Toxicarioside H induces ferroptosis in triple-negative breast cancer cells through Nrf2/HO-1 pathway

Recent studies have identified novel cardiac glycosides from natural sources with potential anti-tumor properties. Toxicarioside H (ToxH) is a novel cardiac glycoside isolated by our collaborative research team. However, its ability to induce ferroptosis in triple-negative breast cancer (TNBC) cells has not been investigated. Therefore, this study evaluates whether ToxH has the capability of inducing ferroptosis and elucidates the underlying molecular mechanisms. Treatment with ToxH led to dose- and time-dependent growth inhibition in BT-549 and MDA-MB-468 cells. Flow cytometry analysis and lactate dehydrogenase assay revealed that ToxH induced various forms of cell death in both BT-549 and MDA-MB-468 cells. Examination through transmission electron microscopy, along with flow cytometry analysis of 7-AAD-stained dead cells and ferroptosis markers BODIPY-C11 and Fe²⁺ ions, identified various forms of cell death induced by ToxH, including apoptosis, autophagy, apoptotic necrosis, and ferroptosis. Co-treatment with the ferroptosis inhibitor Fer-1 significantly reduced ToxH-induced cell death, indicating that ToxH primarily inhibits TNBC cell growth by inducing ferroptosis. Further investigation into the molecular mechanisms revealed upregulation of Nrf2 and HO-1 expression by ToxH. Effective inhibition of ToxH-induced ferroptosis was achieved through shRNA-mediated knockdown of HO-1 expression. Animal experiments demonstrated that ToxH treatment markedly suppressed tumor growth compared to the control group, while co-administration of Fer-1 led to an increase in tumor growth. These findings suggest that ToxH suppresses TNBC cell growth by modulating the Nrf2/HO-1 signaling pathway to induce ferroptosis. ToxH presents itself as a promising cardiac glycoside compound for TNBC treatment, warranting further translational research.