Glutathione Peroxidase 4 Knockdown Research Articles

Glutathione peroxidase 4 restrains temporomandibular joint osteoarthritis progression by inhibiting ferroptosis.

There are few effective therapeutic strategies for temporomandibular joint osteoarthritis (TMJOA) due to the unclear pathology and mechanisms. We aimed to confirm the roles of GPX4 and ferroptosis in TMJOA progression. ELISA assay was hired to evaluate concentrations of ferroptosis-related markers. The qRT-PCR assay was hired to assess gene mRNA level. Western blot assay and immunohistochemistry were hired to verify the protein level. CCK-8 assay was hired to detect cell viability. Human fibroblast-like synoviocytes (FLSs) were cultured to confirm the effects of GPX4 and indicated inhibitors, and further verified the effects of GPX4 and ferroptosis inhibitors in TMJOA model rats. Markers of ferroptosis including 8-hidroxy-2-deoxyguanosine (8-OHdG) and iron were notably increased in TMJOA tissues and primary OA-FLSs. However, the activity of the antioxidant system including the glutathione peroxidase activity, glutathione (GSH) contents, and glutathione/oxidized glutathione (GSH/GSSG) ratio was notably inhibited in TMJOA tissues, and the primary OA-FLSs. Furthermore, the glutathione peroxidase 4 (GPX4) expression was down-regulated in TMJOA tissues and primary OA-FLSs. Animal and cell experiments have shown that ferroptosis inhibitors notably inhibited ferroptosis and promoted HLS survival as well as up-regulated GPX4 expression. Also, GPX4 knockdown promoted ferroptosis and GPX4 overexpression inhibited ferroptosis. GPX4 also positively regulated cell survival which was the opposite with ferroptosis. In conclusion, GPX4 and ferroptosis regulated the progression of TMJOA. Targeting ferroptosis might be an effective therapeutic strategy for TMJOA patients in the clinic.

Jolkinolide B synergistically potentiates the antitumor activity of GPX4 inhibitors via inhibiting TrxR1 in cisplatin-resistant bladder cancer cells

Glutathione peroxidase 4 (GPX4) is a promising anticancer therapeutic target; however, the application of GPX4 inhibitors (GPX4i) is limited owing to intrinsic or acquired drug resistance. Hence, understanding the mechanisms underlying drug resistance and discovering molecules that can overcome drug resistance are crucial. Herein, we demonstrated that GPX4i killed bladder cancer cells by inducing lipid reactive oxygen species-mediated ferroptosis and apoptosis, and cisplatin-resistant bladder cancer cells were also resistant to GPX4i, representing a higher half-maximal inhibitory concentration value than that of parent bladder cancer cells. In addition, thioredoxin reductase 1 (TrxR1) overexpression was responsible for GPX4i resistance in cisplatin-resistant bladder cancer cells, and inhibiting TrxR1 restored the sensitivity of these cells to GPX4i. In vitro and in vivo studies revealed that Jolkinolide B (JB), a natural diterpenoid and previously identified as a TrxR1 inhibitor, potentiated the antiproliferative efficacy of GPX4i (RSL3 and ML162) against cisplatin-resistant bladder cancer cells. Furthermore, GPX4 knockdown and inhibition could augment JB-induced paraptosis and apoptosis. Our results suggest that inhibiting TrxR1 can effectively improve GPX4 inhibition-based anticancer therapy. A combination of JB and GPX4i, which is well-tolerated and has several anticancer mechanisms, may serve as a promising therapy for treating bladder cancer.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Effect of Ferroptosis Inducers and Inhibitors on Cell Proliferation in Acute Leukemia.

Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation. Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated. Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines. Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL.

Circ_UTRN inhibits ferroptosis of ARJ21 cells to attenuate acute pancreatitis progression by regulating the miR-760-3p/FOXO1/GPX4 axis.

To explore the function of circ_UTRN in acute pancreatitis (AP). After exposing AR42J cells to caerulein, the levels of circ_UTRN, miR-760-3p, and glutathione peroxidase 4 (GPX4) were determined by quantitative polymerase chain reaction. Additionally, GPX4 and forkhead box O1 (FOXO1) protein levels were assessed by western blot. The levels of oxidative stress and ferroptosis in the supernatant of the treated AR42J cells were also assessed using commercial kits. circ_UTRN inhibited caerulein-induced oxidative stress and ferroptosis by binding with miR-760-3p. Additionally, miR-760-3p directly targeted FOXO1, thereby regulating GPX4 levels. Furthermore, GPX4 knockdown abolished the effect of miR-760-3p downregulation in AP. circ_UTRN inhibited oxidative stress and ferroptosis by regulating the miR-760-3p/FOXO1/GPX4 axis. This is a potential new treatment strategy for AP.

Nanoparticle Targeting in Chemo-Resistant Ovarian Cancer Reveals Dual Axis of Therapeutic Vulnerability Involving Cholesterol Uptake and Cell Redox Balance.

Platinum (Pt)-based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt-R). This work shows that Pt-R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B-1 (SR-B1). SR-B1 blockade using synthetic cholesterol-poor HDL-like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR-B1 and re-sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2downregulation leads to decreased SR-B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR-B1 expression through SREBF2. Targeting SR-B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt-R OC.

Glutathione peroxidase 4 knock-down triggers ferroptosis in Penaeus vannamei hepatopancreas during hypoxia and reoxygenation

The white shrimp Penaeus vannamei during its life cycle is commonly exposed to environmental stressors including hypoxia and reoxygenation that can affect their growth and survival. Hypoxia inducible Factor-1 (HIF-1) is a very important transcription factor involved in the responses to hypoxia and participates in other processes, including ferroptosis. Glutathione peroxidase 4 (GPx4) is a key ferroptosis component. In mammals, GPx4 has important biological functions beyond its antioxidant role. We studied the changes of HIF-1α and the ferroptosis process components, heme oxygenase −1 (HO-1), acyl-CoA synthetase long chain family member 4 (ACSL4), and catalase (CAT) in GPx4 knock-down shrimp exposed to hypoxia and reoxygenation. Malondialdehyde (MDA) content and CAT activity were also evaluated. Changes in HIF-1α, CAT, ACSL4, and HO-1 expression occurred 6 and 12 h after hypoxia and reoxygenation in shrimp hepatopancreas. HIF-1α and CAT expression were reduced during hypoxia and reestablished in reoxygenation at 6 and 12 h, while HO-1 did not change at 6 h, but increased during hypoxia at 12 h. Also, ACSL4 expression decreased during hypoxia and reoxygenation at 12 h, while MDA content was not affected by hypoxia and reoxygenation. GPx4 knock-down increased ACSL4 expression, MDA content, and CAT activity indicating that the ferroptosis process is induced by the intrinsic pathway in hepatopancreas during GPx4 knock-down.

Methyltransferase-like 3 silenced inhibited the ferroptosis development via regulating the glutathione peroxidase 4 levels in the intracerebral hemorrhage progression

ABSTRACT This study examined the effects of methyltransferase-like 3 (METTL3) on ferroptosis during intracerebral hemorrhage (ICH) progression. The brain microvascular endothelial cells (BMVECs) were stimulated with oxygen and glucose deprivation (OGD) and hemin to establish an ICH model. Cell viability was tested using a CCK8 assay. The levels of Fe2+, glutathione, reactive oxygen species, LPO, and MDA were determined using the corresponding commercial kits. Cell death was analyzed using TUNEL and propidium iodide staining. The correlation between METTL3 and glutathione peroxidase 4 (GPX4) was analyzed using Spearman’s correlation test and further confirmed using the CHIP assay. Western blotting and RT-qPCR were performed to measure the relative expression levels. Mice were injected with 0.2 units collagenase IV to establish an ICH model in vivo. We found that the Fe2+, reactive oxygen species, LPO, and MDA levels were enhanced, and glutathione was depleted in OGD/H-treated BMVECs as well as in ICH mice. Additionally, cell viability and SLC7A11 protein levels decreased, and cell death and TFR1 protein levels increased in OGD/H-treated BMVECs. METTL3 silencing relieves OGD/H-induced injury in BMVECs. In addition, METTL3 was significantly negatively related to GPX4, which was further confirmed by the CHIP assay. Silencing of METTL3 decreased the N6-methyladenosine levels of GPX4 and increased its mRNA levels of GPX4. GPX4 knockdown neutralized the role of METTL3 in OGD/H-treated BMVECs. These results implied that ferroptosis occurred in the ODG/H-treated BMVECs and ICH mouse models. METTL3 silencing effectively suppressed ferroptosis by regulating N6-methyladenosine and mRNA levels of GPX4.

RXRγ attenuates cerebral ischemia-reperfusion induced ferroptosis in neurons in mice through transcriptionally promoting the expression of GPX4.

Cerebral ischemia is a common cerebrovascular disease with high mortality and disability rate. Exploring its mechanism is essential for developing effective treatment for cerebral ischemia. Therefore, this study aims to explore the regulatory effect and mechanism of retinoid X receptor γ (RXRγ) on cerebral ischemia-reperfusion (I/R) injury. A mouse intraluminal middle cerebral artery occlusion model was established, and PC12 cells were exposed to anaerobic/reoxygenation (A/R) as an in vitro model in this study. Cerebral I/R surgery or A/R treatment induced ferroptosis, downregulated RXRγ and GPX4 (glutathione peroxidase 4) levels, upregulated cyclooxygenase-2 (COX-2) level and increased ROS (reactive oxygen species) level in A/R induced cells or I/R brain tissues in vivo or PC12 cells in vitro. Knockdown of RXRγ downregulated GPX4 and increased COX-2 and ROS levels in A/R induced cells. RXRγ overexpression has the opposite effect. GPX4 knockdown reversed the improvement of RXRγ overexpression on COX-2 downregulation, GPX4 upregulation and ferroptosis in PC12 cells. Furthermore, chromatin immunoprecipitation (ChIP) and luciferase reporter gene assays revealed that RXRγ bound to GPX4 promoter region and activated its transcription. Overexpression of RXRγ or GPX4 alleviated brain damage and inhibited ferroptosis in I/R mice. In conclusion, RXRγ-mediated transcriptional activation of GPX4 might inhibit ferroptosis during I/R-induced brain injury.

RSL3 Drives Ferroptosis through NF-κB Pathway Activation and GPX4 Depletion in Glioblastoma

Glioblastoma, the most aggressive form of malignant glioma, is very difficult to treat because of its aggressively invasive nature and high recurrence rates. RAS-selective lethal 3 (RSL3), a well-known inhibitor of glutathione peroxidase 4 (GPX4), could effectively induce oxidative cell death in glioblastoma cells through ferroptosis, and several signaling pathways are involved in this process. However, the role of the nuclear factor kappa-B (NF-κB) pathway in glioblastoma cell ferroptosis has not yet been investigated. Therefore, we aimed to clarify the underlying mechanism of the NF-κB pathway in RSL3-induced ferroptosis in glioblastoma cells. We found that RSL3 led to an increase in lipid ROS concentration and downregulation of ferroptosis-related proteins such as GPX4, ATF4, and SLC7A11 (xCT) in glioblastoma cells. Additionally, the NF-κB pathway was activated by RSL3, and its inhibition by BAY 11-7082 could alleviate ferroptosis. The murine xenograft tumor model indicated that NF-κB pathway inhibition could mitigate the antitumor effects of RSL3 in vivo. Furthermore, we found that GPX4 knockdown could not effectively induce ferroptosis. However, NF-κB pathway activation coupled with GPX4 silencing induced ferroptosis. Additionally, ATF4 and xCT expression might be regulated by the NF-κB pathway. Collectively, our results revealed that the NF-κB pathway plays a novel role in RSL3-induced ferroptosis in glioblastoma cells and provides a new therapeutic strategy for glioblastoma treatment.

Dauricine alleviated secondary brain injury after intracerebral hemorrhage by upregulating GPX4 expression and inhibiting ferroptosis of nerve cells

Intracerebral hemorrhage (ICH) is a severe stroke subtype with high disability and mortality, and no effective treatment is available. Previous research on intracerebral hemorrhage secondary brain injury drugs mainly targeted at cell apoptosis, inflammation and oxidative stress, but did not achieve good effects. In recent years, ferroptosis has become a focus concern in neurological diseases. Ferroptosis is a new type of programmed cell death caused by iron-dependent accumulation of lipid peroxides, in which glutathione peroxidase 4 (GPX4) is a key protein affecting ferroptosis. In this study, we used the STRING protein database to predict the proteins that may be co-expressed with GPX4, and studied the ability of Dauricine(Dau) to up-regulate the expression of GPX4 against ferroptosis and neuroprotection after intracerebral hemorrhage in normal cells in vitro, glutathione peroxidase 4 (GPX4) knockdown cells and collagenase injection in vivo in mouse models of intracerebral hemorrhage. The results showed that glutathione reductase (GSR) was a possible co-expression protein with GPX4. Dau could up-regulate the expression of glutathione peroxidase 4 (GPX4) in intracerebral hemorrhage(ICH) model, normal cells and GPX4 knockdown cells in vitro, and simultaneously up-regulate the expression of GSR in ICH mice. Dau could also reduce the levels of iron and lipid peroxidation, and have a neuroprotective effect on intracerebral hemorrhage(ICH) mice. It was tesified that Dauricine(Dau) could inhibit ferroptosis of nerve cells and alleviate brain injury after intracerebral hemorrhage by upregulating glutathione peroxidase 4 (GPX4) and glutathione reductase (GSR) co-expression. Therefore, Dau may be an effective drug for inhibiting ferroptosis and treating intracerebral hemorrhage.

Down-regulation of Glutathione Peroxidase 4 in Oral Cancer Inhibits Tumor Growth Through SREBP1 Signaling.

This study aimed to elucidate the role of glutathione peroxidase 4 (GPX4) on the sterol regulatory element binding proteins (SREBPs)-proliferation pathway in oral cancer cells, and determine its protein expression in oral cancer tissues. Quantitative RT-PCR and immunoblot analysis were carried out. Cell viability assay, apoptosis detection assay, immunohistochemistry and GPX4 knockdown were performed. The levels of both GPX4 mRNA and protein were highest in SAS cells. GPX4 knockdown in SAS cells, a human oral squamous cell carcinoma cell line, using GPX4 siRNA resulted in a reduction in cell number, which appeared to be due to non-apoptotic cell death such as ferroptosis. Furthermore, SREBP was clearly down-regulated by GPX4 knockdown in SAS cells. Immunopositivity for GPX4 was revealed on the membrane of human oral squamous cell carcinoma cells, and this was correlated with p53 immunoreactivity. GPX4 appears to play an important role in oral cancer proliferation.

A new role of glutathione peroxidase 4 during human erythroblast enucleation

AbstractThe selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.

Abstract 3026: Targeting GPX4 in tumor-associated stromal cells increases inflammatory-cell infiltration

Abstract 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

Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 (‘BAY’) triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.

Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition.Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation.In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

Glutathione peroxidase 4 plays an important role in oxidative homeostasis and wound repair in corneal epithelial cells.

Oxidative stress is involved in the pathologies of corneal epithelial cells. However, the importance of specific antioxidant enzymes in corneal epithelial cells is not fully understood. The purpose of this study is to elucidate the role of glutathione peroxidase 4 (GPx4) in corneal epithelial cells. For in vitro experiments, an immortalized human corneal epithelial cell line was used. Cytotoxicity measured through LDH activity, lipid peroxidation immunostained for 4‐hydroxynonenal, cell viability, and cell death were compared between cells transfected with either GPx4 siRNA or scrambled control siRNA. In addition, the rescue effects of α‐tocopherol and ferrostatin‐1, a ferroptosis inhibitor, were examined in the cells with deficient GPx4 expression. For in vivo experiments, we applied n‐heptanol on the cornea of GPx4+/+ and GPx4+/− mice to create corneal epithelial wound. The epithelial defect area size was measured up to 48 h after epithelial wound creation. Knockdown of GPx4 strongly induced cytotoxicity and cell death in human corneal epithelial cells. Cell death induced by GPx4 knockdown was characterized by positive staining for both annexin V and propidium iodide, nuclear translocation of AIF, and without activation of caspase 3, and was rescued by α‐tocopherol and ferrostatin‐1. The delayed wound healing of GPx4 siRNA‐transfected cells were ameliorated by α‐tocopherol in vitro. In addition, loss of one GPx4 allele was sufficient to significantly delay the healing of experimental corneal epithelial wounds in vivo. Our results suggest that the antioxidant enzyme GPx4 plays an important role in oxidative homeostasis, cell survival, and wound healing in corneal epithelial cells.

Abstract B11: Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition

Abstract Fibroblast-like mesenchymal cells are the most abundant component of tumor stroma and have been shown to promote tumor progression. However, there are no therapeutic agents that target these cells. Developing therapeutics against mesenchymal cells has been challenging because they lack clear “druggable” targets. Here we used a phenotypic screening approach to identify therapeutic targets in bone-marrow mesenchymal stem cells (MSCs), precursors of cancer-associated fibroblasts, that are reported to induce a metastatic phenotype in breast cancer cells (Karnoub et al, Nature 2007). Using the in vitro phenotype of enhanced migration of breast cancer cells (GFP-labeled MDA-MB-231; MDA) induced by MSCs (3-fold faster than MDA alone), we identified inflammatory signaling and glutathione peroxidase 4 as potential targets in MSCs. To validate this screening model, we first confirmed that MSCs can enhance metastasis using orthotopic xenografts of luciferase-labeled MDA cells co-injected with primary human MSCs in NOD-SCID mice. Mice with MSC+MDA tumors had 5-fold greater thoracic bioluminescence (corresponding to lung metastasis) than mice with MDA tumors alone. We then performed gene-expression profiling of MSC+MDA co-cultures in vitro compared to cells grown alone. The top pathway upregulated in co-cultures was the interferon pathway, suggesting that an inflammatory response follows MSC-MDA interactions. Using publicly available gene-expression datasets, we prioritized transcripts that are expressed in patient stroma and correlate with poor survival in a meta-analysis of 20 whole-tumor datasets. To determine if these transcripts are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown and measured effects on in vitro migration of MSC+MDA co-cultures compared to normal endothelial cells. Knockdown of 9 genes was specific for MSC+MDA migration but the effects were weak. This suggested functional redundancy and indicated that targeting individual upregulated genes is insufficient to block the phenotype. We then performed a small-molecule screen on the MSC+MDA migration phenotype, counter-screening hits on endothelial cell migration. Only one compound, RSL3, showed a large and selective inhibition of MSC+MDA migration. RSL3 was selectively toxic to MSCs with no effect on MDA cells. The target of RSL3 was recently identified to be glutathione peroxidase 4 (GPX4) that metabolizes lipid peroxides. To identify lipid mediators of RSL3 toxicity, we profiled lipid levels in RSL3-treated MSC+MDA co-cultures and found that the top changes were lipoxygenase products of arachidonic acid. Pre-treatment with zileuton or PD146176, that inhibit 5- and 15-lipoxygenase respectively, abrogated RSL3's toxic effects on MSCs. We found that MSCs contain >10-fold higher lipoxygenase products than MDA cells, highlighting an inflammatory role of MSCs. Fibroblasts from different tissues (lungs, spleen, breast, skin) were similarly sensitive to RSL3, identifying a previously unrecognized vulnerability in these mesenchymal cells. Since RSL3 is a tool compound with poor plasma stability, we used GPX4 shRNA knockdown (KD) to determine in vivo effects of depleting MSCs in MSC+MDA xenografts. Unexpectedly, we found accelerated tumor growth in GPX4 KD tumors compared with controls. However, this is consistent with previous reports showing that mesenchymal cells restrain tumor growth and can have both pro- and anti-tumor effects. Histological examination revealed increased myeloid cell infiltration into GPX4 KD tumors reflecting increased inflammation mediated by lipoxygenase products like leukotriene B4. In summary, phenotypic screening has identified GPX4 inhibition as a novel approach to target mesenchymal cells. This approach may be particularly effective at recruiting immune/ inflammatory cells into tumors with the exciting possibility of synergizing with cancer immunotherapy. Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Ninib Baryawno, Nicolas Severe, Vasanthi Viswanathan, Zarko Boskovic, Siddhartha Mukherjee, Jeff Gentry, Ben Wittner, Sridhar Ramaswamy, Alykhan Shamji, David Scadden, Stuart Schreiber. Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B11.

Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells

ABSTRACTPurpose: To investigate the role of glutathione peroxidase 4 (GPx4) in corneal endothelial cells.Materials and Methods: An immortalized human corneal endothelial cell line was used. Cells were transfected with either siRNA specifically silencing GPx4 or scrambled control siRNA. Knockdown was confirmed by Real-time RT-PCR and immunoblotting. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal immunostaining. Cytotoxicity, cell death, and cell proliferation were evaluated using a lactate dehydrogenase (LDH) activity assay, Annexin V staining, and WST-8, respectively. Furthermore, cells transfected with GPx4 siRNA or control siRNA were treated with hydrogen peroxide or ferrous sulfate, and cytotoxicity was evaluated using the LDH activity assay.Results: The treatment of siRNA decreased the expression of GPx4 at both mRNA and protein levels. The knockdown of GPx4 significantly increased the levels of lipid oxidation and LDH activity. Annexin V-positive cells increased in GPx4 siRNA-treated cells. The proliferation of GPx4 siRNA-treated cells was downregulated compared with that of control siRNA-treated cells. GPx4 knockdown enhanced hydrogen peroxide- and ferrous sulfate-induced cytotoxicity.Conclusion: These results suggest that GPx4 is an important antioxidant enzyme for maintaining redox status and protecting corneal endothelial cells from oxidative stress.

Glutathione peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells

Intake of the micronutrient selenium, which is incorporated into 25 selenoproteins in humans, has been implicated in affecting risk of colorectal cancer. A genetic variant in the gene encoding the selenoprotein glutathione peroxidase 4 (GPX4) has been reported to influence colorectal cancer risk. In this study GPX4 expression was knocked down by 60% using RNA silencing and the effects were investigated using an unbiased transcriptomic analysis. Microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips and the data were validated by real-time PCR. Ingenuity Pathway Analysis showed that the major canonical pathways affected by GPX4 knockdown were oxidative phosphorylation, ubiquinone biosynthesis, and mitochondrial dysfunction and the top two toxicological lists were mitochondrial dysfunction and oxidative stress. Western blotting and real-time PCR confirmed that knockdown affected target genes encoding components of respiratory complexes I, IV, and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knockdown increased levels of mitochondrial reactive oxygen species and oxidized lipid and decreased mitochondrial adenosine triphosphate levels and mitochondrial membrane potential. Time-course experiments showed that changes in AIF expression preceded those in the respiratory complexes. We conclude that in Caco-2 gut epithelial cells GPx4, through effects on AIF, plays a major role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage.