Andrographolide disrupts SDHA–SDHAF4 assembly-mediated mitochondrial metabolic homeostasis to promote ferroptosis and doxorubicin sensitivity in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains highly refractory to chemotherapy, largely owing to apoptosis-associated resistance to anthracyclines such as doxorubicin (DOX). Ferroptosis, an iron-dependent and lipid peroxidation-driven form of regulated cell death, has emerged as a promising strategy to circumvent apoptotic escape. However, clinically actionable ferroptosis-inducing targets in TNBC remain poorly defined. In particular, whether mitochondrial complex II assembly contributes to ferroptosis and chemoresistance has not been elucidated, and no small-molecule compound has been reported to directly target succinate dehydrogenase complex flavoprotein subunit A (SDHA), the catalytic core component of mitochondrial complex II, or disrupt its interaction with succinate dehydrogenase complex assembly factor 4 (SDHAF4). Ferroptosis responses were assessed by transmission electron microscopy, lipid peroxidation assays, and measurements of intracellular Fe 2+ , malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Target identification was performed using drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), mass spectrometry, pull down and surface plasmon resonance (SPR) assays, and molecular docking. Disruption of the SDHA–SDHAF4 complex was examined by co-immunoprecipitation and molecular dynamics simulation. Nuclear protein isolation and confocal microscopy were used to determine nuclear localization, and protein succinylation was analyzed by co-immunoprecipitation. We identified andrographolide (AG), a natural diterpenoid isolated from Andrographis paniculata , as the first reported small-molecule compound that directly targets SDHA and disrupts SDHA–SDHAF4 assembly. AG potently induced ferroptosis, inhibited TNBC cell proliferation, and enhanced DOX sensitivity in TNBC 2D cell lines and 3D patient-derived organoids. AG increased lipid peroxidation, Fe 2+ accumulation, ROS, and MDA levels, while ferroptosis inhibitors or ROS scavengers attenuated these effects. In vivo, AG synergized with DOX to suppress TNBC tumor growth without detectable systemic toxicity. Mechanistically, SDHA—the catalytic subunit of mitochondrial complex II—was identified as a direct molecular target of AG. Structural and biochemical analyses revealed that AG binding at Arg451 and His407 disrupted the SDHA–SDHAF4 interaction, impairing SDHA maturation and destabilizing complex II assembly. Importantly, this study is the first to demonstrate that SDHA–SDHAF4 assembly integrity is a critical metabolic checkpoint governing ferroptosis sensitivity and chemoresistance. Disruption of this assembly triggered two convergent ferroptotic signals: excessive ROS generation and succinate accumulation-mediated Keap1 succinylation, leading to activation of the Nrf2/HO-1 pathway. Clinically, high SDHA expression correlated with poor TNBC prognosis and reduced DOX sensitivity in lung and stomach cancers, consistent with chemosensitizing effects of AG in these models. Notably, SDHA knockdown further amplified AG-induced ferroptosis. This study identifies AG as the first natural small-molecule inhibitor that targets SDHA and disrupts SDHA–SDHAF4 assembly. More importantly, it reveals for the first time that SDHA–SDHAF4 complex integrity governs ferroptosis susceptibility and anthracycline resistance, uncovering a previously unrecognized SDHA-centered metabolic vulnerability that can be therapeutically exploited to induce ferroptosis and overcome DOX resistance.