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

Abstract

Sulfasalazine (SAS), a clinically utilized anti-inflammatory drug, has been shown to induce ferroptosis by inhibiting system Xc ^- activity, thereby causing cellular glutathione depletion. Recently, protein disulfide isomerase (PDI) was shown to be an upstream mediator of the oxidative cell death (oxytosis/ferroptosis) induced by glutamate, erastin, RSL3 and SAS. The present study aims to further characterize the detailed biochemical and cellular mechanisms of SAS-induced ferroptosis in two cell lines, i. e., H9C2 rat cardiomyocytes and BRL-3A rat hepatocytes, with a focus on elucidating the critical role of PDI in mediating SAS-induced toxicity. We find that SAS can induce ferroptosis in H9C2 and BRL-3A cells, which is accompanied by a sequential increase in the buildup of cellular nitric oxide (NO), reactive oxygen species (ROS) and lipid-ROS. SAS activates PDI-mediated dimerization of inducible NO synthase (iNOS) and cellular accumulation of NO, and these effects are followed by ROS and lipid-ROS accumulation. Furthermore, SAS markedly upregulates the iNOS protein levels in these cells. Knockdown of PDI or pharmacological inhibition of PDI catalytic activity effectively suppresses SAS-induced iNOS dimerization, abrogates SAS-induced accumulation of NO, ROS and lipid-ROS, and prevents ferroptosis. On the other hand, PDI activation through the use of TrxR1 inhibitors sensitizes these cells to SAS-induced ferroptosis. These findings provide further experimental support for a pivotal role of PDI in SAS-induced cytotoxicity through the activation of the PDI-NOS-NO axis, which then leads to the accumulation of cellular ROS and lipid-ROS and ultimately the induction of oxidative cell death.