A fin-loop-like structure in GPX4 underlies neuroprotection from ferroptosis.

TRPV1 as an anti-ferroptotic target in osteoarthritis.

GPX4 deficiency-dependent phospholipid peroxidation drives motor deficits of ALS

The mechanisms of ferroptosis and its role in atherosclerosis

Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma

Neuroprotective role of glutathione peroxidase 4 in experimental subarachnoid hemorrhage models

Diverse age-related effects of Bacopa monnieri and donepezil in vitro on cytokine production, antioxidant enzyme activities, and intracellular targets in splenocytes of F344 male rats

Regulators of system xc--glutathione-glutathione peroxidase 4 antioxidant pathway: Recent advances and challenges in targeting ferroptosis for cancer treatment.

Oxidative stress plays a critical role in postmenopausal osteoporosis, yet its impact on osteoblasts remains underexplored, limiting therapeutic advances. Our study identifies phospholipid peroxidation in osteoblasts as a key feature of postmenopausal osteoporosis. Estrogen regulates the transcription of glutathione peroxidase 4 (GPX4), an enzyme crucial for reducing phospholipid peroxides in osteoblasts. The deficiency of estrogen reduces GPX4 expression and increases phospholipid peroxidation in osteoblasts. Inhibition or knockout of GPX4 impairs osteoblastogenesis, while the elimination of phospholipid peroxides rescues bone formation and mitigates osteoporosis. Mechanistically, 4-hydroxynonenal, an end-product of phospholipid peroxidation, binds to integrin-linked kinase and triggers its protein degradation, disrupting RUNX2 signaling and inhibiting osteoblastogenesis. Importantly, we identified two natural allosteric activators of GPX4, 6- and 8-Gingerols, which promote osteoblastogenesis and demonstrate anti-osteoporotic effects. Our findings highlight the detrimental role of phospholipid peroxidation in osteoblastogenesis and underscore GPX4 as a promising therapeutic target for osteoporosis treatment.

The lack of a T-cell inflamed microenvironment in tumors limits responsiveness to many immunotherapies. T-cell exclusion is often mediated by a dense infiltration of fibroblast-like stromal cells. Up to 55% of triple-negative breast cancers have ‘stroma-rich’ tumors with markedly lower T-cell inflammation. Here we report a therapeutic strategy that can potentially convert stroma-rich tumors into T-cell inflamed tumors by forcing stromal cells to secrete 5-lipoxygenase products which are powerful chemo-attractants for T cells. We were initially interested in identifying selective inhibitors of stromal-cell function. To achieve this, we used phenotype-based small-molecule screening in which the phenotype of stroma-induced cancer cell migration in vitro was a surrogate for stroma-induced metastasis in vivo. We identified a compound, RSL3 that inhibited this migration. RSL3 was selectively cytotoxic to stromal cells over cancer cells, in comparisons of immortalized cell lines as well as comparisons of patient-derived primary breast cancer cells to cancer-associated fibroblasts (CAFs). We therefore undertook studies to identify its mechanism of action. RSL3 was recently reported to target the redox enzyme glutathione peroxidase 4 (GPX4). GPX4 metabolizes lipid peroxides so we performed metabolomic profiling of RSL3-treated stroma-cancer co-cultures and found elevated arachidonic acid products of lipoxygenase enzymes. Stromal cells were found to contain >10-fold higher levels of lipoxygenase products than carcinoma cells. Blocking either 5-lipoxygenase (5-LO) or 15-lipoxygenase (15-LO) with selective inhibitors abrogated RSL3’s cytotoxicity to stromal cells. Thus, high lipoxygenase activity in stromal cells increases their susceptibility to GPX4 inhibition. Because 5-LO products like leukotriene B4 are powerful chemo-attractants for myeloid cells and T cells, we studied the impact of GPX4 knockdown in vivo using xenografts of cancer cells co-injected with stromal cells. GPX4 knockdown resulted in a large increase in myeloid-cell infiltration into tumors but, surprisingly, T-cell infiltration was suppressed. We reasoned that 15-LO products are immunosuppressive based on recent findings that 15-LO gene amplifications are inversely correlated with T-cell infiltration in breast cancers in The Cancer Genome Atlas. Consistent with this, GPX4 inhibition of stroma-cancer co-cultures suppressed T-cell chemotaxis but combined inhibition of GPX4 and 15-LO significantly enhanced T-cell chemotaxis over untreated controls in vitro. We are undertaking in vivo testing of this combination. In summary, our unbiased chemical biology approach has revealed a therapeutic strategy to promote T-cell inflammation. We envision this to be a priming strategy for stroma-rich cancers to become responsive to a variety of different immunotherapies thereby unleashing their full curative potential. Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Ninib Baryawno, Nicolas Severe, Vasanthi Viswanathan, Carlotta Costa, David Scadden, Stuart Schreiber. Targeting GPX4 in tumor-associated stromal cells increases inflammatory-cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2017-3026

Ferroptosis is a nonapoptotic type of cell death that is dependent on iron and involves the accumulation of reactive oxygen species. Ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) are ferroptosis regulators that inhibit ferroptosis through independent pathways. This study assessed the prognostic value of GPX4 and FSP1 expression in colorectal cancer (CRC). We also examined the effects of FSP1 and GPX4 inhibition on cell survival of CRC cells. This study included 206 surgical specimens from Stage II or III CRC patients. FSP1 and GPX4 expression was analyzed immunohistochemically, and the association of their expression levels with clinical outcome was evaluated. We also examined the effects of FSP1 and GPX4 inhibitors on the cell proliferative capacity of CRC cell lines. Overall survival and recurrence-free survival were reduced in patients with high expression of FSP1 or GPX4, and those with both GPX4 and FSP1 expression showed worse prognosis. Positivity of both FSP1 and GPX4 was an independent poor prognostic factor for CRC patients. In CRC cells, the combination of GPX4 and FSP1 inhibitors led to more effective cell death than either inhibitor alone. High expression of both GPX4 and FSP1 is a significant poor prognostic factor for CRC. Simultaneous inhibition of GPX4 and FSP1 to induce ferroptosis may be a novel therapeutic strategy in CRC.

BackgroundFerroptosis (iron‐mediated phospholipid peroxidation) has been implicated in neuronal loss associated with Alzheimer's disease (AD). Glutathione Peroxidase‐4 (GPX4) mitigates ferroptosis in healthy neurons, but the activity of this pathway declines with age. Menaquinone‐4 (MK4), a natural form of vitamin K, can be enzymatically reduced by FSP1, VKORC1 and VKORC1L1 to generate the potent ferroptosis suppressor MK4‐H. We hypothesize that vitamin K suppression of ferroptosis becomes critical to neuronal survival as GPX4 and its substrate glutathione decline in aging brain. In these studies we examined whether MK4 acts as a ferroptosis suppressor in murine‐derived HT22 neuronal cells grown as adherent monolayers or as neurospheres.MethodHT22 cells were acutely exposed to MK4 (≤1µM) in the presence/absence of ferroptosis inducers RSL3 and FIN56 (GPX4 inhibitors), sulfasalazine (xCT inhibitor) and glutamate (NMDAR agonist). Cell density, imaging, flow cytometry and Western blotting were used to assess survival, morphology, ferroptosis‐specific phospholipid peroxidation (Liperfluo) and subcellular expression of FSP1, VKORC1 and VKORC1L1. Sensitivity to RSL3 was also assessed in HT22 cells after differentiation in neurobasal media supplemented with N2, Gln, EGF, bFGF and B27 lacking antioxidants, and when cultured as neurospheres under low attachment conditions.ResultFerroptosis was induced in HT22 cells at concentrations of RSL3 as low as 0.01µM (65% reduction in cell density within 20hrs). MK4 (0.05µM) abrogated RSL3 mediated cell loss and phospholipid peroxidation as measured by Liperfluo. Similar protective effects of MK4 were observed in neurospheres and in differentiated HT22 cells. MK4 also protected HT22 cells from sulfasalazine, glutamate and FIN56 which induce ferroptosis via mechanisms distinct from RSL3. GPX4 was detected in both cytosolic (CYT) and non‐nuclear membrane (NNM) fractions of HT22 cells, indicating that it mediates cellular‐wide protection against ferroptosis. FSP1 was strongly expressed in the CYTO fraction whereas VKORC1 and VKORC1L1 were only detected in the NNM fraction.ConclusionMK4 (vitamin K2) promotes survival of both proliferating and differentiated HT22 cells via suppression of ferroptosis induced by multiple pathways. GPX4 and the MK4 metabolizing enzymes (FSP1, VKORC1, VKORC1L1) are expressed in HT22 cells and likely cooperate to suppress ferroptosis in distinct cellular compartments.

Introduction: Reactive oxygen species (ROS) play a pivotal role in TGF-b signaling and Idiopathic pulmonary fibrosis (IPF) pathogenesis. Glutathione peroxidase 4 (GPx4) can directly reduce phospholipid peroxidation, a representative oxidative modification by ROS. We attempted to elucidate the involvement of GPx4-regulated lipid peroxidation in modulating TGF-b signaling in terms of IPF pathogenesis. Methods: Using human lung tissues and lung fibroblasts (LFB) isolated from normal lungs and IPF lungs, immunohistochamical staining and western blotting (WB) were performed to evaluate GPx4 expression levels. SiRNA-mediated knockdown experiments were conducted to elucidate the role of GPx4 in TGF-b-induced myofibroblast differentiation. To clarify the role of GPx4 in physiological conditions, we used a bleomycin (BLM)-induced lung fibrosis model in GPx4+/-, GPx4+/+, GPx4 transgenic mice. Results: Immunohistochamical staining demonstrated decreased GPx4 and increased 4 hydroxynonenal (4HNE) expression levels, a major end product of lipid peroxidation, in IPF lungs. Reduced GPx4 expression levels were also detected in LFB isolated from IPF lungs. GPx4 siRNA increased TGF-b-induced lipid peroxidation and myofibroblast differentiation with concomitantly enhanced SMAD2/SMAD3 phosphorylation, which were efficiently inhibited by N-acetylcystein treatment. GPx4+/- mice showed marked enhancement of BLM-induced lung fibrosis development but significant attenuation was demonstrated in GPx4 transgenic mice. Conclusion: These findings suggest that GPx4 expression levels were reduced in IPF and GPx4-regulated lipid peroxidation can be involved in IPF pathogenesis via modulating TGF-b-induced myofibroblast differentiation.

Gold salt phospholipid inhibits glutathione peroxidase 4 (GPx4) and causes cell death in Mantle Cell Lymphoma: Demonstration of single agent activity and synergy with ibrutinib

Methylmercury (MeHg), an environmental neurotoxicant, induces site-specific toxicity in the brain. Although oxidative stress has been demonstrated with MeHg toxicity, the site-specific toxicity is not completely understood. Among the cerebellar neurons, cerebellar granule cells (CGCs) appear vulnerable to MeHg, whereas Purkinje cells and molecular layer neurons are resistant. Here, we use a MeHg-intoxicated rat model to investigate these cerebellar neurons for the different causes of susceptibility to MeHg. Rats were exposed to 20ppm MeHg for 4weeks and subsequently exhibited neuropathological changes in the cerebellum that were similar to those observed in humans. We first isolated the three cerebellar neuron types using a microdissection system and then performed real-time PCR analyses for antioxidative enzymes. We observed that expression of manganese-superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1), and thioredoxin reductase 1 (TRxR1) was significantly higher in Purkinje cells and molecular layer neurons than in CGCs. Finally, we performed immunohistochemical analyses on the cerebellum. Immunohistochemistry showed increased expression of Mn-SOD, GPx1, and TRxR1 in Purkinje cells and molecular layer neurons, which was coincident with the mRNA expression patterns. Considering Mn-SOD, GPx1, and TRxR1 are critical for protecting cells against MeHg intoxication, the results indicate that low expression of these antioxidative enzymes increases CGCs vulnerability to MeHg toxicity.

This review provides a comprehensive overview of hereditary spastic paraplegias (HSPs) and summarizes the recent progress on the role of glial cells in the pathogenesis of HSPs. HSPs are a heterogeneous group of neurogenetic diseases characterized by axonal degeneration of cortical motor neurons, leading to muscle weakness and atrophy. Though the contribution of glial cells, especially astrocytes, to the progression of other motor neuron diseases like amyotrophic lateral sclerosis (ALS) is well documented, the role of glial cells and the interaction between neurons and astrocytes in HSP remained unknown until recently. Using human pluripotent stem cell-based models of HSPs, a study reported impaired lipid metabolisms and reduced size of lipid droplets in HSP astrocytes. Moreover, targeting lipid dysfunction in astrocytes rescues axonal degeneration of HSP cortical neurons, demonstrating a non-cell-autonomous mechanism in axonal deficits of HSP neurons. In addition to astrocytes, recent studies revealed dysfunctions in HSP patient pluripotent stem cell-derived microglial cells. Increased microgliosis and pro-inflammation factors were also observed in HSP patients' samples, pointing to an exciting role of innate immunity and microglia in HSP. Building upon these recent studies, further investigation of the detailed molecular mechanism and the interplay between glial cell dysfunction and neuronal degeneration in HSP by combining human stem cell models, animal models, and patient samples will open avenues for identifying new therapeutic targets and strategies for HSP.