Ferroptosis necessitates caspase 5 dependent GSDME cleavage in ovarian cancer

Ferroptosis, an intracellular iron-catalyzed form of programmed cell death (PCD) driven by lethal lipid reactive oxygen species (ROS)-induced membrane damage, is mechanistically uncharacterized in its execution process as a newly discovered PCD pathway. This study aimed to elucidate the molecular execution cascade of ferroptosis in ovarian cancer and to determine whether it uses common effector proteins shared with other PCD mechanisms. Mesenchymal-like ovarian cancer cells (OCC1, OVCAR8, ES2) were exposed to Erastin and ML162 with pathway-specific inhibitors, providing evidence that only the pyroptosis inhibitor YVAD reduced ferroptosis-induced death, supporting the association of pyroptotic executors. Lactate dehydrogenase (LDH) release and IL-1beta secretion indicated that ferroptosis activates a pore-forming execution process, while both YVAD and gasdermin inhibitor disulfiram protected membrane damage. Ferroptotic cells exhibited cleavage of Gasdermin E (GSDME), whereas Gasdermin D was not processed. GSDME knockdown protected ovarian cancer cells from ferroptotic death, confirming its essential role in the execution phase. Our study further illustrated that ferroptosis does not follow the canonical inflammasome pathway by showing that caspase-1 remained inactive, and both NLRP3 inhibition (MCC950) and ASC knockdown did not affect ferroptotic sensitivity. Noncanonical pyroptotic PCD executors, specifically caspase-5 was activated during ferroptosis, but not caspase-4. Additionally, the siRNA Knockdown and CRISPR-Cas9 knockout of caspase-5 significantly suppressed ferroptotic death. The cell-free biochemical assay demonstrated that active caspase-5 directly cleaves full-length GSDME, confirming the interaction between caspase-5 activation and pore formation. Oxidized lipidomic profiling identified the accumulation of oxidized phosphatidylethanolamine (PE) species in ferroptotic cells, suggesting a potential upstream trigger for caspase-5/GSDME activation. Our data define a novel ferroptosis execution pathway mediated by caspase-5 activation and GSDME cleavage, revealing a mechanism with potential therapeutic relevance in ovarian cancer.