Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025100
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.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2024165
Protein disulfide isomerase (PDI) was recently shown to be an upstream mediator of erastin-induced, glutathione depletion-associated ferroptosis through its catalysis of nitric oxide synthase (NOS) dimerization and nitric oxide (NO) accumulation. A recent study reported that RSL3, a known ferroptosis inducer and glutathione peroxidase 4 (GPX4) inhibitor, can inhibit thioredoxin reductase 1 (TrxR1). The present study seeks to test the hypothesis that RSL3 may, through its inhibition of TrxR1, facilitate PDI activation (i.e., in a catalytically active, oxidized state), thereby enhancing RSL3-induced ferroptosis through NOS dimerization and NO accumulation. Using HT22 mouse neuronal cells as an in vitro model, we show that treatment of these cells with RSL3 strongly increases NOS protein levels and that PDI-mediated NOS dimerization is activated by RSL3, resulting in NO accumulation. Mechanistically, we find that PDI is activated in cells treated with RSL3 because of its inhibition of TrxR1, and the activated PDI then catalyzes NOS dimerization, which is followed by the accumulation of cellular NO, ROS and lipid-ROS and ultimately ferroptotic cell death. Genetic or pharmacological inhibition of PDI or TrxR1 partially abrogates RSL3-induced NOS activation and the subsequent accumulation of cellular NO, ROS/lipid-ROS, and ultimately ferroptosis in HT22 cells. The results of this study clearly show that PDI activation resulted from RSL3 inhibition of TrxR1 activity contributes crucially to RSL3-induced ferroptosis in a cell culture model through the PDI→NOS→NO→ROS/lipid-ROS pathway, in addition to its known inhibition of GPX4 activity.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025100
Sulfasalazine (SAS), a clinically utilized anti-inflammatory drug, induces ferroptosis by inhibiting system Xc− and depleting glutathione. This study characterizes the biochemical and cellular mechanisms of SAS-induced ferroptosis in H9C2 rat cardiomyocytes and BRL-3A rat hepatocytes, focusing on protein disulfide isomerase (PDI). SAS induced ferroptosis with sequential increases in cellular nitric oxide (NO), reactive oxygen species (ROS), and lipid-ROS. SAS activated PDI-mediated dimerization of inducible NO synthase (iNOS) and NO accumulation, followed by ROS and lipid-ROS buildup. SAS also upregulated iNOS protein levels. PDI knockdown or pharmacological inhibition suppressed iNOS dimerization, abrogated NO, ROS, and lipid-ROS accumulation, and prevented ferroptosis. Conversely, PDI activation via TrxR1 inhibitors sensitized cells to SAS-induced ferroptosis. These findings support a pivotal role of the PDI-NOS-NO axis in SAS-induced cytotoxicity, leading to oxidative cell death. The study provides mechanistic insights and suggests strategies for sensitizing cancer cells to SAS-induced ferroptosis.