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, it was shown that protein disulfide isomerase (PDI) is an upstream mediator of 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, focusing 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 the inducible NO synthase (iNOS) and cellular NO accumulation, and these effects are followed by ROS and lipid-ROS accumulation. Furthermore, SAS markedly upregulates the expression of iNOS protein in these cells. Knockdown of PDI or pharmacological inhibition of its catalytic activity each effectively suppresses SAS-induced iNOS dimerization, along with abrogation of SAS-induced accumulation of NO, ROS and lipid-ROS, and prevention of ferroptosis. On the other hand, PDI activation through the use of TrxR1 inhibitors sensitizes these cells to SAS-induced ferroptosis. These experimental 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 cellular ROS and lipid-ROS accumulation, and ultimately induction of oxidative cell death.