Since the discovery of ferroptosis in 2012 [1], a variety of defense mechanisms have been reported to protect against ferroptosis. At the center of ferroptosis defense are the core antioxidants pathways, including glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein 1 (FSP1), GTP cyclohydrolase 1 (GCH1), and dihydroorotate dehydrogenase (DHODH) [2-4]. Furthermore, regulation of lipid and iron metabolism is also heavily involved in modulating ferroptosis [5-9]. However, despite these significant findings, our understanding of ferroptosis remains incomplete. In a recent study by our group, we uncovered another pathway that protects cells from ferroptosis without modulating the core anti-oxidant pathways as well as the lipid peroxidation process. With RU486 as a tool, we found that the NRF2/GSH/GST/RLIP76&MRP1 axis is not dependent on the classical anti-ferroptosis pathway such as GPX4/DHODH/FSP1, and can function independently to prevent ferroptosis. RU486 is the most widely used antiprogestational agent in clinical practice, and given its relative safety profile and unique pharmacologic effects, the use of RU486 should not be limited to the termination of early pregnancies, but also needs to be investigated in different areas of action. In this recent study, RU486 has been demonstrated to exhibit potent ferroptosis-inhibitory effects. However, it was found to be ineffective in rescuing lipid peroxidation induced by the ferroptosis inducer RSL3, unlike widely used ferroptosis inhibitors such as Liproxstatin-1 [10,11]. Information retrieved from the Pharmmapper database suggests that GST is a potential target for RU486. The GST protein family exhibits structural and functional similarities and has been shown to be associated with ferroptosis [12-14]. GST catalyzes the binding of lipid peroxidation products (e.g., 4-HNE) to GSH, forming glutathione conjugates (e.g., GSH-4-HNE). As an efflux transporter, multidrug-resistance proteins (MRPs)/ RalA-binding protein 16 encoded 76-kDa splice variant (RLIP76) facilitates the export of intracellular toxic substances (such as glutathione conjugates) from the cell, thereby reducing cytotoxic levels. Subsequently, these conjugates are transported out of the cell via RLIP76 and MRPs, preventing lipid peroxidation product-induced cellular damage and thus inhibiting the development of ferroptosis [10,15]. Previous reports have established nuclear factor erythroid 2-related factor 2 (NRF2), a master transcriptional regulator of cellular antioxidant responses, as a key antioxidant signaling pathway [2,16-19]. NRF2 coordinates the expression of cytoprotective genes (e.g., GSTs) under oxidative stress by binding to antioxidant response elements (AREs), thereby maintaining redox homeostasis. Multiple GST isoforms, including GSTM1, GSTM2, and GSTM3, are regulated by NRF2 [20]. However, our study revealed an interesting phenomenon: the activity of GST was not significantly affected in the context of NRF2 knockout, and RU486 was still able to upregulate its activity. This finding is different from the expected results, suggesting that the regulatory mechanism of GST may be more complex than expected. Further studies showed that knockout of NRF2 upregulated the expression of GSTM1 in response to stress induction, whereas GSTM2/GSTM3/GSTT2 were significantly downregulated. In addition, the ability of RU486 to upregulate these mRNAs was diminished. Meanwhile, knockout of NRF2 significantly downregulated the expression of MRP1 and RLIP76, which is consistent with previous reports [21,22]. Moreover, RU486 was unable to upregulate their expression levels. Based on the above results, we hypothesized that RU486 mediates the function of GST only partially through NRF2, suggesting that there may be other signaling pathways or machines in the regulatory network of ferroptosis that are parallel to or independent of NRF2 that are also important in influencing the activity of GST and the process of ferroptosis.

There are many members in the GST family, which can be categorized into several subfamilies according to their structure and function, including GSTA, GSTM, GSTP, GSTT, GSTS, GSTK, GSTZ, GSTO, and MGST [23,24]. The regulation of ferroptosis by GST is complex, with different subfamily members playing different roles in the ferroptosis process. This complexity may be related to a variety of factors, including tumor type, cellular microenvironment, and the expression level and activity of GST subfamily members. Specific GST subfamily members exhibit inhibitory effects on ferroptosis initiation: GSTA1 has been found to promote sorafenib resistance in hepatocellular carcinoma and to enhance resistance by inhibiting ferroptosis. Up-regulation of GSTA1 expression is mediated by the transcription factor Catenin Beta 1 (CTNNB1), which forms a GSTA1-CTNNB1 complex that promotes the nuclear translocation of CTNNB1 and establishes a positive feedback loop, thereby inhibiting ferroptosis [25]. Signal transducer and activator of transcription 3 (STAT3) binds to the MGST2 promoter, promotes its transcription and facilitates proliferation, migration and invasion of osteosarcoma cells, whereas naringenin inhibits STAT3, blocks MGST2 expression, and induces ferroptosis in osteosarcoma cells [26]. GSTO2 was found to protect neurons from ferroptosis injury by upregulating GPX4 expression in intracerebral hemorrhage [27]. GSTP1 inhibits ferroptosis by catalyzing the binding of GSH to lipid peroxidation products, such as 4-HNE, and exerting antioxidant effects independent of GPX4 and FSP1. Smad ubiquitin regulatory factor 2 (SMURF2) promotes the ubiquitination and degradation of GSTP1, thereby attenuating its inhibitory effect on ferroptosis [28]. MGST1 has been found to inhibit ferroptosis in pancreatic cancer. MGST1 expression is positively correlated with NRF2 expression, which upregulates MGST1 expression to counteract ferroptosis. MGST1 reduces lipid peroxidation by binding to arachidonic acid 5-lipoxygenase (ALOX5), thereby inhibiting ferroptosis [29]. Inhibition of peroxisome proliferator γ-activated receptor coactivator 1-α (PGC-1α) suppresses GSTK1 protein expression via nuclear respiratory factor 1 (NRF1), which increases ROS accumulation and promotes ferroptosis [30]. GSTM1 and GSTT1 deficiency exacerbate cisplatin-induced renal dysfunction and oxidative stress [31]. Conversely, other members of the GST subfamily act as pro-ferroptosis drivers: Overexpression of GSTZ1 was able to downregulate GPX4 and glutathione levels and increase iron, MDA, and ROS levels, thereby inducing ferroptosis, and in sorafenib-resistant hepatocellular carcinoma cells, downregulation of GSTZ1 was able to augment the activation of the NRF2 pathway and the rise in GPX4 levels [32]. GSTM3 stabilizes ubiquitin-specific peptidase 14 (USP14), effectively blocking ubiquitination and consequently preventing the degradation of fatty acid synthase (FASN). Furthermore, GSTM3 engages in an interaction with GPX4, leading to the inhibition of GPX4 expression [33]. The GST family exhibits a complex double-sided nature in the regulation of ferroptosis, and its different members may play diametrically opposed roles under specific conditions. Given this, functional interventions targeting specific isoforms (e.g., activation of inhibitory isoforms or blockade of promoter isoforms) are expected to provide new strategies for the treatment of ferroptosis-related diseases.

In recent years, the role of GST in a variety of diseases has received increasing attention, demonstrating its great potential as a biomarker. GST activity is strongly associated with the severity of chronic kidney disease [34-37]. GST activity is often abnormally altered in patients with renal disease compared to healthy people, increased GST activity, or even specific isoform deletions, which strongly suggests that GST plays a critical role in the pathogenesis and progression of renal disease [36,38,39]. Particularly in diabetic nephropathy, changes in GST activity may be associated with the early development of the disease, and therefore, GST is expected to be a biomarker for monitoring the progression of this diabetic complication [40]. Furthermore, GST also plays an important role during general anesthesia [41-43]. It is responsible for the detoxification process of anesthetics; therefore, measurements of GST in plasma and urine reflect hepatocyte integrity and renal injury. This property makes GST a useful indicator for assessing toxicity in patients undergoing general anesthesia and helps clinicians make timely adjustments to treatment regimens [44]. In addition, GST gene variants are strongly associated with the onset and progression of a variety of diseases. In particular, polymorphisms in the GSTP1, GSTT1, and GSTM1 genes have been shown to be associated with an increased risk of diabetes, cardiovascular disease vascular complications of diabetes, and oral leukoplakia. Therefore, analysis of the status of these genes may contribute to the early diagnosis of disease and the development of personalized treatment plans [45-48]. Studies have shown that anti-GSTO1-1 antibodies can be detected in several types of inflammatory diseases, such as autoimmune rheumatoid arthritis and infectious SARS-CoV-2 infection. This suggests that GSTO1-1 may be a marker of tissue damage/inflammation [49]. Notably, GST-Pi is abundantly expressed in tumor cells and is an important biomarker for cancer diagnosis. It can participate in the regulation of cellular metabolism, signal transduction, and other processes, and is associated with chemotherapy resistance in cancer. The expression of GST-Pi is regulated by epigenetic factors such as gene polymorphisms, methylation, etc., and its expression level and activity are closely related to the pathogenesis of many cancers. Therefore, GST-Pi can not only be used as a biomarker for cancer but may also be a target for cancer therapy [50-53]. GST is also strongly associated with APAP-induced liver injury. After APAP treatment, the level of GSTA1 in cell culture supernatant responded rapidly and increased significantly at 6 hours, which was earlier than the changes of traditional liver injury markers, such as ALT and AST (these markers showed differences only 8 hours after APAP exposure). The increase of GSTA1 is also dose-dependent, that is, it has increased significantly under the treatment of low concentration of APAP, while the significant changes of ALT, AST, and other indexes require a higher concentration of APAP, which makes it possible to be an early sensitive marker of APAP liver injury [54]. On the other hand, RU486 treatment significantly upregulated the expression of several proteins, including MRP1/RLIP76, GSTM1, and GSTM2, while APAP downregulated the levels of some of the same proteins. Notably, GSTM2 expression was significantly up-regulated in the combined treatment group, which may be an important target for RU486 treatment. Meanwhile, APAP treatment resulted in the upregulation of GSTM1 expression, which may be related to its detoxification role as an antioxidant enzyme in the oxidative stress response. In conclusion, GSTA1, GSTM1, and GSTM2 all exhibited properties as potential biomarkers in APAP-induced liver injury.It is important to note that GST status is usually assessed by measuring its level in the blood [55-59], which does not exist in isolation, but is influenced by a complex of multiple factors such as genetic background, environmental exposures, and the status of a particular disease. Therefore, when using GST as a biomarker, other relevant factors must be taken into account for a comprehensive assessment and diagnosis.