Study on Mitochondrial Apoptosis and Ferroptosis Induced by Huqi Powder(虎七散)in EC9706 Esophageal Cancer Cells
Objective:The purpose of this study was to investigate whether Huqi Powder(虎七散)could induce mitochondrial apoptosis and ferrop-tosis by regulating redox and mitochondrial homeostasis,and to further analyze its effect on p53 protein expression.Methods:In the mitochon-drial apoptosis experiment,the esophageal cancer EC9706 cells were divided into control group,Huqi Powder 2 mg/mL,4 mg/mL and 8 mg/mL groups.In ferroptosis assay,the cells were divided into control group,ferrostatin-1 10 μmol/L group,combination group(ferrosta-tin-1 10 pmoVL+Huqi Powder 8 mg/mL),and Huqi Powder 8 mg/mL group.Cell viability was measured by CCK-8 assay.The levels of intracellular GSH and MDA,and SOD activity were measured by the assay kits.The fluorescence probes such as DCFH-DA,C11-BODIPY 581/591,JC-1,and Phen Green SK(PGSK)were used to measure the fluorescence intensity of reactive oxygen species(ROS),lipid peroxi-dation of ROS,mitochondrial membrane potential,and iron ions,respectively.Cell invasion and migration were measured by the Transwell in-vasion and migration assay.One step TUNEL apoptosis assay kit was used to detect apoptosis.Expression of mitochondrial apoptosis-related proteins(Bcl-2,Bax,CytC,active caspase-3),ferroptosis-related proteins(xCT,ACLS4,GPX4)and p53 protein were measured by Western Blot.Results:In mitochondrial apoptosis assays,compared with control group,8 mg/mL Huqi Powder effectively inhibited EC9706 cell pro-liferation,decreased intracellular GSH content and SOD activity,increased the fluorescence intensities of ROS and mitochondrial membrane potential,increased cell apoptosis,and inhibited cell invasion and migration,up regulated the expressions of Bax,CytC,active caspase-3 and p53,and down regulated the Bcl-2 expression(P＜0.05 or P＜0.01).Ferroptosis assays showed that compared with control group,8 mg/mL Huqi Powder reduced GSH level and increased MDA level,increased the fluorescence intensities of ROS,lipid peroxidation of ROS and mito-chondrial membrane potential,decreased iron ion fluorescence,up regulated the expressions of ACSL4 and p53,down regulated the protein ex-pressions of xCT and GPX4(P＜0.05).Compared with 8 mg/mL Huqi Powder group,the combination treatment(Ferrostatin-1 10 µmol/L+Huqi Powder 8 mg/mL)increased intracellular GSH level,decreased MDA level,decreased the fluorescence intensities of ROS,lipid peroxi-dation of ROS and mitochondrial membrane potential,while the fluorescence intensity of ferric ions was enhanced.The expressions of xCT and GPX4 were up regulated,while the expressions of ACSL4 and p53 were down regulated(P＜0.05).Conclusion:Huqi Powder has a pro-oxi-dant effect and can regulate redox and mitochondrial homeostasis by promoting the over-accumulation of ROS and lipid-based ROS,which in-duces mitochondrial apoptosis and ferroptosis,and positively up regulates p53.The drugs targeting redox,mitochondrial homeostasis and p53,as well as synergistic mitochondrial apoptosis and ferroptosis could be potential medicine for cancer therapy.
Huqi Powder(虎七散)RedoxMitochondriaMitochondrial apoptosisFerroptosisp53 protein
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