Expression Of Glutathione Peroxidase 4 Research Articles

GPX4 Plays a Crucial Role in Fuzheng Kang'ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis.

Background: Fuzheng Kang’ai decoction (FZKA) has been widely used to treat Non-Small Cell Lung Cancer (NSCLC) patients in China for decades, showing definitively curative effects in clinic. Recently, we found that FZKA could induce NSCLC cell ferroptosis, another type of programmed cell death (PCD), which is totally different from cell apoptosis. Therefore, in the present study, we aim to discover the exact mechanism by which FZKA induces NSCLC cell ferroptosis, which is rarely studied in Traditional Chinese Medicine (TCM). Methods: Cell proliferation assay were performed to detect the cell viability. Cell ferroptosis triggered by FZKA was observed by performing lipid peroxidation assay, Fe2+ Ions assay, and mitochondrial ultrastructure by transmission electron microscopy. Ferroptosis inhibitors including liproxstatin-1 and UAMC 3203 were used to block ferroptosis. The ratio of GSH/GSSG was done to measure the alteration of oxidative stress. Western blot and qRT-PCR were carried out to detect the expression of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and glutathione peroxidase 4 (GPX4) at protein and mRNA levels, respectively. Lentivirus transfection was performed to overexpress GPX4 stably. Animal model was done to verify the effect of FZKA-induced ferroptosis in NSCLC in vivo and immunohistochemistry was done to detect the expression of SLC7A11, SLC3A2 and GPX4 at protein level. Results: First of all, in vitro experiments confirmed the inhibition effect of FZKA on NSCLC cell growth. We then, for the first time, found that FZKA induced NSCLC cell ferroptosis by increasing lipid peroxidation and cellular Fe2+ Ions. Moreover, characteristic morphological changes of NSCLC cell ferroptosis was observed under transmission electron microscopy. Mechanistically, GPX4, as a key inhibitor of lipid peroxidation, was greatly suppressed by FZKA treatment both at protein and mRNA levels. Furthermore, system xc− (SLC7A11 and SLC3A2) were found to be suppressed and a decreased GSH/GSSG ratio was observed at the same time when treated with FZKA. Notably, overexpressing GPX4 reversed the effect of FZKA-induced NSCLC cell ferroptosis significantly. Finally, the above effect was validated using animal model in vivo. Conclusion: Our findings conclude that GPX4 plays a crucial role in FZKA-induced NSCLC cell ferroptosis, providing a novel molecular mechanism by which FZKA treats NSCLC.

A novel anticancer property of Lycium barbarum polysaccharide in triggering ferroptosis of breast cancer cells.

Breast cancer is one of the most malignant tumors and is associated with high mortality rates among women. Lycium barbarum polysaccharide (LBP) is an extract from the fruits of the traditional Chinese herb, L. barbarum. LBP is a promising anticancer drug, due to its high activity and low toxicity. Although it has anticancer properties, its mechanisms of action have not been fully established. Ferroptosis, which is a novel anticancer strategy, is a cell death mechanism that relies on iron-dependent lipid reactive oxygen species (ROS) accumulation. In this study, human breast cancer cells (Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast-231 (MDA-MB-231)) were treated with LBP. LBP inhibited their viability and proliferation in association with high levels of ferroptosis. Therefore, we aimed to ascertain whether LBP reduced cell viability through ferroptosis. We found that the structure and function of mitochondria, lipid peroxidation, and expression of solute carrier family 7 member 11 (SLC7A11, also known as xCT, the light-chain subunit of cystine/glutamate antiporter system Xc-) and glutathione peroxidase 4 (GPX4) were altered by LBP. Moreover, the ferroptosis inhibitor, Ferrostatin-1 (Fer-1), rescued LBP-induced ferroptosis-associated events including reduced cell viability and glutathione (GSH) production, accumulation of intracellular free divalent iron ions and malondialdehyde (MDA), and down-regulation of the expression of xCT and GPX4. Erastin (xCT inhibitor) and RSL3 (GPX4 inhibitor) inhibited the expression of xCT and GPX4, respectively, which was lower after the co-treatment of LBP with Erastin and RSL3. These results suggest that LBP effectively prevents breast cancer cell proliferation and promotes ferroptosis via the xCT/GPX4 pathway. Therefore, LBP exhibits novel anticancer properties by triggering ferroptosis, and may be a potential therapeutic option for breast cancer.

Tetrandrine Ameliorates Traumatic Brain Injury by Regulating Autophagy to Reduce Ferroptosis.

Traumatic brain injury (TBI) is the leading cause of death and disability in trauma patients. However, the effects and mechanism of autophagy after TBI remain unclear. This study aimed to investigate whether tetrandrine could ameliorate TBI through autophagy to reduce ferroptosis. A mice model for TBI was implemented. Behavioral and histomorphological experiments were performed to evaluate outcomes of the mice. The ferroptosis levels was detected by Perls staining. Enzyme-linked immunosorbent assay (ELISA) was applied to detect malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase 4 (GPX4) levels in the brain tissue. Western blot test was performed to detect Beclin 1, light chain 3 (LC3) II/I, p62, GPX4, SCL7A11, and ferritin heavy chain 1 (FTH1) levels, and the expression of LC3B, Beclin 1, GPX4, and FTH1 in the brain tissue was detected by immunofluorescence (IF). The behavioral and histomorphological results demonstrated that tetrandrine improved the neurological function and cerebral edema on days 1, 3, and 7 after TBI. The ELISA results suggested that tetrandrine reduced the MDA concentration and increased GSH concentration on days 1, 3, and 7 after TBI. IF staining and Perls staining reflected that tetrandrine promoted autophagy and inhibited ferroptosis on days 1, 3, and 7 after TBI, respectively. Tetrandrine further improved the neurological function, cerebral edema, autophagy, and ferroptosis on days 1, 3, and 7 after TBI after adding the autophagy inducer rapamycin. The effect of TET in alleviating TBI increased with the increase of time and dose. Tetrandrine ameliorated TBI by regulating autophagy to reduce ferroptosis, providing a new therapeutic strategy for TBI.

Effects of aerobic exercise on Nrf2/GPX4/Ferroptosis pathway in myocardial injury in high-fat diet mice

Objective: To illuminate the protective effects of pathway in inhibiting ferroptosis by glutathione peroxidase 4 (GPX4) activated by nuclear factor erythroid 2-related factor 2 (Nrf2) during aerobic exercise against myocardial injury in high-fat diet mice. Methods: Forty 5-week-old SPF C57BL/6 male mice were randomly divided into the control group (NC), the exercise group (NE), the high fat group (HC) and the high fat diet with exercise group (HE, began at the same time). There were 10 mice in each group. The mice in the high fat diet group were fed with 60% Kcal SPF high fat model diet. Aerobic exercise was performed using increasing load platform exercise, 5 days /week, 60 min/d, the speed started from 13m/min, and increased by 1m/min every two weeks. Myocardium and blood samples were collected after 14 weeks. Structural changes of myocardial tissues were observed by HE staining. Western blot was used to detect the expressions of Nrf2/GPX4/Ferroptosis related proteins in myocardium. Myocardial peroxide concentration and antioxidant enzyme activity were measured by spectrophotometry. Myocardial mitochondrial 8-hydroxy-2 deoxyguanosine (8-OHdG) and serum insulin were measured by ELISA. Results: Compared with the NC group, there was more lipid accumulation in the myocardial fiber space in the HC group, and the levels of FBG and FINS were increased significantly, while ISI was decreased significantly (P＜0.01). Compared with the HC group, the lipid concentration was decreased in the HE group, and the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione (GSH) were increased significantly, while the levels of mitochondrial 8-OHdG and myocardial iron content were decreased (P＜0.01). The expression levels of Ferroportin1 (FPN1), ferritin heavy chain 1 (FTH1), GPX4, glucose transporter (GLUT1) and Nrf2 in the HE group were significantly higher than those in the HC group (P＜0.01). Conclusion: The expression of GPX4 was enhanced by more Nrf2 transposition into the nuclear during aerobic exercise, which inhibited the occurrence of myocardial ferroptosis. The activities of antioxidant enzymes were promoted and inhibited the peroxidation damage of myocardial mitochondria.

The RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) modulates glioma cells sensitivity to temozolomide by regulating ferroptosis

Temozolomide (TMZ) is a first-line chemotherapeutic agent for the treatment of glioma. However, at least 50% of glioma patients do not respond to TMZ, and the exact mechanism leading to TMZ resistance is still unclear. In the present study, we investigated molecular mechanisms underlying the resistance to TMZ in glioma cells. Glioma cell lines A172 and U251 were maintained in medium with increasing doses of TMZ for 12 months to induce the TMZ-resistance. Cells were then transduced with different adenoviral vectors to overexpress or inhibit RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) and glutathione peroxidase 4 (GPX4), which has been associated with the ferroptosis mechanism. Cell viability and cell death were analysed using cell counting Kit-8 (CCK-8) and Annexin V-FITC staining, respectively. RT-PCR, RNA-seq analysis, and RNA immunoprecipitation were used to analyse RNA expression; Western blot was used for protein expression. We discovered that RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) was upregulated in TMZ-resistance glioma. Knockdown of FXR1 could overcome TMZ-resistance by promoting ferroptosis. Mechanically, FXR1 could bind with GPX4 mRNA and positively regulate the expression of GPX4. Inhibition of GPX4 further increased the sensitivity to TMZ in glioma cells with upregulated FXR1. Our data suggest that targeting FXR1-GPX4 might be a potential strategy to overcome chemoresistance to TMZ in glioma cells.

Dexmedetomidine post-conditioning alleviates myocardial ischemia-reperfusion injury in rats by ferroptosis inhibition via SLC7A11/GPX4 axis activation.

The SLC7A11/GPX4 axis plays an important role in ferroptosis during cardiac ischemia/reperfusion injury (IRI). The present study was designed to evaluate the impact of dexmedetomidine (DEX) post-conditioning on cardiac IRI and to explore whether the effect was achieved by SLC7A11/GPX4 signaling pathway regulation. Rat myocardial IRI was established by occluding the left anterior descending artery for 30min followed by 2-h reperfusion. The infarct area was detected by diphenyltetrazolium chloride (TTC) staining; the cardiac function was evaluated by echocardiography. The levels of lipid peroxide biomarkers were measured to estimate the injury caused by lipid peroxide. HE staining and Sirius staining were utilized to assess myocardial damage and fibrosis. The mitochondrial morphology was observed by electron micrography. Western blot and quantitative real-time polymerase chain reaction were employed to measure the relative molecular characteristics. Our results showed that DEX administration at the beginning of reperfusion attenuated IRI-induced myocardial injury, alleviated mitochondrial dysfunction, decreased the level of reactive oxygen species (ROS), alleviated mitochondrial dysfunction, inhibited the activation of SLC7A11/GPX4, and modulated the expression of ferroptosis-related proteins, including SLC7A11, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH), and cyclooxygenase-2 (COX-2). Conversely, the ferroptosis activator erastin partly suppressed the DEX-mediated cardio protection. Altogether, these results reveal that DEX inhibits ferroptosis by enhancing the expression of SLC7A11 and GPX4, thereby preventing cardiac I/R injury.

Ferrostatin-1 alleviates cytotoxicity of cobalt nanoparticles by inhibiting ferroptosis

ABSTRACT Cobalt is the main component of metal prostheses in hip arthroplasty. Studies have shown that metal particles mainly composed of cobalt nanoparticles (CoNPs) can cause systemic and local toxic reactions due to various physical and chemical factors. Therefore, elucidating the underlying mechanisms of metal prosthesis action, coupled with identification of effective detoxification drugs are imperative to minimizing postoperative complications and prolonging the service life of these clinical tools. In this study, we treated Balb/3T3 mouse fibroblast cell line with CoNPs and ferrostatin-1, then measured cell viability via the CCK-8 assay. Next, we determined levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), cobalt and iron contents, as well as glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) expression in each group. Finally, we employed transmission electron microscopy (TEM) to detect changes in the ultrastructure of each group of cells. Exposure of cells to CoNPs significantly suppressed their viability, and downregulated expression of GSH, GPX4, and SLC7A11 proteins. Conversely, this treatment mediated a significant increase in ROS, MDA, cobalt, and iron levels in the cells. TEM images revealed a marked increase in density of the mitochondrial membrane of cells in the CoNPs group, while the outer membrane was broken. Notably, treatment with ferroptosis inhibitor Ferrostatin-1 alleviated the cytotoxic response caused by CoNPs. These findings suggest that CoNP-induced cytotoxicity may be closely related to ferroptosis, indicating that inhibition of ferroptosis is a potential therapeutic strategy for reducing CoNP toxicity.

Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis

Endometriosis (EMs) is one of the most frequent diseases of reproductive-age women and is characterized by the growth of endometrial tissues beyond the uterus. The enhanced proliferative and migratory potential of endometrial stromal cells (ESCs) plays an important role in the progression of EMs. Mounting studies have demonstrated that long noncoding RNAs (lncRNAs) exert an important role in regulating the development and progression of EMs. Given the aberrant expression of lncRNA ADAMTS9-AS1 in ectopic endometrium (ecEM), we investigated the biological effect of ADAMTS9-AS1 on ESC proliferation and migration and explored the underlying mechanism. The current data showed that ADAMTS9-AS1 expression was significantly upregulated in ecEM compared with eutopic endometrium (euEM) in patients with EMs and in a murine model of EMs. Functionally, ADAMTS9-AS1 knockdown in ectopic ESCs (EESCs) decreased cell viability and migration, whereas ADAMTS9-AS1 overexpression in normal ESCs (NESCs) enhanced cell viability and migration. More importantly, the effect of ADAMTS9-AS1 inhibition on decreasing ESC viability was significantly blocked by ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and ADAMTS9-AS1 overexpression repressed erastin (a ferroptosis activator)-induced cell death. Furthermore, the regulatory role of ADAMTS9-AS1 in ferroptosis was defined and evidenced by increased reactive oxygen species (ROS) levels and malonyl dialdehyde (MDA) content and decreased expression of glutathione peroxidase 4 (GPX4) after ADAMTS9-AS1 inhibition. Mechanistically, ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4, the critical repressor of ferroptosis. Taken together, these results demonstrate that upregulated ADAMTS9-AS1 accelerates ESC proliferation and migration by regulating miR-6516-5p/GPX4-dependent ferroptosis and may be a potential target for the treatment of EMs.

Glycine increased ferroptosis via SAM-mediated GPX4 promoter methylation in rheumatoid arthritis.

Over-proliferation of synovium is a key event of invasive pannus formation and cartilage damage in the progression of RA disease. At the same time, ferroptosis may play a pivotal role in maintaining the balance of proliferation and death of synovium. In this study, we firstly evaluated the ferroptosis level in RA fibroblast-like synoviocytes (FLS) and then explored the role of glycine in ferroptosis. Ferroptosis was evaluated in RA synovium and FLS. The therapeutic effect of glycine on RA was evaluated by clinical and histopathological score and cytokine level in a CIA mouse model. The influence of glycine on ferroptosis was evaluated by mitochondrial morphology observation and membrane potential assay in RA FLS. Methylase expression was detected to explore the mechanism behind the effect of glycine on glutathione peroxidase 4 (GPX4) methylation. Compared with healthy controls, ferroptosis decreased in the RA synovium and FLS, with a decrease in Acyl Coenzyme A Synthetase Long Chain 4 (ACSL4) and an increase in Ferritin heavy chain 1 (FTH1), GPX4 and cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11). Although both oxidation and antioxidation levels of lipids were higher in RA FLS than in healthy controls, the increase in antioxidation was slightly higher than oxidation. RNA-seq and verification showed that glycine regulated the ferroptosis pathway through increase S-adenosylmethionine (SAM) concentration and decrease the expression of GPX4 and FTH1 by promoting SAM-mediated GPX4 promoter methylation and reducing FTH1 expression in RA FLS. In summary, we confirmed a decline in ferroptosis in RA and explored that glycine enhanced ferroptosis via SAM-mediated GPX4 promoter methylation and ferritin decrease.

Contribution of ferroptosis and GPX4's dual functions to osteoarthritis progression.

SummaryBackgroundOsteoarthritis (OA) is the most common joint disease and is the leading cause of chronic disability among older people. Chondrocyte death and extracellular matrix (ECM) degradation was involved in OA pathogenesis. Ferroptosis was an iron-dependent cell death associated with peroxidation of lipids. Here, we proved that ferroptosis exists in OA and identified glutathione peroxidase 4 (GPX4) as an important regulator of OA.MethodsFerroptosis-related alterations were analyzed in human OA and undamaged cartilage. Expression of GPX4 was examined in 55 paired human OA samples. Ferrostatin-1 (Fer-1) and Deferoxamine (DFO) were used to treat OA, in vitro and in vivo. Alterations of GPX4-mediated signaling pathway were identified by RNA-seq analysis. AAV-Gpx4-shRNA were used to downregulate GPX4 expression in vivo.FindingsTranscriptomic, biochemical, and microscopical analyses indicated that ferroptosis was closely associated with OA. Expression of GPX4 in the OA cartilage from 55 OA patients were significantly lower than undamaged cartilage. Fer-1 and DFO could protect OA in a necroptosis-independent manner, suggesting that ferroptosis exists in OA prog. Importantly, GPX4 downregulation could increase the sensitivity of chondrocytes to oxidative stress and aggravate ECM degradation through the MAPK/NFκB pathway. Furthermore, downregulation of GPX4 expression by AAV-Gpx4 shRNA aggravated OA in vivo.InterpretationFerroptosis contributes to OA pathogenesis and GPX4 was the intersection of two mechanisms in regulating OA progression: ferroptosis and ECM degradation.

Ophiopogonin B induces gastric cancer cell death by blocking the GPX4/xCT-dependent ferroptosis pathway.

Ophiopogonin B (OP-B) is extensively applied as a treatment for pulmonary disease and is reported to suppress lung cancer. However, further study is needed to determine whether OP-B suppresses gastric cancer (GC). The mRNA levels of prostaglandin-endoperoxide synthase 2 (Ptgs2) and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (Chac1) were determined using quantitative PCR. Ptgs2 and Chac1 mRNA levels were significantly increased in GC cancer tissues compared with those of adjacent normal controls. The CCK-8 assay revealed that OP-B suppressed GC cell viability in a time- and dose-dependent manner. The administration of OP-B in combination with different cell death inhibitors showed that only the ferroptosis inhibitor, ferrostatin-1 (Fer-1), abolished the OP-B-induced death of both AGS and NCI-N87 cells, but not other inhibitors. Western blot analysis indicated that OP-B reduced the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11, xCT) but had no effects on the expression of nuclear receptor coactivator 4 (NCOA4) and ferritin heavy chain 1 (FTH1) in AGS and NCI-N87 cells. In vivo administration of OP-B reduced the volume and weight of AGS tumors. In addition, the expression of GPX4 and xCT was reduced in nude mice treated with OP-B compared with control mice. In summary, results of the present study suggest that OP-B induces ferroptosis in gastric cancer cells by blocking the GPX4/xCT system.

A “Closed‐Loop” Therapeutic Strategy Based on Mutually Reinforced Ferroptosis and Immunotherapy

AbstractThe immunosuppression and immune escape of current immunotherapy result in low efficacy, and ferroptosis is greatly restricted by the low reactive oxygen species (ROS) production efficiency. Here, for the first time a “closed‐loop” therapy based on photothermal enhancement of ferroptosis and immunotherapy stimulated by each other on a multifunctional nanoplatform is reported. This platform is composed of copper silicate and iron silicate mesoporous hollow nanospheres, followed by in situ growth of Au nanoparticles and loading of an immune adjuvant resiquimod R848. The laser irradiation‐mediated heat and the introduction of copper ions significantly enhance ROS generation, leading to the simultaneous depletion of glutathione peroxidase 4 (GPX4) and glutathione (GSH). The onset of ferroptosis in tumor cells is thus enhanced and an immune response with immunogenic cell death (ICD) is triggered, promoting the dendritic cells (DCs) maturation and T cell infiltration. Interferon γ (IFN‐γ) released from CD8+ T cells downregulates the expression of SLC7A11 and GPX4, which in turn enhances ferroptosis expression, thus constituting a “closed‐Loop” therapy. Importantly, this system is effective in both killing the primary tumor and inhibiting tumor metastasis. The proposed “closed‐loop” therapeutic strategy may provide a guidance for the design of future antitumor nanoplatforms.

Effects of moxibustion on p53， SLC7A11， and GPX4 expression in synovial tissues of rats with adjuvant arthritis

To observe the effects of moxibustion on p53, cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in synovial tissues of rats with adjuvant arthritis (AA), so as to explore the mechanism of moxibustion in alleviating rheumatoid arthritis. Eighty rats were randomly divided into normal, model, moxibustion, and medication groups (n=20 in each group). The AA model was established by exposure to wind, cold, and damp environmental factors combined with injection of complete Freund's adjuvant. Rats in the moxibustion group received suspended moxibustion at "Shenshu" (BL23) and "Zusanli" (ST36) alternately, while those in the medication group were treated with tripterygium glycoside tablet suspension (8 mg/kg) by gavage, once a day, for 15 successive days. The pathological change in synovial tissue of rat right knee joint was observed by HE staining. The protein and mRNA expression levels of p53, SLC7A11, and GPX4 in the synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. The se-rum glutathione (GSH) and reactive oxygen species (ROS) contents were measured by colorimetry and fluorescence probe me-thod. Compared with the normal group, the model group exhibited synovial hyperplasia of the right knee joint, massive inflammatory cell infiltration, up-regulated mRNA and protein expression of p53 in synovial tissue, elevated serum ROS content (P<0.01), down-regulated mRNA and protein expression of SLC7A11 and GPX4 in synovial tissue, and lowered serum GSH content (P<0.01). Comparison with the model group showed that the synovial injuries in the moxibustion and medication groups were obviously alleviated. The mRNA and protein expression levels of p53 in the synovial tissues and the serum ROS content declined significantly (P<0.01), while the mRNA and protein expression of SLC7A11 and GPX4 in the synovial tissues and the se-rum GSH content increased (P<0.01). There was no significant difference in histopathological change of synovial tissue between the moxibustion group and medication group. However, the p53 protein expression in the synovial tissue and the level of serum ROS were significantly higher in the medication group than in the moxibustion group (P<0.05), while the GPX4 protein expression and serum GSH content were down-regulated (P<0.05). Moxibustion improves the inflammatory response in synovial tissue of AA model rats, which may be closely related to its regulation of the expression of ferroptosis-related factors.

Bufotalin induces ferroptosis in non-small cell lung cancer cells by facilitating the ubiquitination and degradation of GPX4

Ferroptosis is a new form of regulated cell death that is dependent on iron- and lipid reactive oxygen species. Emerging evidence indicate that induction of ferroptosis could inhibit the proliferation of diverse cancer cells, which functions as a potent tumor suppressor in cancer. Here, we firstly reported Bufotalin (BT), a natural small molecule, was a novel glutathione peroxidase 4 (GPX4) inhibitor, which could trigger the ferroptosis in non-small cell lung cancer cells. In vitro, BT significantly inhibited the proliferation of A549 cells and induced the ferroptosis, whereas ferroptosis inhibitor or iron chelator significantly reversed the cytotoxicity of BT on A549 cells. Moreover, BT also increased the intracellular Fe2+. Subsequently, immunoblotting showed that BT could inhibit the protein expression of GPX4. Notably, BT dramatically accelerated the degradation of GPX4 in A549 cells. Immunoprecipitation assay further certified the increased ubiquitination of GPX4 induced by BT. Nevertheless, BT could not further increase the lipid ROS after silencing of GPX4, suggesting the induction of ferroptosis by BT was dependent on GPX4. Furthermore, BT also observably inhibited tumor growth and promoted lipid peroxidation in vivo. In conclusion, our findings indicated that BT could induce ferroptosis and cause lipid peroxidation by accelerating the degradation of GPX4 and raising the intracellular Fe2+, and BT will hopefully serve as a lead compound in developing anti-tumor agents for targeting ferroptosis.

Lipopolysaccharide-induced splenic ferroptosis in goslings was alleviated by polysaccharide of atractylodes macrocephala koidz associated with proinflammatory factors

ABSRACTFerroptosis is a newly discovered form of cell death due to iron-dependent lipid peroxidation. In animal breeding, many environmental factors could lead to oxidative stress, which in turn reduce animal immunity and production performance. Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has antioxidation, immunomodulatory, and inflammatory modulating effects. For investigating the effect of PAMK on splenic ferroptosis in gosling caused by lipopolysaccharide (LPS), 40 one-day-old Magang goslings were randomly divided into 4 groups (CON group, LPS group, PAMK group, and LPS+PAMK group). The protein expression of the ferroptosis marker Glutathione Peroxidase 4 (GPX4), the relative mRNA expression of ferroptosis-related genes and cytokines, and the oxidative stress and iron content of spleen tissues were examined. The correlation between ferroptosis and inflammatory factors was further analyzed by principal component analysis. The results showed that, compared with CON group, LPS caused alterations in the expression of the ferroptosis pathway genes and cytokines, which could upregulate levels of ferroptosis and inflammation. However, after treated with PAMK, the inflammation and ferroptosis was alleviated. Meanwhile, PAMK restored the expression and distribution of GPX4. In addition, PAMK alleviated the oxidative stress caused by LPS and reduced the iron content in spleen. Principal component analysis showed that cytokines were more closely related to antioxidant indexes. The CON, PAMK and LPS+PAMK groups had similar effects on the four components, with the LPS and PAMK groups showing the furthest difference in results. The result indicated that PAMK could reduce the level of oxidative stress and inflammatory cytokines in spleen of gosling caused by LPS, and jointly alleviate ferroptosis by regulating genes related to the ferroptosis pathway.

Proteome Analysis of Camellia nitidissima Chi Revealed Its Role in Colon Cancer Through the Apoptosis and Ferroptosis Pathway.

Colon cancer is the third most common cancer in the world with a high mortality rate. At present, surgery combined with radiotherapy and chemotherapy is the primary treatment, but patient prognosis remains poor. Traditional Chinese medicine (TCM) has become a complementary and alternative source of anti-cancer drugs. Camellia nitidissima Chi (CNC) is a TCM used to treat a variety of cancers. However, the role of CNC in cancer remains elusive, and its effect and mechanism on colon cancer have not been reported. Here, we show that CNC exerts an excellent inhibitory effect on colon cancer proliferation and apoptosis induction in vitro and in vivo. We performed label free-based quantitative proteomic analysis to evaluate the HCT116 cells treated with CNC. Our data revealed a total of 363 differentially expressed proteins, of which 157 were up-regulated and 206 down-regulated. Gene Ontology enrichment analysis showed that these proteins were involved in tumor occurrence and development through multiple biological processes such as cell proliferation, cell apoptosis, cell cycle, and cell death. Interestingly, we also found significant changes in ferroptosis pathways. The role of essential proteins glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HMOX1) were verified. CNC decreased the expression of GPX4 and increased the expression of HMOX1 at the mRNA and protein levels in vivo and in vitro. Collectively, these findings reveal that CNC regulates colon cancer progression via the ferroptosis pathway and could be an attractive treatment for colon cancer.

Prognostic and Immunological Role of Key Genes of Ferroptosis in Pan-Cancer.

Solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and apoptosis inducing factor mitochondria associated 2 (AIFM2) are the key regulators in ferroptosis. However, the expression patterns and prognostic roles of these genes in pan-cancer are still largely unclear. The expression patterns and prognostic roles of SLC7A11, GPX4, and AIFM2 and the relationships between the expression levels of these genes and immune infiltration levels in pan-cancer were analyzed by using TIMER, gene expression profiling interactive analysis (GEPIA), Oncomine, and Kaplan–Meier databases. Our results showed that both SLC7A11 and GPX4 were overexpressed in colorectal cancer, and SLC7A11 was overexpressed in lung cancer. High levels of SLC7A11 and AIFM2 were significantly linked with the shortened disease-free survival and overall survival (OS) in adrenocortical carcinoma (ACC), respectively. And high expression of SLC7A11, GPX4, and AIFM2 were significantly correlated with the shortened OS of acute myeloid leukemia patients. In esophageal carcinoma (ESCA), GPX4 expression was significantly associated with the infiltration of macrophage and myeloid dendritic cell, and AIFM2 expression was significantly associated with the infiltration of CD4+ T cell. Importantly, GPX4 expression was positively correlated with the expression levels of monocyte markers (CD14 and CD115) and M2 macrophage markers (VSIG4 and MS4A4A) both in ESCA and in head and neck squamous cell carcinoma (HNSC). In summary, SLC7A11, GPX4, and AIFM2 are dysregulated in many types of cancers, and are candidate prognostic biomarkers for many types of cancers, and can be used to evaluate the infiltration of immune cells in tumor tissues.

Activation of Nrf2 signaling protects hypoxia-induced HTR-8/SVneo cells against ferroptosis.

This article aims to investigate the possible mechanism by which nuclear factor erythroid 2-related factor 2 (Nrf2) signaling exerts protective effects on the progression of preeclampsia (PE). HTR-8/SVneo cells were cultured under hypoxic conditions to establish PE cell model. Cell viability and invasion were respectively examined by CCK-8 assay and transwell assay. The detection of oxidative stress and Fe2+ content was carried out by corresponding commercial kits, and the expression of corresponding proteins and genes was detected by western blot and qPCR. After Nrf2 was overexpressed in HTR-8/SVneo cells, the above assays were conducted to confirm the exact effects of Nrf2 activation on ferroptosis. Hypoxia reduced the invasion of HTR-8/SVneo cells, induced oxidative stress (upregulated levels of glutathione [GSH], malondialdehyde [MDA], and reactive oxygen species [ROS]), and induced ferroptosis which exhibited by upregulated Fe2+ and downregulated expression of solute carrier family 7 member 11/System xCT (SCL7A11), glutathione peroxidase 4 (GPX4), and ferroportin1 (FPN1). Meanwhile, hypoxia promoted the translocation of Nrf2 to the nucleus, leading to Nrf2/HO-1 signaling activation. These changes upon hypoxia induction were strengthened by ferric ammonium citrate (FAC) but were abolished by deferoxamine (DFO). Moreover, Nrf2 overexpression in hypoxia-induced HTR-8/SVneo cells exerted inhibitory effects on levels of GSH, MDA, ROS, and Fe2+ , and promotive effects on Nrf2/HO-1 signaling activation and expression of SCL7A11, GPX4, and FPN1, indicating that Nrf2 overexpression decreased oxidative stress and ferroptosis in hypoxia-induced HTR-8/SVneo cells. Collectively, activation of Nrf2 signaling alleviated hypoxia-induced invasion lacking, oxidative stress, and ferroptosis in hypoxia-induced HTR-8/SVneo cells, suggesting that Nrf2 signaling activation play a protective role in PE, which can provide instructions for the targeted treatment of PE.

Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4.

BackgroundLiver cancer ranks the top four malignant cancer type worldwide, which needs effective and safe treatment. Ferroptosis is a novel form of regulated cell death driven by iron-dependent lipid peroxidation and has been regarded as a promising therapeutic target for cancers. In this work, we aimed to study the effects of anesthetic ketamine on proliferation and ferroptosis of liver cancer.MethodsCell viability and proliferation were detected by cell counting kit 8 (CCK-8), colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) assay. Ferroptosis was determined by levels of Fe2+, lipid reactive oxygen species (ROS), and malondialdehyde (MDA). RNA levels of lncPVT1, miR-214-3p, and glutathione peroxidase 4 (GPX4) were checked by real-time PCR assay. Clinical liver tumor samples were collected to detect the levels of long noncoding RNA lncPVT1, miR-214-3p, and GPX4, and their correlation was evaluated by Pearson comparison test. Luciferase reporter gene assay and RNA pulldown were conducted to determine the binding between lncPVT1, miR-214-3p, and GPX4 3ʹUTR.ResultsKetamine significantly suppressed viability and proliferation of liver cancer cells both in vitro and in vivo, as well as stimulated ferroptosis, along with decreased expression of lncPVT1 and GPX4. LncPVT1 directly interacted with miR-214-3p to impede its role as a sponge of GPX4. Depletion of lncPVT1 accelerated the ferroptosis of live cancer cells, whereas miR-214-3p inhibition and GPX4 overexpression reversed this effect. Ketamine-induced cell growth suppression and ferroptosis were also suppressed by miR-214-3p inhibition and GPX4 overexpression.ConclusionIn this work, we determined that ketamine suppressed viability of liver cancer cells and induced ferroptosis and identified the possible regulatory mechanism of lncPVT1/miR-214-3p/GPX4 axis.

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.