Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity.

The linkage between neutrophil death and the development of autoimmunity has not been thoroughly explored. Here, we show that neutrophils from either lupus-prone mice or patients with systemic lupus erythematosus (SLE) undergo ferroptosis. Mechanistically, autoantibodies and interferon-α present in the serum induce neutrophil ferroptosis through enhanced binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (Gpx4, the key ferroptosis regulator) promoter, which leads to suppressed expression of Gpx4 and subsequent elevation of lipid-reactive oxygen species. Moreover, the findings that mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate key clinical features of human SLE, including autoantibodies, neutropenia, skin lesions and proteinuria, and that the treatment with a specific ferroptosis inhibitor significantly ameliorates disease severity in lupus-prone mice reveal the role of neutrophil ferroptosis in lupus pathogenesis. Together, our data demonstrate that neutrophil ferroptosis is an important driver of neutropenia in SLE and heavily contributes to disease manifestations.

Selenium-GPX4 axis protects follicular helper T cells from ferroptosis.

Follicular helper T (TFH) cells are a specialized subset of CD4+ T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of TFH cell survival remains unclear. Here we report that TFH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for TFH cell survival. The deletion of GPX4 in T cells selectively abrogated TFH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased TFH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium-GPX4-ferroptosis axis in regulating TFH homeostasis, which can be targeted to enhance TFH cell function in infection and following vaccination.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

Unraveling ETC complex I function in ferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

Ferroptosis: A flexible constellation of related biochemical mechanisms.

It is common to think about and depict biological processes as being governed by fixed pathways with specific components interconnected by concrete positive and negative interactions. However, these models may fail to effectively capture the regulation of cell biological processes that are driven by chemical mechanisms that do not rely absolutely on specific metabolites or proteins. Here, we discuss how ferroptosis, a non-apoptotic cell death mechanism with emerging links to disease, may be best understood as a highly flexible mechanism that can be executed and regulated by many functionally related metabolites and proteins. The inherent plasticity of ferroptosis has implications for how to define and study this mechanism in healthy and diseased cells and organisms.

3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

USP8-governed GPX4 homeostasis orchestrates ferroptosis and cancer immunotherapy.

Ferroptosis is an iron-dependent type of regulated cell death resulting from extensive lipid peroxidation and plays a critical role in various physiological and pathological processes. However, the regulatory mechanisms for ferroptosis sensitivity remain incompletely understood. Here, we report that homozygous deletion of Usp8 (ubiquitin-specific protease 8) in intestinal epithelial cells (IECs) leads to architectural changes in the colonic epithelium and shortens mouse lifespan accompanied by increased IEC death and signs of lipid peroxidation. However, mice with heterozygous deletion of Usp8 in IECs display normal phenotype and become resistant to azoxymethane/dextran sodium sulfate-induced colorectal tumorigenesis. Mechanistically, USP8 interacts with and deubiquitinates glutathione peroxidase 4 (GPX4), leading to GPX4 stabilization. Thus, USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis in vitro. Notably, USP8 inhibition in combination with ferroptosis inducers retards tumor growth and enhances CD8+ T cell infiltration, which potentiates tumor response to anti-PD-1 immunotherapy in vivo. These findings uncover that USP8 counteracts ferroptosis by stabilizing GPX4 and highlight targeting USP8 as a potential therapeutic strategy to boost ferroptosis for enhancing cancer immunotherapy.

Lymph protects metastasizing melanoma cells from ferroptosis.

Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.

Melatonin suppresses atherosclerosis by ferroptosis inhibition via activating NRF2 pathway.

Melatonin (MLT), a conserved small indole compound, exhibits anti-inflammatory and antioxidant properties, contributing to its cardioprotective effects. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with atherosclerosis disease risk, and is known as an atherosclerosis risk biomarker. This study aimed to investigate the impact of MLT on Lp-PLA2 expression in the atherosclerotic process and explore the underlying mechanisms involved. In vivo, ApoE-/- mice were fed a high-fat diet, with or without MLT administration, after which the plaque area and collagen content were assessed. Macrophages were pretreated with MLT combined with ox-LDL, and the levels of ferroptosis-related proteins, NRF2 activation, mitochondrial function, and oxidative stress were measured. MLT administration significantly attenuated atherosclerotic plaque progression, as evidenced by decreased plaque area and increased collagen. Compared with those in the high-fat diet (HD) group, the levels of glutathione peroxidase 4 (GPX4) and SLC7A11 (xCT, a cystine/glutamate transporter) in atherosclerotic root macrophages were significantly increased in the MLT group. In vitro, MLT activated the nuclear factor-E2-related Factor 2 (NRF2)/SLC7A11/GPX4 signaling pathway, enhancing antioxidant capacity while reducing lipid peroxidation and suppressing Lp-PLA2 expression in macrophages. Moreover, MLT reversed ox-LDL-induced ferroptosis, through the use of ferrostatin-1 (a ferroptosis inhibitor) and/or erastin (a ferroptosis activator). Furthermore, the protective effects of MLT on Lp-PLA2 expression, antioxidant capacity, lipid peroxidation, and ferroptosis were decreased in ML385 (a specific NRF2 inhibitor)-treated macrophages and in AAV-sh-NRF2 treated ApoE-/- mice. MLT suppresses Lp-PLA2 expression and atherosclerosis processes by inhibiting macrophage ferroptosis and partially activating the NRF2 pathway.

PITX2 functions as a transcription factor for GPX4 and protects pancreatic cancer cells from ferroptosis.

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.

Inhibition of Gpx4-mediated ferroptosis alleviates cisplatin-induced hearing loss in C57BL/6 mice.

Cisplatin-induced hearing loss is a common side effect of cancer chemotherapy in clinics; however, the mechanism of cisplatin-induced ototoxicity is still not completely clarified. Cisplatin-induced ototoxicity is mainly associated with the production of reactive oxygen species, activation of apoptosis, and accumulation of intracellular lipid peroxidation, which also is involved in ferroptosis induction. In this study, the expression of TfR1, a ferroptosis biomarker, was upregulated in the outer hair cells of cisplatin-treated mice. Moreover, several key ferroptosis regulator genes were altered in cisplatin-damaged cochlear explants based on RNA sequencing, implying the induction of ferroptosis. Ferroptosis-related Gpx4 and Fsp1 knockout mice were established to investigate the specific mechanisms associated with ferroptosis in cochleae. Severe outer hair cell loss and progressive damage of synapses in inner hair cells were observed in Atoh1-Gpx4-/- mice. However, Fsp1-/- mice showed no significant hearing phenotype, demonstrating that Gpx4, but not Fsp1, may play an important role in the functional maintenance of HCs. Moreover, findings showed that FDA-approved luteolin could specifically inhibit ferroptosis and alleviate cisplatin-induced ototoxicity through decreased expression of transferrin and intracellular concentration of ferrous ions. This study indicated that ferroptosis inhibition through the reduction of intracellular ferrous ions might be a potential strategy to prevent cisplatin-induced hearing loss.

Selenium-SelK-GPX4 axis protects nucleus pulposus cells against mechanical overloading-induced ferroptosis and attenuates senescence of intervertebral disc.

Intervertebral disc degeneration (IVDD) is one of the most prevalent spinal degenerative disorders and imposes places heavy medical and economic burdens on individuals and society. Mechanical overloading applied to the intervertebral disc (IVD) has been widely recognized as an important cause of IVDD. Mechanical overloading-induced chondrocyte ferroptosis was reported, but the potential association between ferroptosis and mechanical overloading remains to be illustrated in nucleus pulposus (NP) cells. In this study, we discovered that excessive mechanical loading induced ferroptosis and endoplasmic reticulum (ER) stress, which were detected by mitochondria and associated markers, by increasing the intracellular free Ca2+ level through the Piezo1 ion channel localized on the plasma membrane and ER membrane in NP cells. Besides, we proposed that intracellular free Ca2+ level elevation and the activation of ER stress are positive feedback processes that promote each other, consistent with the results that the level of ER stress in coccygeal discs of aged Piezo1-CKO mice were significantly lower than that of aged WT mice. Then, we confirmed that selenium supplementation decreased intracellular free Ca2+ level by mitigating ER stress through upregulating Selenoprotein K (SelK) expression. Besides, ferroptosis caused by the impaired production and function of Glutathione peroxidase 4 (GPX4) due to mechanical overloading-induced calcium overload could be improved by selenium supplementation through Se-GPX4 axis and Se-SelK axis in vivo and in vitro, eventually presenting the stabilization of the extracellular matrix (ECM). Our findings reveal the important role of ferroptosis in mechanical overloading-induced IVDD, and selenium supplementation promotes significance to attenuate ferroptosis and thus alleviates IVDD, which might provide insights into potential therapeutic interventions for IVDD.

A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

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.

Orexin-A mediates glioblastoma proliferation inhibition by increasing ferroptosis triggered by unstable iron pools and GPX4 depletion.

Glioblastoma (GBM) represents a prevalent form of primary malignant tumours in the central nervous system, but the options for effective treatment are extremely limited. Ferroptosis, as the most enriched programmed cell death process in glioma, makes a critical difference in glioma progression. Consequently, inducing ferroptosis has become an appealing strategy for tackling gliomas. Through the utilization of multi-omics sequencing data analysis, flow cytometry, MDA detection and transmission electron microscopy, the impact of orexin-A on ferroptosis in GBM was assessed. In this report, we provide the first evidence that orexin-A exerts inhibitory effects on GBM proliferation via the induction of ferroptosis. This induction is achieved by instigating an unsustainable increase in iron levels and depletion of GPX4. Moreover, the regulation of TFRC, FTH1 and GPX4 expression through the targeting of NFE2L2 appears to be one of the potential mechanisms underlying orexin-A-induced ferroptosis.

LRRK2 regulates ferroptosis through the system Xc-GSH-GPX4 pathway in the neuroinflammatory mechanism of Parkinson's disease.

Parkinson's disease (PD) is the most prevalent neurodegenerative disorder. Neuroinflammation mediated by activated microglia and apoptosis of dopaminergic (DA) neurons in the midbrain are its primary pathological manifestations. Leucine-rich repeat protein kinase 2 (LRRK2) kinase has been observed to increase expression during neuroinflammation, however, the effect of LRRK2 on microglia activation remains poorly understood. In this study, we have established lipopolysaccharide (LPS) treated BV2 cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models for both in vivo and in vitro investigation. Our data in vivo reveal that LRRK2 can promote microglia activation by regulating ferroptosis and activating nuclear factor-κB. Inhibition of LRRK2 expression effectively suppressed the LPS-induced pro-inflammatory cytokines and facilitated the secretion of neuroprotective factors. Importantly, by co-overexpressing LRRK2 and glutathione peroxidase 4 (GPX4), we identified the system Xc-GSH-GPX4 pathway as a crucial component in LRRK2-mediated microglial ferroptosis and inflammatory responses. Using a microglial culture supernatant (MCS) transfer model, we found that inhibiting LRRK2 or downregulating ferroptosis in BV2 cells prevented SH-SY5Y cell apoptosis. Additionally, we observed abundant expression of LRRK2 and P-P65 in the midbrain, which was elevated in the MPTP-induced PD model, along with microglia activation. LRRK2 and P-P65 expression inhibition with PF-06447475 attenuated microglia activation in the nigrostriatal dense part of MPTP-treated mice. Based on our findings, it is evident that LRRK2 plays a critical role in promoting the neuroinflammatory response during the pathogenesis of PD by regulating the system Xc-GSH-GPX4 pathway. Taken together, our data highlights the potential research and therapeutic value of targeting LRRK2 to regulate neuroinflammatory response in PD through ferroptosis.

Induction of stearoyl-CoA desaturase confers cell density-dependent ferroptosis resistance in melanoma.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.