Role of protein disulfide isomerase in mediating sulfasalazine-induced ferroptosis in HT22 cells: The PDI−NOS–NO−ROS/lipid-ROS cascade

Ferroptosis is a form of regulated cell death resulting from excessive lipid peroxidation. Sulfasalazine (SAS), an anti-inflammatory drug, can induce ferroptosis through inhibiting the system Xc⁻ and triggering glutathione depletion. SAS has attracted considerable interest in recent years because of its potential for repurposing as an anticancer agent. Our recent studies have shown that protein disulfide isomerase (PDI) is an upstream mediator of chemically-induced ferroptosis through catalyzing the dimerization of nitric oxide synthase (NOS) and NO accumulation in cultured HT22 hippocampal neuronal cells. The present study aims to investigate SAS-induced ferroptotic cell death in HT22 cells with a focus on determining the role of PDI in mediating SAS-induced ferroptosis. We find that SAS induces ferroptotic cell death in HT22 cells, which is accompanied by a time-dependent sequential increase in the accumulation of cellular NO, ROS and lipid-ROS. We find that treatment of HT22 cells with SAS activates PDI-mediated iNOS activation (dimerization) and NO accumulation. In addition, SAS also strongly upregulates iNOS protein levels in HT22 cells. PDI knockdown or pharmacological inhibition of PDI's activity each suppresses SAS-induced iNOS dimerization, which is associated with abrogation of SAS-induced accumulation of NO, ROS and lipid-ROS, and a strong protection against ferroptotic cell death. On the other hand, PDI activation through the use of a TrxR1 inhibitor can strongly sensitize cells to SAS-induced ferroptosis. Together, these experimental observations demonstrate a crucial role of PDI in SAS-induced ferroptosis in a cell culture model through the activation of the PDI → NOS → NO → ROS/lipid-ROS pathway. Insights gained from this study also provide effective strategies to selectively sensitizing human cancer cells to SAS-induced ferroptosis, such as through the use of NO-releasing agents or TrxR1 inhibitors.