The Mechanism Of Cinobufagin-Induced Cell Cycle Arrest And Cell Apoptosis In Human Nasopharyngeal Carcinoma HK-1 Cells

Objective:The purpose of this study was to investigate the effect of cinobufagin on human nasopharyngeal carcinoma(NPC)HK-1 cell proliferation,and to further explore the molecular mechanism on cell cycle arrest and apoptosis induced by cinobufagin.Methods:(1)The effect of different concentrations of cinobufagin on HK-1 cell proliferation was determined by MTT assay.(2)The HK-1 cells treated with cinobufagin were stained by PI single staining method,and the distribution of cell cycle was analyzed by flow cytometry.(3)The morphologic changes of HK-1 cells treated with cinobufagin were observed by Hoechst 33258 staining.(4)The HK-1 cells treated with cinobufagin were stained by Annexin V-FITC/PI double staining method,and the apoptotic rates were measured by flow cytometry.(5)The mitochondrial membrane potential of HK-1 cells was detected by JC-1 method.(6)DCFH-DA probe was used to detect the changes of ROS level in HK-1 cells treated with cinobufagin.(7)The levels of cyclin-associated proteins(e.g.cyclin E and CDK2)and apoptosis-related proteins(Bax,Bcl-2,cytoplasm Cyt-C,Apaf-1,cleaved PARP1,cleaved caspase-3 and cleaved caspase-9)were detected by western blot.Results:The results of MTT assay showed that cinobufagin could significantly inhibit the proliferation of HK-1 cells,and its IC50 was 0.061 μg/mL.Meanwhile,after the treatment with cinobufagin,we found that the HK-1 nucleus were deformed in different degrees under fluorescence microscope.Some of the nucleus are contracted into clusters,characterized by high brightness.In addition,the proportion of HK-1 cells at S phase was significantly increased after treatment with cinobufagin.Meanwhile,the apoptosis rate was increased with the increase of drug concentration.At the same time,the mitochondrial membrane potential was significantly decreased and the level of intracellular reactive oxygen species was remarkably increased in cinobufagin-treated HK-1 cells.Western blot analysis showed that cinobufagin had an regulatory effect on the cell cycle and apoptosis-related proteins in HK-1 cells as follow:down-regulated the levels of cyclin E,CDK2 and Bcl-2,and up-regulated the levels of Bax,cytoplasm Cyt-C,Apaf-1,cleaved PARP1,cleaved caspase-3 and cleaved caspase-9.Conclusion:Cinobufagin inhibited the proliferation of HK-1 cells and arrested them in S phase by down-regulating the expression of cyclin E and CDK2.Meanwhile,cinobufagin increased the level of reactive oxygen species in HK-1 cells,down-regulated the level of Bcl-2,and up-regulated the level of Bax,and further increased the level of cytoplasm Cyt-C,elevated the levels of Apaf-1,cleaved PARP1,cleaved caspase-3 and cleaved caspase-9 in HK-1 cells,and thus induced apoptosis of HK-1 cells through mitochondrial-mediated apoptotic pathway.This study showed that cinobufagin could cause S-phase arrest of HK-1 cells and induce apoptosis,and subsequently inhibited cell proliferation.Therefore,cinobufagin may be helpful for the treatment of NPC.