Diphyllin induces autophagy-mediated ferroptosis in liver cancer

Background: Liver cancer is one of the most prevalent malignancies and a leading cause of cancer-related mortality worldwide, underscoring the urgent need for novel therapeutic strategies. Ferroptosis, a newly discovered form of regulated cell death characterized by iron accumulation and lipid peroxidation, has emerged as a significant mechanism in cancer biology and a potential therapeutic target. Diphyllin, a natural compound and potent V-ATPase inhibitor, displays promising anticancer activity in preliminary liver cancer studies. However, the molecular mechanism underlying the anti-tumor efficacy of diphyllin remains unclear, thus elucidating these mechanisms may not only deepen our understanding of liver cancer pathogenesis but also provide a scientific foundation for developing diphyllin-based therapies. Methods: The proliferative effect and cell death of diphyllin on liver cancer cells were quantitatively assessed using the EdU assay and Annexin V-FITC/PI staining followed by flow cytometry. To visualize the morphological alterations induced by diphyllin, the ultrastructure of treated cells was examined using transmission electron microscopy (TEM). For mechanistic exploration, transcriptomic analysis was performed to capture global gene expression changes. The western blotting and confocal immunofluorescence staining were used to measure the ferroptosis key proteins and autophagy markers. Furthermore, pharmacological autophagy inhibitors were applied to determine the dependency of ferroptosis on autophagy. Results: Treatment with diphyllin significantly suppressed the proliferation activity and increased the cell death rate in liver cancer cell lines. Transcriptomic profiling and western blot analysis revealed that diphyllin triggered ferroptosis, as evidenced by a marked downregulation of GPX4 protein expression. Furthermore, TEM analysis and immunofluorescence staining demonstrated that diphyllin induced autophagy, characterized by an elevated LC3B-II/LC3B-I ratio and a reduction in p62. Notably, the suppression of GPX4 expression was partially reversed upon treatment with an autophagy inhibitor, suggesting a functional link between diphyllin-induced autophagy and ferroptosis. Together, these results indicate that diphyllin promotes ferroptosis in liver cancer cells through an autophagy-dependent pathway. These findings provide a mechanistic basis supporting further preclinical investigation of diphyllin as a potential therapeutic agent for liver. Conclusions: This study demonstrates that diphyllin suppresses liver cancer by inducing both ferroptosis and autophagy. Mechanistic investigations revealed that diphyllin induced ferroptosis is an autophagy-dependent process, evidenced by the restoration of GPX4 upon autophagy inhibition. These findings establish autophagy-mediated ferroptosis as the novel mechanism underlying diphyllin's anti-tumor efficacy.