Study On The Effect And Mechanism Of AZT Combined With As₂O₃ On Pyroptosis In Hepatocellular Carcinoma

Objective:The target is to clarify the effect of AZT combined with As₂O₃on pyroptosis of hepatocellular carcinoma HepG2 and MHCC97-H cells,and to clarify the role of AQP9and FOXO1/Wnt-β-catenin signaling pathways in the process of pyroptosis induced by AZT combined with As₂O₃.The aim is to further understand the molecular mechanism of AZT combined with As₂O₃against hepatocellular carcinoma,which has important theoretical guiding significance for the clinical application of AZT combined with As₂O₃,an effective anti-liver cancer combination scheme.Methods:HepG2 and MHCC97-H cells of human hepatoma were routinely cultured.The effects of 20μmol/L AZT,2μmol/L As₂O₃and the combination of 20μmol/L AZT and2μmol/L As₂O₃on the proliferation of HepG2 and MHCC97-H cells were detected by CCK-8assay.ELISA kit was used to measure the release of inflammatory factor IL-18 in each group of cells after intervention.HepG2 and MHCC97-H cells were then transfected with AQP9si RNA and FOXO1 si RNA using liposomal GP as the vector,and the cells were divided into three groups:blank control group,non-specific control group（transfected with non-specific sequences）and target si RNA group（transfected with AQP9 or FOXO1 si RNA）.The expression of AQP9,FOXO1,β-catenin and pyroptosis-related genes NLRP3,GSDMD and IL-18 mRNA and proteins were examined by RT-qPCR and WB in each group of cells after the intervention,respectively.Results:1.In the CCK8 experiment,the proliferation rate of hepatocellular carcinoma cells in each group declined more remarkably（P<0.01）at 72h after drug treatment compared with the negative control group（equal amount of DMEM with the drug administration group）,and the proliferation rate of cells in the combined drug application group decreased more significantly（P<0.01）than that in the drug usage group alone.2.ELISA experiments showed that the release of IL-18 was obviously higher in both cell dosing groups compared with the white group（P<0.01）,more pronounced in HepG2 cells compared with the As₂O₃group（P<0.05）,and more marked in MHCC97-H cells compared with the AZT and As₂O₃groups（P<0.01 or 0.05）.3.After 72h of drug administration alone or in combination,RT-qPCR and WB results showed that NLRP3,GSDMD,IL-18 mRNA and protein levels were significantly higher in the combination group in comparison with the blank control group（P<0.01）.The NLRP3,IL-18 mRNA and protein expression of HepG2 cells were greatly increased and GSDMD protein expression was especially higher（P<0.01 or 0.05）in the combination group compared with AZT and As₂O₃groups,respectively;NLRP3,GSDMD mRNA and protein expression of MHCC97-H cells were significantly elevated（P<0.01 or 0.05）.4.Relevant mRNA and proteins were assayed by RT-qPCR and WB assays after cells were transfected with AQP9 or FOXO1 and co-intervened with drugs.The AQP9-NC group showed higher expression of NLRP3,GSDMD and IL-18 mRNA and protein in both cell lines in comparison with the MOCK group（P<0.01 or 0.05）.In the si-AQP9 group,compared with the MOCK group,HepG2 cells showed high expression of NLRP3 and IL-18 mRNA together with increased levels of NLRP3 and GSDMD proteins（P<0.01 or 0.05）;MHCC97-H cells showed a significantly high exposure to NLRP3 mRNA,while GSDMD mRNA was notably lowered,and meanwhile NLRP3,GSDMD and IL-18 protein all had increased expression（P<0.01 or 0.05）.The si-AQP9 group showed lower expression of FOXO1 mRNA and protein and higher expression ofβ-catenin protein in both cell lines compared with the NC group（P<0.01）;GSDMD mRNA exposure was dramatically reduced in HepG2 cells,but IL-18 mRNA expression was significantly increased,and NLRP3,GSDMD and IL-18 protein expression were remarkably declined（P<0.01）;MHCC97-H cells showed considerably higher NLRP3 mRNA and protein expression,while GSDMD and IL-18 mRNA and protein levels were notably depressed（P<0.01 or 0.05）.The NLRP3,GSDMD and IL-18 mRNA and protein were highly expressed in the si-FOXO1 and NC groups,respectively,compared with the MOCK group in both strains（P<0.01 or 0.05）.The si-FOXO1 group showed elevatedβ-catenin protein production in both strains of cells compared with the NC group（P<0.01）;NLRP3,GSDMD,and IL-18 mRNA contents were greatly improved in HepG2 cells,and NLRP3 and GSDMD protein expression were significantly increased,while IL-18 protein was markedly decreased（P<0.01）;NLRP3 and GSDMD mRNA expression were seriously raised in MHCC97-H cells,and GSDMD protein expression was obviously elevated,while IL-18 protein exposure was significantly decreased（P<0.01）.Conclusion:（1）AZT combined with As₂O₃may promote pyroptosis of hepatocellular carcinoma HepG2 and MHCC97-H cells through FOXO1/Wnt-β-catenin signal path;（2）AZT combined with As₂O₃may restrain the proliferation of hepatocellular carcinoma cells by inducing pyroptosis.