Toxicarioside H induces cytoprotective autophagy by hindering the progression of necroptosis in triple-negative breast cancer cells.

Recent studies have shown that cardiac glycosides can induce protective autophagy, but the precise mechanism is unclear. Our research focused on Toxicarioside H (ToxH), a newly discovered cardiac glycoside, to explore its ability to induce cytoprotective autophagy in triple-negative breast cancer (TNBC) cell lines and investigate the underlying mechanism. Cell growth and proliferation were measured using CCK-8 and EdU assays, while cell death was evaluated by flow cytometry and lactate dehydrogenase release assay. Autophagy and necroptosis markers were detected by Western blotting, and immunofluorescence was conducted for the autophagy marker LC3B and phosphorylated RIPK3 complex. Immunoprecipitation was used to detect the RIPK1/RIPK3 complex. Additionally, a mouse TNBC tumor model was established to assess the therapeutic effect of ToxH combined with CQ and the occurrence of autophagy and necroptosis. Our research showed that ToxH inhibited cell growth, increased cell death, and caused full autophagic flux in three TNBC cell lines. Co-treatment with CQ augmented ToxH's cytotoxicity both in vitro and in vivo. Autophagic degradation of the necroptosis-related proteins was observed in the TNBC cells treated with ToxH, as evidenced by the reduction of RIPK1/RIPK3 necrosomes and phosphorylated MLKL oligomers. This was restored by co-treatment with CQ. Autophagy and inhibition of necroptosis were also observed in TNBC tumor tissues. Our findings point to the conclusion that ToxH induces cytoprotective autophagy, leading to the breakdown of necrosomes and suppression of necroptosis in TNBC cells. Thus, a combination therapy involving ToxH and an autophagy inhibitor may be a potential treatment option for TNBC.