Autophagy Contributes To Erastin-induced Ferroptosis In Breast Cancer Cells Via Promotion Of Intracellular Iron And Hydrogen Peroxide

Background:Breast cancer is the most common malignant tumor among women worldwide.It is the leading cause of cancer related death in most countries.In China,the incidence and mortality rate of breast cancer are rising every year,which is a serious threat to women’s life and health.Breast cancer is a highly heterogeneous systemic disease.Individualized combined treatment strategy should be formulated according to its pathological classification.In order to mitigate drug resistance and side effects while reduce the recurrence and metastasis rate of breast cancer,new therapeutic targets need to be explored.Ferroptosis,characterized by iron-dependent lipid peroxidation,is a research hotspot in recent years.The interventions targeting ferroptosis have a wide application prospect in the treatment of malignant tumors.Small molecule compound Erastin can trigger ferroptosis through multiple signal pathways and produce synergistic effects when combined with a variety of antitumor drugs.Conceivably,it is expected to be used as a sensitizer for chemotherapy and radiotherapy.It has been speculated that there is a network organization between ferroptosis and other programmed death such as autophagy.In our previous study,we found that Erastin could induce autophagy in breast cancer cells.However,there is no specific definition of how autophagy regulates ferroptosis at present.Therefore,we studied the role and mechanism of autophagy in Erastin-induced ferroptosis in breast cancer cells,in order to provide a theoretical basis for its clinical application.Objective:To clarify the role of autophagy in Erastin-induced ferroptosis in breast cancer cells and explore its mechanism.Methods:1.Detection of cell viability and cell death ratio:CCK-8 assay detects the inhibitory rate of Erastin on proliferation of breast cancer cells;LDH release assay was used to detect the toxicity of Erastin to tumor cells and the killing effect of Erastin after added inhibitors.2.Detection of iron content:Calcein staining method was used to detect the change of intracellular unstable Erastin pool（LIP）in Erastin-treated breast cancer cells and effect by inhibitor DFO.Iron colorimetric method detection kit was used to evaluate H₂O₂-treated breast cancer cells and effect by inhibitor GSH.3.Detection of Cellular protein expression level:Western Blot was used to detect the ferroptosis and autophagy-related protein levels of the samples of Erastin time-gradient and the samples of Erastin+inhibitors.4.Detect the intracellular concentration of H₂O₂ in Erastin-treated breast cancer cells and effect by inhibitors include GSH.5.Detect the concentration of intracellular cysteine in Erastin-treated breast cancer cells and effect by inhibitors include 3-MA、Baf-A1.6.Detect the concentration of GSH in Erastin-treated breast cancer cells and effect by inhibitors include 3-MA、Baf-A1.7.Detect the level of lipid peroxidation using C11-BODIPY^581/591 in Erastin-treated breast cancer cells and effect by inhibitor 3-MA.8.Detection of cell gene expression level:Test the effect of silencing ATG5 by si RNA on Erastin-induced ferroptosis process in breast cancer cells.Results:1.Erastin can inhibit the viability of breast cancer cells and increase the level of intracellular iron and ferroptosis related proteins in a dose-dependent manner.This process can be salvaged by iron scavenger DFO.2.Erastin promoted hydrogen peroxide accumulation in breast cancer cells in a time and concentration dependent manner.Hydrogen peroxide promoted iron accumulation and ferroptosis related protein levels in a dose-dependent manner,and the process could be inhibited by antioxidant GSH.3.Erastin promotes the expression of autophagy related proteins in breast cancer cells in a time-dependent manner.Autophagy inhibitors 3-MA and Baf-A1 can inhibit the expression of ferroptosis related protein,accumulation of hydrogen peroxide and cell death in Erastin-treated breast cancer cells.4.Erastin induces consumption of cysteine,GSH,x CT and catalase in breast cancer cells in a time and dose dependent manner,and this process can be inhibited by autophagy inhibitor 3-MA and Baf-A1.Knockdown of ATG5 prevented Erastin-triggered expressional downregulation of catalase and x CT in breast cancer cells.Conclusion:1.Erastin inhibited viabilities and induced death in breast cancer cells.2.Erastin caused hydrogen peroxide accumulation and glutathione depletion in human breast cancer cells.3.Autophagy contributed to Erastin-induced ferroptosis via improving H₂O₂.4.Hydrogen peroxide promoted Erastin-induced ferroptosis by improving intracellular iron.5.Autophagy contributed to Erastin-induced accumulation of intracellular H₂O₂via clearance of x CT and catalase.