini injection different concentrations of, cell proliferation of HepG-2 cells was assessed by MTT assay,cell morphologic was observed by the inverted microscopy, Anne-xinV/PI stain was used to detect the apoptosis and necrosis of the tumor cells,Flow cytometry (FCM) was emplayed to detect tumor cell cycle distribution,Immunohistochemistry assay was used to analyse expression of apoptosis-related genes Bcl-2/Bax.RT-PCR was used to analyse expression of CyclinA,CDK2 mRNA levels in HepG-2 cells,and the expression of TOPOⅠmRNA and TOPOⅡmRNA were also examined by RT-PCR. Quantitative colorimetric assay was used to analyse cyclinA/CDK2 activity in HepG-2 cells.Result:1.Cinobufacini injection with the concentration of 0.006μg/ml or more can significantly inhibited HepG-2 cells proliferation in doses and time dependent ways.2.After Cinobufacini injection intervention,HepG-2 cells showed typical apoptotic morphological changes:the partial cell become smaller volume than before,and the chromatin looseness in doses and time-dependent ways (P<0.05)3. FCM analysis showed cinobufacin injection induced cell cycle arrest at S phase in time-dependent ways (P< 0.05) 4.Cinobufacin injection can inhibited the expression of apoptosis-related genes Bcl-2 and promote the expression of genes Bax in doses-dependent way.5.Cinobufac ininjection down-regulated CDK2,CyclinA,TOPOⅠand TOPOⅡexpression at mRNA levels in doses-dependent ways.6.Cinobufacin injection deceased cyclinA/CDK2 activity in HepG-2 cells in doses-dependent way (P<0.05)Conclusion:Cinobufacini injection can inhibit human hepotocarcinoma HepG-2 cell growth, induce tumor cell apoptosis, and induce cell cycle arrest at S phase, the mechanism might be partly related to the down-regulation of CDK2, CyclinA, TOPOⅠand TOPOⅡexpression at mRNA level, inhibition of cyclinA/CDK2 activity and influence the expression of genes Bcl-2 and Bax.