6″-methyl-2″,3″-dihydroochnaflavone induces ferroptosis in breast cancer cells via adaptive glutathione remodeling and stress-mediated antioxidant system collapse

This study aimed to elucidate the anti-breast cancer effects and underlying molecular mechanisms of (2″S)-6″-methyl-2″,3″-dihydroochnaflavone (DHO) in MCF-7 and MDA-MB-231 cells. Functional validation revealed that DHO significantly suppressed the proliferation of both breast cancer cell lines, with IC 50 values of 6.5 μM and 16.8 μM, respectively, and triggered canonical ferroptosis phenotypes including lipid ROS/MDA buildup and mitochondrial injury, which was fully rescued by ferroptosis inhibitors. Integrated transcriptomic and metabolomic profiling revealed a convergent disruption of glutathione, cysteine, and methionine metabolism, alongside purine and fatty acid pathways. Molecular dynamics simulations predicted a stable binding interaction between DHO and GPX4, accompanied by significant downregulation of GPX4 functional activity. The rescue experiments demonstrated that the cytotoxic effect of DHO could be partially counteracted by co-treatment with the GPX4 activator 1d4. Mechanistically, DHO suppressed GPX4 activity to initiate ferroptosis damage; in addition, DHO treatment triggered an adaptive but dysfunctional upregulation of the ferroptosis defense mediators SLC7A11 and GCLM through activation of the integrated stress response pathway marked by elevated ATF4, CHAC1 and DDIT3, yet this compensatory antioxidant program ultimately failed to reverse severe GSH depletion and subsequent antioxidant system collapse, leading to robust ferroptosis in breast cancer cells. Collectively, these results demonstrate that DHO triggers ferroptosis in breast cancer cells by remodeling adaptive glutathione metabolism and disrupting cellular antioxidant homeostasis, thereby leading to irreversible oxidative damage and tumor cell death.