Key Takeaways & Executive Findings
- •• • 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.
China Biomedical & Cell Therapy Radar
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Abstract
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.
1. Introduction
Sulfasalazine (SAS) is a well-established anti-inflammatory drug for Crohn's disease and ulcerative colitis, but its clinical utility is limited by dose-dependent toxicity, including ferroptosis induction via system Xc− inhibition and glutathione depletion. Existing ferroptosis inducers lack specificity, and SAS's mechanism involves a poorly understood cascade that hampers the development of safer or more effective analogs.
This study addresses the bottleneck by elucidating the PDI-NOS-NO axis in SAS-induced ferroptosis. Using H9C2 cardiomyocytes and BRL-3A hepatocytes, the authors demonstrate that PDI activation mediates iNOS dimerization and NO accumulation, preceding ROS and lipid-ROS buildup. Genetic and pharmacological interventions confirm PDI's pivotal role, and TrxR1 inhibition sensitizes cells to SAS. These findings provide a mechanistic framework for optimizing SAS-based therapies and mitigating off-target toxicity.
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JIA Yi-Chen, ZHONG Jia-Ling, HAO Xiangyu, ZHU Bao Ting (2025). Characterization of the mechanisms underlying sulfasalazine-induced ferroptotic cell death: role of protein disulfide isomerase-mediated NOS activation and NO accumulation. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2025100
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Frequently Asked Questions
What is the temporal sequence of NO, ROS, and lipid-ROS accumulation in SAS-induced ferroptosis, and how does it inform intervention timing?
The study reports a sequential increase: NO accumulation precedes ROS and lipid-ROS buildup. This order suggests that early intervention with NO scavengers or iNOS inhibitors could block the downstream oxidative cascade, potentially preventing ferroptosis more effectively than targeting later events.
How does PDI inhibition compare to genetic knockdown in preventing SAS-induced ferroptosis, and what are the translational implications?
Both PDI knockdown and pharmacological inhibition effectively suppress iNOS dimerization, abrogate NO, ROS, and lipid-ROS accumulation, and prevent ferroptosis. Pharmacological inhibition offers a more feasible therapeutic strategy, but off-target effects and bioavailability must be optimized for clinical use.
What is the role of TrxR1 inhibitors in sensitizing cells to SAS-induced ferroptosis, and what are the potential clinical applications?
TrxR1 inhibitors activate PDI, accelerating iNOS dimerization and ferroptosis. This sensitization could be exploited in cancer therapy to enhance SAS efficacy, particularly in tumors with high TrxR1 expression, but requires careful dosing to avoid systemic toxicity.
Does SAS directly activate PDI, and what are the implications for drug design?
The study rules out direct PDI activation by SAS; instead, PDI oxidation occurs due to glutathione depletion. This indirect mechanism suggests that drugs modulating glutathione levels could influence SAS-induced ferroptosis, offering a target for combination therapies.
What are the limitations of using H9C2 and BRL-3A cell lines to model SAS-induced ferroptosis, and how might they affect clinical translation?
These rat cell lines provide mechanistic insights but may not fully replicate human tissue responses. Species-specific differences in PDI, iNOS, and glutathione metabolism could affect translation. Further validation in human primary cells or animal models is necessary to confirm the PDI-NOS-NO axis as a therapeutic target.
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