Effect Of Cinobufagin On Cell Cycle And Apoptosis Of Esophageal Carcinoma Kyse-520 Cells And Its Mechanism

Objective To observe the effect of cinobufagin on cell cycle and apoptosis of esophageal carcinoma Kyse-520 cells,and to explore its possible molecular mechanism.Methods Esophageal cancer Kyse-520 cells were treated with different concentrations of cinobufagin.CCK-8 assay,cell growth curve and clone formation assay were used to detect the cell proliferation.The changes of cells morphology were detected by Hoechst 33342 fluorescent staining.The changes of cell cycle distribution were detected by flow cytometry.The m RNA expression levels of cell cycle related genes(p21,Wee1,Cyclin B1 and Cdc2)in each group were analyzed by fluorescence quantitative PCR(RT-q PCR).Western blotting was used to measure the expression levels of apoptosis-related proteins(Bax and Bcl-2)and cycle-related proteins [ p21,Wee1,Cyclin B1,Cdc2 and p-Cdc2(Tyr15)].Results 1.The results of CCK-8 assay showed that cinobufagin could inhibit cell proliferation(P<0.05,R2>0.8)in a time-and concentration-dependent manner.2.The results of clone formation showed that cinobufagin could inhibit cell colony formation.3.The results of growth curve showed that cinobufagin could significantly cause growth inhibition.4.The results of PI staining by flow cytometry showed that cinobufagin could cause the cell cycle G2/M phase arrest.5.The results of Hoechst 33342 fluorescence staining showed that the morphological changes of cells were observed after 24 h intervention of cinobufagin,and the cells showed apoptotic morphological characteristics of nuclear chromatin condensation.6.The results of RT-q PCR showed that cinobufagin could up-regulate the m RNA expressions of p21 and Wee1 and down-regulate the m RNA expressions of Cyclin B1 and Cdc2(P<0.05).7.The results of Western blotting showed that cinobufagin could up-regulate the expression levels of p21,Wee1 and phosphorylated Cdc2(Tyr15),down-regulate the expression levels of Cyclin B1 and Cdc2(P<0.05)and up-regulate the ratio of Bax/Bcl-2(P<0.05).Conclusions 1.Cinobufagin can significantly inhibit the growth of esophageal cancer Kyse-520 cells in a dose-and time-dependent manner.2.Cinobufagin can arrest Kyse-520 cell in G2/M phase.3.Cinobufagin can induce apoptosis of esophageal cancer Kyse-520 cells.