Study On The Mechanism Of 8-Azaguanine-Induced Autophagy And Apotosis In Hepatic Cancer Cells

8-Azaguanine(8-AG),a purine analog that competes with guanine in metabolism,interfering with DNA replication and protein synthesis,has an anti-tumor activity.However,8-AG resistance has limited its development as a cancer drug,and the mechanism of resistance is not fully understood.Autophagy,a conservative degradation pathway,is an important process for maintaining intracellular homeostasis in eukaryotic cells.It is closely related to the development of tumor.Autophagy is used by tumor cells to prevent apoptosis induced by anti-tumor drugs,thus promoting tumor resistance.In this study,the effect of 8-AG on autophagy and apoptosis was detected in hepatic cancer cells,and its mechanism of action was studied by the molecular and cellular biology techniques.The aim of the study is to reveal the mechanism of 8-AG drug resistance.The main results were as follows:(1)Using HeLa cell stably expressing EGFP-LC3 to study the effect of 8-AG on autophagy,the results showed that 8-AG induces EGFP-LC3 puncts in a time-and dose-dependent manner.(2)The protein levels of autophagy marker LC3 and p62 were detectedin hepatic carcinoma cell lines HepG2 and SMMC-7721.Western blot results showed that 8-AG induced the accumulation of LC3-Ⅱ in a time-and dose-dependent manner,and promoted the degradation of p62.Co-treatment with the autophagy inhibitor Bafilomycin A1(Baf Al)and 8-AG promoted the accumulation of LC3-Ⅱ,indicating that 8-AG promotes autophagy in hepatic cancer cells.(3)The molecular mechanism of 8-AG-induced autophagy was detected by Western blot and fluorescence staining.The results showed that 8-AG treatment significantly inhibited the activity of Akt and mTOR downstream protein p70s6k and increased the levels of ULK1 dephosphorylation.8-AG treatment also induced an increase in intracellular ROS levels,indicating that 8-AG might induce autophagy in hepatic cancer cells through the Akt/mTOR/ULK1 signaling and the ROS pathway.(4)The effects of 8-AG on cell viability and apoptosis in HepG2 and SMMC-7721 cells were detected by MTT and flow cytometry techniques.The results showed that 8-AG treatment significantly inhibited hepatic cancer cell viability in a dose-and time-dependent manner and promotes mitochondrial membrane permeability.Moreover,8-AG induces the increases of the pro-apoptotic protein BimS,the cleaved-caspase 9 and cleaved-caspase 3 protein,thereby inducing endogenous apoptosis of hepatic cancer cells.(5)The effect of autophagy on 8-AG-induced apoptosis was detected by RNAi technology and inhibitor assays.The results showed that shRNA knockdown of ATG7 significantly increased 8-AG-induced cell viability inhibition and apoptosis.The same results were obtained by the combination of 8-AG and autophagy inhibitors(Baf Al or CQ).The data showed that inhibiting autophagy in hepatic cancer cells can significantly increase cytotoxicity of 8-AG.In summary,8-AG induces autophagy through the Akt/mTOR/ULK1 signaling and the ROS pathway.Inhibiting autophagy significantly increases the cytotoxicity of 8-AG in hepatic cancer cells.These data reveal the molecular mechanism of 8-AG-induced autophagy,deepening our understanding of 8-AG chemoresistance.Our study provides a theoretical basis for the anti-cancer drug development of 8-AG.