The Role Investigation Of Acid Ceramidase Regulates Apoptosis Of HepG2 Cells In Hypoxia

Objective:An analogue of hypoxia called cobalt chloride（CoCl₂）was taken to simulate a hypoxic microenvironment of liver tumor of solid in vitro,investigating the roles and their mutual relationship of acid ceramidase which called ASAH1 and Caspase-3 in the apoptosis of HepG2 cells which induced by hypoxia.Methods:A human liver cancer cell line marked HepG2 was chose for this study,thawing and culturing for one week by conventional method.Then,we put to use the cells which are proliferating during the Logarithmic phase to carry out the subsequent experiments.According to our study scheme,for the experiment groups,treating the cells by corresponding solution which contains media and different reagents for 24 h.The proliferation of HepG2 cells was detected by MTT assay,the apoptosis by Flow cytometry,the protein expression of ASAH1 and active caspase-3 by western blot,and the obtained results would be digitized by gray scanning（Image-J software）and statistical analysis.Results:（1）The proliferation and apoptosis（ASAH1inhibitors treated free）:As the concentrations of CoCl₂ elevation,that is to say,the hypoxic degree of liver cancer get more and more severe,the proliferation displays more and more decrease,on the contrary,the apoptosis suggests a tendency of increase,the viability/apoptosis of those groups have a statistically significant difference（p<0.05）.（2）The proliferation and apoptosis（ASAH1inhibitors treated）:the inhibitors of ASAH1 combine with CoCl₂（200μM）respectively,the proliferation of combinations have a trend of decline compared to the other three groups,apparently,the apoptosis holds the opposite results,the viability/apoptosis of those groups have a statistically significant difference（p<0.05）.（3）The expression of ASAH1 and active Caspase-3 protein（ASAH1inhibitors treated free）:when the hypoxia status gets harder,the expression of ASAH1 gets lower and lower compared to the control,Akt gets lower and lower compared to the control,and active Caspase-3 gets higher and higher,however,the group of CoCl₂（200μM）higher than the control,the expression of the two proteins of those groups have a statistically significant difference（p<0.05）.（4）The expression of ASAH1 and active Caspase-3protein（ASAH1inhibitors treated）:The inhibitors of ASAH1 that B13,C6-cer combines with CoCl₂（200μM）respectively,the expression of active Caspase-3protein higher obviously than the control and inhibitors only treated,the differences have a statistical significance（p<0.05）,and the group which treated with NOE（an inhibitor of ASAH1）and CoCl₂（200μM）holds a lower expression of active Caspase-3 protein than the control and the NOE treated only.Conclusions:（1）The analogue of hypoxia named cobalt chloride（CoCl₂）can simulate a hypoxic microenvironment of liver tumor of solid in vitro,so a kind of cell patterns of hypoxia were established.（2）Under the status of hypoxia,ASAH1 can regulate the apoptosis of HepG2 cells.（3）Under the state of hypoxia,active Caspase-3 can induce the apoptosis of HepG2 cell.（4）In hypoxic condition,ASAH1 may be through the Akt protein function with the mitochondrial pathway to active Caspase-3 protease,resulting in cell apoptosis.（5）These data will give a guiding role for tumors intervention which are in hypoxia microenvironment,and will provide some help for the treatment of hepatic solid tumors.