The Mechanism Of Bufotalin-induced Cell Cycle Arrest And Cell Apoptosis In Human Malignant Melanoma A375 Cells

Objective:In this study,we attempted to examine the effect of bufotalin on human malignant melanoma A375 cell proliferation and to explore its molecular mechanism.Methods:Inhibition of proliferation of A375 cells by different concentrations of bufotalin by MTT assay.The A375 cells treated with bufalin were stained by PI single staining method,and the DNA content in the cells was determined by flow cytometry,and the distribution of cell cycle was analyzed.Observation of nuclear morphologic changes of A375 cells treated with bufotalin by Hoechst 33258 fluorescence staining.The A375 cells treated with bufotalin were stained by AnnexinV-FITC/PI double staining method,and the total apoptosis rate was measured by flow cytometry.The expression of apoptosis-related proteins was detected by Western blot.For example:AKT?p-AKT?Caspase-3?Caspase-9?Bcl-2 and BAX.Simultaneous detection of cyclin-associated protein expression.For example,the changes of Cyclin B?CDK1?CDC25C?ATM and Chk1.Results:The results of MTT assay showed that bufotalin could effectively inhibit the proliferation of A375 cells,and the inhibitory effect was significantly increased with the increase of administration concentration and time.In addition,the proportion of A375 cells in G2/M phase was significantly increased after treated with bufotalin for 24 h.In addition,it was observed under fluorescence microscope that the nucleus morphology of A375 treated with Bufotalin showed obvious changes,such as nuclear concentration and nuclear fragmentation.At the same time,the results of flow cytometry showed that the total apoptosis rate of A375 cells was increased.Finally,at the level of protein expression,bufotalin could change the expression of cell cycle related protein and apoptosis-related protein in A375 cells.The expression of ATM,Chk2,BAX,Cleaved Caspase-9 and Cleaved Caspase-3 was upregulated.At the same time,the expression of AKT,p-AKT,BCL-2 and CDC25C was down-regulated.Conclusion:The results showed that the contents of ATM,Chk2 and CDC25C in A375 cells changed after treatment with toadin,which resulted in the decrease of the total amount of Cyclin B/CDK2 complex and the stagnation of A375 cells in G2/M phase.In addition,toadin can down-regulate the expression of anti-apoptosis factors p-AKT and BCL-2 in A375 cells,and up-regulate the expression of BAX,Cleaved Caspase-3 and Cleaved Caspase-9 in A375 cells,thus promoting apoptosis.The results of this study provide substantial evidence for the anticancer activity of toadin and reveal the mechanism of the role of toadin in human malignant melanoma A375 cells.