Study On The Improvement Of Cell Sensitivity Of Human Hepatocellular Carcinoma BEL-7402/ADM By The Regulation Of Autophagy By Gambogenic Acid

Autophagy is a self-degradation process of cells,which can maintain cell homeostasis by degrading damaged organelles and macromolecular substances and recycling decomposition products,especially under the conditions of chemotherapy drugs and growth factor deprivation.Autophagy is a double-edged sword.On the one hand,the body can kill or remove tumor cells through autophagy,on the other hand,it also provides necessary nutrients and energy for the growth of tumor cells.When chemotherapy drugs,radiation and other external pressure act on tumor cells,tumor cells can resist the killing effect of anticancer drugs or radiation by regulating the autophagy level of cells,so as to enhance the survival rate and tolerance of tumor cells.The relationship between autophagy and drug resistance has been widely concerned.As an active component of traditional Chinese medicine,Gambogenic acid(GNA)has been shown to have a good anti-tumor effect,and its mechanism is related to the regulation of mitochondrial oxidative stress,the induction of cell apoptosis and the inhibition of protective autophagy of tumor cells.Low dose chemotherapeutic drugs can reverse tumor resistance,but the pathway and target of the reversal action are still unknown.Based on the relationship between autophagy of tumor cells and drug resistance,this study investigated the correlation between GNA reversal of drug resistance and its regulation of autophagy of tumor cells,and the preliminary localization of related action targets.This study is expected to provide a theoretical basis for the further development and utilization of GNA.Objective:This study examined the basic autophagy level of human hepatocellular carcinoma resistant cell line BEL-7402 /ADM and its parent BEL-7402 cell line,identified the relationship between autophagy and cell resistance,and investigated whether GNA could improve the sensitivity of BEL-7402 /ADM by regulating the level of autophagy.Methods:1.MTT assay was used to detect the proliferation activity of BEL-7402 and BEL-7402/ADM cells.Western blot method was used to detect the expression level of drug resistance-related protein p-gp.2.GNA increased the sensitivity of drug-resistant cells to adriamycin.The IC90 values of GNA on BEL-7402 /ADM cells at 12 h,24h and 48 h were 7.01 mol/L,3.36 mol/L and 2.17 mol/L,respectively.3.The autophagosomes were observed by fluorescence staining with montan sulfonyl resin(MDC)and transmission electron microscopy.Western blot method was used to detect the expression levels of basic autophagy-related proteins LC3-?/LC3-?,Beclin1 and P62.4.MDC staining and transmission electron microscopy were used to detect autophagy morphology.The expression levels of autophagy-related proteins LC3-?/LC3-?,Beclin1 and P62 were detected by Western blot.5.The cell state was observed by an inverted microscope.DAPI staining fluorescence microscopy was used to observe the nuclear changes.The apoptosis rate was detected by flow cytometry.The expression levels of caspase-3,Bax and Bcl-2 were detected by Western blot.Results:1.The sensitivity of BEL-7402/ADM cells to doxorubicin was low,and the IC50 values of 12 h,24h and 48 h were 16.7,17.0 and 18.53 times that of BEL-7402 cells,respectively.Western blot results showed that p-gp was highly expressed in BEL-7402/ADM cells and poorly expressed in bel-7402 cells.2.GNA increased the sensitivity of drug-resistant cells to adriamycin.The IC90 values of GNA on BEL-7402 ADM cells at 12 h,24h and 48 h were 7.011,3.361 and 2.167mol/L,respectively.3.BEL-7402/ADM cells based autophagy level obviously is higher than the BEL-7402 cells,BEL-7402/ADM cells contain more autophagy bubble fluorescent accumulation and typical autophagy bubble structure,at the same time the BEL-7402/ADM cells Beclin1 protein involved in autophagy and LC3-?/LC3-? ratio is higher than the BEL-7402 cells,P62 protein levels are low the BEL-7402 cells.4.GNA inhibit autophagy levels increase the BEL-7402/ADM cells to adriamycin sensitivity,GNA pretreatment group can enhance autophagy bubble fluorescent accumulation and more typical autophagy bubble structure,at the same time,the BEL-7402/ADM cells Beclin1 protein involved in autophagy and LC3-?/LC3-? ratio is higher than that of BEL-7402 cells,but P62 protein levels are significantly increased the BEL-7402 cells.5.GNA increased adriamycin-induced cell apoptosis.the apoptosis rate was increased by cell flow cytometry.Meanwhile,caspase-3 protein level and Bax/Bcl-2 protein ratio were significantly increased.Compared with BEL-7402 cells,the basis of BEL-7402/ADM cells with high level of autophagy,that autophagy is involved in the formation of the cell resistance,and GNA can reverse the BEL-7402/ADM cells to adriamycin resistance and GNA inhibiting autophagy and increased the level of apoptosis,GNA can inhibit cell autophagy activity to improve the BEL-7402/ADM cells to promote apoptosis of the sensitivity of adriamycin.