Characterization of the mechanisms underlying sulfasalazine-induced ferroptotic cell death: role of protein disulfide isomerase-mediated NOS activation and NO accumulation

• • SAS induces ferroptosis in H9C2 and BRL-3A cells with a time-dependent sequential increase in NO, ROS, and lipid-ROS; this cascade identifies NO as an early mediator, offering a therapeutic window for intervention with NO scavengers or iNOS inhibitors. • • PDI knockdown or pharmacological inhibition abrogates SAS-induced iNOS dimerization and prevents accumulation of NO, ROS, and lipid-ROS, conferring strong protection against ferroptotic cell death; this validates PDI as a druggable target for mitigating SAS toxicity in normal tissues. • • PDI activation by TrxR1 inhibitors sensitizes cells to SAS-induced ferroptosis, suggesting a combination strategy to enhance SAS efficacy in cancer therapy by promoting oxidative cell death. • • SAS upregulates iNOS protein levels, contributing to elevated NO production; this identifies iNOS as a potential biomarker for SAS responsiveness and a target for modulating ferroptosis in inflammatory and malignant conditions.