3’-epi-12β-Hydroxyfroside-Mediated Autophagic Degradation Of RIPK1/RIPK3 Necrosomes Leads To Anergy Of Immunogenic Cell Death In Triple-Negative Breast Cancer Cells

Objective: The study aimed to verifying whether HyFS can induce the occurrence of immunogenic cell death(ICD)in the occurrence of triple negative breast cancer(TNBC)cells,and explore its molecular mechanism to play an anti-tumor role.Methods:1.Flow cytometry,Western blot,ELISA and immunofluorescence tests were used to compare the death modes of triple negative breast cancer cells induced by HyFS and digoxin treatment.2.Under the condition of HyFS treatment,the expression of autophagy markers was detected by Western blot after further treatment with autophagy inhibitors 3-MA and CQ,and the content and location of autophagosomes and autophagolysosomes in TNBC cells were detected by immunofluorescence assay.3.The degree of ICD occurrence in triple negative breast cancer cells under the conditions of HyFS plus autophagy inhibitor and digoxin alone was compared,thus confirming the involvement of HyFS-induced autophagy in the regulation of ICD.4.The expression of ICD markers RIPK1,RIPK3,KLKL,and p MLKL in combination with HyFS plus autophagy inhibitor treatment and digoxin treatment alone clarified the molecular mechanism of HyFS participating in the regulation of ICD through autophagy.5.In vivo animal experiments have verified that the combined treatment of HyFS and autophagy inhibitor CQ induces ICD and can play a powerful anti-tumor role in triple negative breast cancer.Results:1.Although it has a similar structure to DIG,HyFS does not induce ICD in TNBC cells like DIG,but mainly induces autophagy.2.HyFS induces complete autophagy flux in TNBC cells.3.Autophagy induced by HyFS inhibited ICD in TNBC cells.4.HyFS-induced autophagy degradation of RIPK1/RIPK3 necrosomes leads to inactivation of RIPK3/MLKL signaling pathway and ICD inhibition in TNBC cells.5.HyFS combined with autophagy inhibitor CQ can make immunocompetent mice produce ICD and exert strong anti-tumor activity.Conclusions:1.1.Autophagy degradation of RIPK1/RIPK3 necrosomes mediated by HyFS leads to ICD inactivation in TNBC cells.2.HyFS combined with autophagy inhibitor can enhance the anti-tumor activity,suggesting an alternative therapeutic for TNBC treatment.