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.

1. Introduction

Sulfasalazine (SAS), a well-established drug for treating inflammatory disorders such as Crohn’s disease and ulcerative colitis [1–3], has recently been identified as a potent inducer of ferroptosis [4]. SAS inhibits the cystine/glutamate antiporter system Xc− [5,6], which is essential for cystine uptake and intracellular glutathione (GSH) synthesis [7]. By depleting GSH, SAS disrupts the cellular redox balance and triggers oxidative stress, culminating in ferroptotic cell death. Studies have shown that SAS can induce ferroptosis in several cell lines [8–15], and SAS can increase the efficacy of chemotherapeutic agents by increasing oxidative damage [5].

Ferroptosis is a form of regulated cell death often associated with the accumulation of reactive oxygen species (ROS) [16]. Unlike apoptosis or necrosis, ferroptosis has notable morphological and biochemical features, such as reduced mitochondrial size, increased membrane density, and GSH deficiency-associated oxidative damage to cellular lipids [17]. Central to this process is the depletion of cellular GSH and inactivation of glutathione peroxidase 4 (GPX4), and these changes disrupt a cell’s ability to neutralize reactive lipid peroxides (lipid-ROS), subsequently leading to cell death [18].

Protein disulfide isomerase (PDI or PDIA1) is a well-known member of the thioredoxin superfamily and is primarily localized in the endoplasmic reticulum [19]. Functionally, PDI serves as a dithiol/disulfide oxidoreductase and facilitates protein folding by catalyzing the isomerization of the intra- and intermolecular disulfide bonds [20]. Recent studies from our laboratory have shown that PDI is involved in mediating chemically-induced, glutathione (GSH) depletion-associated ferroptosis [21–23]. Specifically, GSH depletion leads to PDI oxidation and activates its catalytic activity for nitric oxide synthase (NOS) dimerization via disulfide bond formation, which subsequently results in the buildup of cellular nitric oxide (NO) and ROS/lipid-ROS and mitochondrial ROS and ultimately ferroptotic cell death [21–23]. Notably, the inhibition of PDI function has been shown to block NOS dimerization and NO accumulation and thus effectively rescues cells from chemically induced ferroptosis [21–23]. These findings position PDI as a pivotal upstream mediator of ferroptosis, linking GSH depletion to oxidative cell death pathways.