The Role Of Autophagy In ADM Resistance In Human Hepatoma Carcinoma HepG2/ADM Cells

Objective : To explore the role of autophagy in ADM(Adriamycin)resistance in human hepatocellular carcinoma Hep G2/ADMcells,and then to detect the role of autophagy in chemoresistance human hepatocellular carcinoma cells.The autophagy was inhibited by the application of chloroquine,and the change of the ADM resistance was observed in human hepatocellular carcinoma HepG2/ADM cells.Methods:Human hepatocellular carcinoma Hep G2 cells and drug-resistant HepG2/ADM cells were cultured in vitro;CCK-8 assay was also used to test the ADM resistance in HepG2/ADM cells;MDC method was used to observe distributions of intracytoplasmic autophagic vesicles after the use of ADM of different concentrationsin Hep G2/ADM cells;WB method was uesd to detect the expressions of LC-3 and P62 in HepG2/ADM cells at different concentrations of ADM;CCK8 assay was used to test the inhibitory effect of autophagy inhibitor chloroquine(CQ)on HepG2/ADM cells;MDC method was used to observe the distributions of intracytoplasmic autophagic vesicles in HepG2/ADM cells in ADM group,CQ group and CQ+ADM group;Western Blot method was used to detect the expressions of LC-3 and P62 in ADM group,CQ group and CQ+ADM group in HepG2/ADM cells.CCK-8 assay was also used to test the ADM resistance in HepG2/ADM cells and changes of drug sensitivity to ADM before and after inhibiting autophagy.Results:1.The half-maximal inhibitory concentration(IC50)value was 5.01±1.43mg/L in HepG2/ADM cells,and 0.77±0.11mg/L in Hep G2 cells,respectively;The resistance indexes were 6.512.Via MDC method the accumulation of autophagic vacuoles was observed in cytoplasm of HepG2/ADM cells after the ADM treatment.The expression of LC3-II increased with elevated ADM concentration,but the expression of P62 decreased with increased ADM concentration.3.Chloroquine,which can effectively inhibit autophagic flux,with a concentration of 10-50 umol/ml,had no apparent cytotoxic effects on the HepG2 or HepG2/ADM cell line and its growth inhibitory rates were lower than 10%.4.By MDC method the accumulation of autophagic vacuoles was observed in the cytoplasm in ADM group,CQ group and ADM+CQ group.The most significant were in group ADM+CQ,and there was no significant accumulation of autophagic vacuoles in negative control group.Western Blot method,LC3-II expression ratio increased,compared with the control group.In ADM Group P62 expression decreased and did not change significantly,and LC3-II expression rate increased.In CQ+ADM group the expression of LC3-II significantly increased,the expression of P62 was slightly lower than group CQ,and little higher than group ADM.5.After autophagy inhibited by CQ,the IC50 of ADM in HepG2/ADM cells was decreased from 5.01±1.43mg/L to 2.83±0.14mg/L,and the reversal index was 1.77 times.Conclusions:1.After Hep G2 / ADM was treated with ADM,the drug resistance of liver cancer cell lines HepG2 / ADM induced autophagy.Autophagy level increases with the raised concentration of ADM.2.Inhibition of autophagy partial reversal the resistance of ADM in HepG2 / ADM.