TrxR1 inhibitors(Shenyang Pharmaceutical University)

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.

Disulfidptosis is a recently identified form of cell death characterized by the aberrant accumulation of cellular disulfides. This process primarily occurs in glucose-starved cells expressing higher levels of SLC7A11 and has been proposed as a therapeutic strategy for cancers with hyperactive SCL7A11. However, the potential for inducing disulfidptosis through other mechanisms in cancers remains unclear. Here, we found that inhibiting thioredoxin reductase 1 (TrxR1), a key enzyme in the thioredoxin system, induces disulfidptosis in glioblastoma (GBM) cells. TrxR1 expression is elevated in GBM with activated transcriptional coactivator with PDZ-binding motif (TAZ) and correlates with poor prognosis. TrxR1 inhibitors induced GBM cell death that can be rescued by disulfide reducers but not by ROS scavengers or inhibitors of apoptosis, ferroptosis, or necroptosis. Glucose-starved cells, but not those deprived of oxygen or glutamine, increased TrxR1 expression in an NRF2-dependent manner and were more sensitive to TrxR1 inhibition-induced cell death. The dying cells initially exhibited highly dynamic lamellipodia, followed by actin cytoskeleton collapse. This process involved the accumulation of cytosolic peroxisomes and micropinocytic caveolae, as well as small gaps in the plasma membrane. Depletion of the WAVE complex component NCKAP1 partially rescued the cells, whereas Rac inhibition enhanced cell death. In an orthotopic xenograft GBM mouse model, TrxR1 depletion inhibited tumor growth and improved survival. Furthermore, cells undergoing TrxR1 inhibition exhibited features of immunogenic cell death. Therefore, this study suggests the potential of targeting TrxR1 as a therapeutic strategy in GBM.