GPX1 Expression Research Articles

Depletion of SLC7A11 Sensitizes Nasopharyngeal Carcinoma Cells to Ionizing Radiation.

Radiotherapy is the primary treatment choice for Nasopharyngeal Carcinoma (NPC). However, its efficacy is compromised due to radioresistance. Ferroptosis, a novel iron-dependent regulated cell death induced by Ionizing Radiation (IR), plays a role in promoting cancer cell death. Yet, the relationship between enhanced ferroptosis and increased sensitivity of NPC cells to IR remains poorly understood. This study aimed to explore the association between IR and ferroptosis in NPC, as well as the role of the ferroptosis repressor SLC7A11 in IR-treated NPC cells. CNE1 and HNE-2 NPC cells were subjected to IR treatment. We performed qPCR and western blotting to evaluate the expression of ferroptosis-related genes in both control and IR-treated NPC cells. Additionally, we used the MTT assay to measure the viability of these NPC cells. JC-1 and DCFH-DA staining were employed to assess mitochondrial membrane potential and Reactive Oxygen Species (ROS) levels in both control and IR-treated NPC cells. Furthermore, we examined the levels of Fe2+, Malondialdehyde (MDA), reduced Glutathione (GSH), and oxidized glutathione (GSSG) in these cells. Moreover, we depleted SLC7A11 in IR-treated NPC cells to investigate its impact on the ferroptosis of these cells. IR upregulated the expression of ferroptosis-related genes, including SLC7A11, ACSL4, COX2, FTH1, and GPX4, in CNE1 and HNE-2 cells. IR treatment also resulted in decreased cell viability, disrupted mitochondrial membrane potential, increased ROS levels, altered glutathione levels, and elevated Fe2+ levels. Knockdown of SLC7A11 enhanced the sensitivity of NPC cells to IR. IR may induce ferroptosis in NPC cells, and stimulating ferroptosis could potentially serve as a therapeutic strategy to enhance the efficacy of IR in treating NPC patients.

Turmeric Extract-loaded Selenium Nanoparticles Counter Doxorubicin-induced Hepatotoxicity in Mice via Repressing Oxidative Stress, Inflammatory Cytokines, and Cell Apoptosis.

Doxorubicin (DOX) is an antitumor anthracycline used to treat a variety of malignancies; however, its clinical use is associated with noticeable hepatotoxicity. Therefore, the current study was designed to delineate if biosynthesized SeNPs with turmeric extract (Tur-SeNPs) could alleviate DOX-induced hepatic adverse effects. Mice were orally post-treated with Tur extract, Tur-SeNPs, or N-acetyl cysteine after the intraperitoneal injection of DOX. Our findings have unveiled a remarkable liver attenuating effect in DOX-injected mice post-treated with Tur-SeNPs. High serum levels of ALT, AST, ALP, and total bilirubin induced by DOX were significantly decreased by Tur-SeNPs therapy. Furthermore, Tur-SeNPs counteracted DOX-caused hepatic oxidative stress, indicated by decreased MDA and NO levels along with elevated levels of SOD, CAT, GPx, GR, GSH, and mRNA expression levels of Nrf-2. Noteworthily, decreased hepatic IL-1β, TNF-α, and NF-κB p65 levels in addition to downregulated iNOS gene expression in Tur-SeNPs-treated mice have indicated their potent antiinflammatory impact. Post-treatment with Tur-SeNPs also mitigated the hepatic apoptosis evoked by DOX injection. A liver histological examination confirmed the biochemical and molecular findings. In brief, the outcomes have demonstrated Tur loaded with nanoselenium to successfully mitigate the liver damage induced by DOX via blocking oxidative stress, and inflammatory and apoptotic signaling.

Significance of dietary quinoa husk (Chenopodium quinoa) in gene regulation for stress mitigation in fish

The persistent challenges posed by pollution and climate change are significant factors disrupting ecosystems, particularly aquatic environments. Numerous contaminants found in aquatic systems, such as ammonia and metal toxicity, play a crucial role in adversely affecting aquaculture production. Against this backdrop, fish feed was developed using quinoa husk (the byproduct of quinoa) as a substitute for fish meal. Six isonitrogenous diets (30%) and isocaloric diets were formulated by replacing fish meal with quinoa husk at varying percentages: 0% quinoa (control), 15, 20, 25, 30 and 35%. An experiment was conducted to explore the potential of quinoa husk in replacing fish meal and assess its ability to mitigate ammonia and arsenic toxicity as well as high-temperature stress in Pangasianodon hypophthalmus. The formulated feed was also examined for gene regulation related to antioxidative status, immunity, stress proteins, growth regulation, and stress markers. The gene regulation of sod, cat, and gpx in the liver was notably upregulated under concurrent exposure to ammonia, arsenic, and high-temperature (NH3 + As + T) stress. However, quinoa husk at 25% downregulated sod, cat, and gpx expression compared to the control group. Furthermore, genes associated with stress proteins HSP70 and DNA damage-inducible protein (DDIP) were significantly upregulated in response to stressors (NH3 + As + T), but quinoa husk at 25% considerably downregulated HSP70 and DDIP to mitigate the impact of stressors. Growth-responsive genes such as myostatin (MYST) and somatostatin (SMT) were remarkably downregulated, whereas growth hormone receptor (GHR1 and GHRβ), insulin-like growth factors (IGF1X, IGF2X), and growth hormone gene were significantly upregulated with quinoa husk at 25%. The gene expression of apoptosis (Caspase 3a and Caspase 3b) and nitric oxide synthase (iNOS) were also noticeably downregulated with quinoa husk (25%) reared under stressful conditions. Immune-related gene expression, including immunoglobulin (Ig), toll-like receptor (TLR), tumor necrosis factor (TNFα), and interleukin (IL), strengthened fish immunity with quinoa husk feed. The results revealed that replacing 25% of fish meal with quinoa husk could improve the gene regulation of P. hypophthalmus involved in mitigating ammonia, arsenic, and high-temperature stress in fish.

Metabolomics analysis revealed the neuroprotective role of 2-phosphoglyceric acid in hypoxic-ischemic brain damage through GPX4/ACSL4 axis regulation

Hypoxic-ischemic brain damage (HIBD) is a cerebral injury resulting from the combination of ischemia and hypoxia in neonatal brain tissue. Presently, there exists no efficacious remedy for HIBD. A mounting body of evidence indicates that dynamic metabolites formed during metabolic procedures assume a vital role in neuronal maturation and recuperation. However, it remains unclear whether any endogenous metabolites are involved in the pathogenesis of HIBD. Here, an untargeted metabolomics analysis was conducted by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry (GC/LC-MS) in OGD/R (oxygen-glucose deprivation/reoxygenation)-induced HT-22 cells. We observed that ferroptosis signaling plays an essential role in HI-induced neuronal injury. Interestingly, we also found that the differentially expressed metabolite, 2-phosphoglyceric acid, significantly improved the neuronal cell survival of OGD/R HT-22 cells by inhibiting ferroptosis. Moreover, 2-phosphoglyceric acid effectively rescued the cell activity of HT-22 cells treated with the ferroptosis inducer RSL-3. Furthermore, 2-phosphoglyceric acid alleviated cerebral infarction and reduced HIBD-induced neuronal cell loss of the central nervous system in neonatal rats by regulating GPX4 expression. Taken together, we found that 2-phosphoglyceric acid, which was downregulated in HT-22 cells induced by OGD/R, exerted neuronal protective effects on OGD/R-treated HT-22 cells and HIBD-induced neonatal rats by inhibiting hypoxic-ischemic-induced ferroptosis through the regulation of the GPX4/ACSL4 axis.

Lipoxin A4 ameliorates knee osteoarthritis progression in rats by antagonizing ferroptosis through activation of the ESR2/LPAR3/Nrf2 axis in synovial fibroblast-like synoviocytes

BackgroundOur previous studies have shown that lipoxin A4 (LXA4) can serve as a potential biomarker for assessing the efficacy of exercise therapy in knee osteoarthritis (KOA), and fibroblast-like synoviocytes (FLSs) may play a crucial role in KOA pain as well as in the progression of the pathology. ObjectiveBy analyzing the GSE29746 dataset and collecting synovial samples from patients with different Kellgren–Lawrence (KL) grades for validation, we focused on exploring the potential effect of LXA4 on ferroptosis in FLSs through the ESR2/LPAR3/Nrf2 axis to alleviate pain and pathological advancement in KOA. MethodsThe association between FLSs ferroptosis and chondrocyte matrix degradation was explored by cell co-culture. We overexpressed and knocked down LPAR3 in vitro to explore its potential mechanism in FLSs. A rat model of monosodium iodoacetate (MIA)-induced KOA was constructed and intervened with moderate-intensity treadmill exercise and intraperitoneal injection of PHTPP to investigate the effects of the LXA4 intracellular receptor ESR2 on exercise therapy. ResultsESR2, LPAR3, and GPX4 levels in the synovium decreased with increasing KL grade. After LXA4 intervention in the co-culture system, GPX4, LPAR3, and ESR2 were upregulated in FLSs, collagen II was upregulated in chondrocytes, and MMP3 and ADAM9 were downregulated. LPAR3 overexpression upregulated the expression of GPX4, Nrf2, and SOD1 in FLSs, while downregulating the expression of MMP13 and MMP3; LPAR3 knockdown reversed these changes. Moderate-intensity platform training improved the behavioral manifestations of pain in KOA rats, whereas PHTPP treatment partially reversed the improvement in synovial and cartilage pathologies induced by platform training. ConclusionLXA4 inhibited FLSs ferroptosis by activating the ESR2/LPAR3/Nrf2 axis, thereby alleviating the pain and pathological progression of KOA. This study brings a new target for the treatment of KOA and also leads to a deeper understanding of the potential mechanisms of exercise therapy for KOA.

Ferroptosis Mediates Pulmonary Fibrosis: Implications for the Effect of Astragalus and Panax notoginseng Decoction.

To investigate the mechanism through which Astragalus and Panax notoginseng decoction (APD) facilitates the treatment of ferroptosis-mediated pulmonary fibrosis. First, the electromedical measurement systems were used to measure respiratory function in mice; the lungs were then collected for histological staining. Potential pharmacologic targets were predicted via network pharmacology. Finally, tests including immunohistochemistry, reverse transcription-quantitative polymerase chain reaction, and western blotting were used to evaluate the relative expression levels of collagen, transforming growth factor β, α-smooth muscle actin, hydroxyproline, and ferroptosis-related genes (GPX4, SLC7A11, ACSL4, and PTGS2) and candidates involved in the mediation of pathways associated with ferroptosis (Hif-1α and EGFR). APD prevented the occurrence of restrictive ventilation dysfunction induced by ferroptosis. Extracellular matrix and collagen fiber deposition were significantly reduced when the APD group compared with the model group; furthermore, ferroptosis was attenuated, expression of PTGS2 and ACSL4 increased, and expression of GPX4 and SLC7A11 decreased. In the APD group, the candidates related to the mediation of ferroptosis (Hif-1α and EGFR) decreased compared with the model group. Discussion and Conclusions. APD may ameliorate restrictive ventilatory dysfunction through the inhibition of ferroptosis. This was achieved through the attenuation of collagen deposition and inflammatory recruitment in pulmonary fibrosis. The underlying mechanisms might involve Hif-1α and EGFR.

Role of ferroptosis in Porphyromonas gingivalis-induced impairment of epithelial junction

ABSTRACT Objectives This study aims to clarify the effect of ferroptosis by P. gingivalis on periodontal epithelium impairment and potential mechanisms. Materials and methods The expression of epithelial junction proteins (CDH1, OCLN, ZO-1), FTL and GPX4 in healthy and periodontitis tissues was analyzed using bioinformatics analysis and validated in vivo. An in vitro model was constructed to evaluate ferroptosis by mitochondria morphology, content of iron and GSH, and level of lipid peroxidation, FTL, GPX4 and SLC7A11. The iron concentration was changed with iron chelator DFO and iron supplementation FAC. The epithelial impairment was assessed by protein expression. To investigate the mechanism, si-MYB (a negative transcription factor of SLC7A11) and GPX4 inhibitor RSL3 were employed. Results CDH1, OCLN, ZO-1 and GPX4 expression was decreased, while FTL expression was elevated in periodontitis tissues. Infected cells showed ferroptosis change of the mitochondria with higher level of lipid peroxidation, iron, FTL and lower level of GPX4, GSH, SLC7A11. FAC augmented ferroptosis and weakened epithelial junction, while DFO exhibited a counteractive effect. Silencing MYB rescued SLC7A11, GPX4 and epithelial junction proteins, which was hindered by RSL3. Conclusions Our study demonstrated that P. gingivalis weakened the oral epithelial barrier by causing ferroptosis via inhibiting SLC7A11/GSH/GPX4 axis.

Camphene as a Protective Agent in Myocardial Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion injury (I/R) and the resulting heart failure is one of the main causes of mortality and morbidity worldwide. Camphene has been shown to have anti-inflammatory and hypolipidemic properties; however, its role in the protection of the heart from ischemia and reperfusion has not been investigated. The cardioprotective role of camphene and the mechanism that mediates its action against I/R injury was evaluated in the present study. A single dose of camphene was administered in adult rats prior to ex vivo I/R induction. Infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining and cardiomyocyte injury was assessed by determining the release of the enzyme lactate dehydrogenase (LDH). Camphene pretreatment provided significant protection reducing myocardial infarct size and cell death after I/R. The effect was correlated with the reduction in oxidative stress as evidenced by the determination of protein carbonylation, GSH/GSSG ratio, the increase in mitochondrial content as determined by CS activity, and the modulation of antioxidant defense mechanisms (expression of Nrf2 and target genes and activities of CAT, MnSOD, and GR). Furthermore, ferroptosis was decreased, as demonstrated by downregulation of GPx4 expression and reduction in lipid peroxidation. The results suggest that camphene can protect the heart against I/R injury by maintaining redox homeostasis and can hold therapeutic potential for mitigating the detrimental effects of I/R in the heart.

Sex-specific vulnerabilities in human astrocytes underpin the differential impact of palmitic acid

Obesity and neurometabolic diseases have been linked to neurodegenerative diseases. Our hypothesis is that the endogenous estrogenic component of human astrocytes plays a critical role in cell response during lipotoxic damage, given that obesity can disrupt hormonal homeostasis and cause brain inflammation. Our findings showed that high concentrations of palmitic acid (PA) significantly reduced cell viability more in male astrocytes, indicating sex-specific vulnerabilities. PA induced a greater increase in cytosolic reactive oxygen species (ROS) production in males, while female astrocytes exhibited higher superoxide ion levels in mitochondria. In addition, female astrocytes treated with PA showed increased expression of antioxidant proteins, including catalase, Gpx-1 and Nrf2 suggesting a stronger cellular defence mechanism. Interestingly, there was a difference in the expression of estrogenic components, such as estrogen, androgens, and progesterone receptors, as well as aromatase and 5α-reductase enzymes, between males and females. PA induced their expression mainly in females, indicating a potential protective mechanism mediated by endogenous hormones. In summary, our findings highlight the impact of sex on the response of human astrocytes to lipotoxicity. Male astrocytes appear to be more susceptible to cellular damage when exposed to high concentrations of fatty acids.

Protective Effect of Selenium-enriched Peptide from Cardamine violifolia on Ethanol-induced L-02 Hepatocyte Injury.

In this study, we investigated the protective effect of selenium (Se)-enriched peptide isolated from Cardamine violifolia (SPE) against ethanol-induced liver injury. Cell proliferation assays show that different concentrations of SPE protect human embryonic liver L-02 cells against ethanol-induced injury in a dose-dependent manner. Treatment with 12 μmol/L Se increases the cell survival rate (82.44%) and reduces the release of alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, and apoptosis rate. SPE treatment with 12 μmol/L Se effectively reduces the concentration of intracellular reactive oxygen species and increases the contents of intracellular superoxide dismutase (51.64 U/mg), catalase (4.41 U/mg), glutathione peroxidase (1205.28 nmol/g), and glutathione (66.67 μmol/g), thereby inhibiting the effect of ethanol-induced oxidative damage. The results of the transcriptomic analysis show that the glutathione metabolism and apoptotic pathway play significant roles in the protection of L-02 hepatocytes by SPE. Real-time qPCR analysis shows that SPE increases the mRNA expression of GPX1 and NGFR. The results of this study highlight the protective effects of SPE against ethanol-induced liver injury.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

Systemic Oxidative Balance Reflects the Liver Disease Progression Status for Primary Biliary Cholangitis (Pbc): The Narcissus Fountain.

Biological antioxidant potential (BAP) and Reactive Oxygen Metabolites (dROMs) are two tests complementarily assessing systemic oxidative statuses (SOSs) that are never applied in chronic liver disorders (CLDs). We enrolled 41 ursodeoxycholic acid (UDCA)-naïve Primary Biliary Cholangitis (PBC) patients [age: 58.61 ± 11.26 years; females (F): 39], 40 patients with metabolic-dysfunction-associated steatotic livers (age: 54.30 ± 11.21; F: 20), 52 patients with HBV (age: 52.40 ± 8.22; F: 34), 50 patients with (age: 56.44 ± 7.79, F: 29), and 10 controls (age: 52.50 ± 9.64; F: 7). Liver fibrosis and the steatosis severity were determined using transient elastography, and the SOS was balanced using d-ROMs and the BAP test. The gene expressions of superoxide dismutase (SOD1; SOD2) and glutathione peroxidase (GPx1) were evaluated using real-time PCR in advanced fibrosis (AF: F3F4) in patients with PBC. In contrast to other CLDs, in PBC the dROMs and BAP levels were, respectively, directly and inversely correlated with hepatic fibrosis (dROMs, R: 0.883; BAP, R: -0.882) and steatosis (dROMs, R: 0.954; BAP, R: -0931) severity (p < 0.0001 all). Patients with PBC also revealed a progressively increasing trend of d-ROMs (F0-F2 vs. F3: p = 0.0008; F3 vs. F4: p = 0.04) and reduction in BAP levels (F0-F2 vs. F3: p = 0.0007; F3 vs. F4 p = 0.04) according to the worsening of liver fibrosis. In AF-PBC, the SOD1, SOD2, and GPx1 expressions were significantly downregulated in patients presenting SOS imbalance (SOD1, p = 0.02; SOD2, p = 0.03; GPx1, p = 0.02). SOS disequilibrium represents a leitmotiv in patients with PBC, perfectly reflecting their liver disease progression status.

Eldecalcitol protected osteocytes against ferroptosis of D-gal-induced senescent MLO-Y4 cells and ovariectomized mice

BackgroundActive vitamin D analog eldecalcitol is clinically applied in treatment of postmenopausal osteoporosis. This study aims to determine the role of eldecalcitol in the protection of osteocytes from senescence and the associated ferroptosis. MethodsThe MLO-Y4 osteocytes were exposed to D-gal inducing senescence. The ovariectomized (OVX) mice treated with D-gal using as an aging inducer were intraperitoneally injected with eldecalcitol. The multiplexed confocal imaging, fluorescence in situ hybridization and transmission electron microscopy were applied in assessing osteocytic properties. Immunochemical staining and immunoblotting were carried out to detect abundance and expression of molecules. ResultsThe ablation of vitamin D receptor led to a reduction in amounts of osteocytes, a loss of dendrites, an increase in mRNA expression of SASP factors and in protein expression of senescent factors, as well as changes in mRNA expression of ferroptosis-related genes (PTGS2 & RGS4). Eldecalcitol reversed senescent phenotypes of MLO-Y4 cells shown by improving cell morphology and density, decreasing β-gal-positive cell accumulation, and down-regulating protein expression (P16, P21 & P53). Eldecalcitol reduced intracellular ROS and MDA productions, elevated JC-1 aggregates, and up-regulated expression of Nrf2 and GPX4. Eldecalcitol exhibited osteopreserve effects in D-gal-induced aging OVX mice. The confocal imaging displayed its improvement on osteocytic network organization. Eldecalcitol decreased the numbers of senescent osteocytes at tibial diaphysis by SADS assay and attenuated mRNA expression of SASP factors as well as down-regulated protein expression of senescence-related factors and restored levels of ferroptotic biomarkers in osteocytes-enriched bone fraction. It reduced 4-HNE staining area, stimulated Nrf2-positive staining, and promoted nuclear translocation of Nrf2 in osteocytes of mice as well as inhibited and promoted protein expression of 4-HNE and Nrf2, respectively, in osteocytes-enriched bone fraction. ConclusionsThe present study revealed the ameliorative effects of eldecalcitol on senescence and the associated ferroptosis of osteocytes, contributing to its preservation against osteoporosis of D-gal-induced senescent ovariectomized mice.

Ferroptosis and iron mineralization involved in the death and survival of orange‐spotted groupers challenged with Pseudomonas plecoglossicida

AbstractPseudomonas plecoglossicida (P. plecoglossicida) is a pathogen in aquaculture that causes considerable economic loss. According to artificial infection experiments, the fish were classified into control group, moribund group, and survival group. Compared to the control group, both the moribund group and the survival group of fish had fewer red blood cells (RBCs) and lower oxygen saturation (SaO2). Furthermore, the fish in the survival group has more RBCs and SaO2 compared to the moribund group. The concentrations of total iron, ferrous iron, ferric iron, and mineralized iron in the fish spleen of the moribund and survival groups were lower compared to those of the control group. Additionally, the concentrations of these iron components in the fish spleen of the survival group were higher than those of the moribund group. The results demonstrated that iron mineralization is involved in the survival of fish challenged with P. plecoglossicida. Compared to the control and survival groups, the fish spleen had several distinguishing features in the moribund group, including less reduced glutathione (GSH), higher mitochondrial complex V activity, more lipid peroxidation, and reactive oxygen species, as well as reduced glutathione peroxidase 4 (gpx4) expression. Moreover, there were intact cell membranes, a normal nucleus size, no chromatin concentration, and disappearance of cristae in the mitochondria of the spleens of the moribund group. The characteristics of spleen cells in the moribund group were consistent with ferroptosis, suggesting that ferroptosis was involved in the death of fish challenged with P. plecoglossicida.

Abstract 4459: Metabolic liabilities of iron-dependent ferroptosis mediated through NRF2/GPX4 axis in osteosarcoma

Abstract Osteosarcoma is an aggressive bone malignancy that predominantly affects adolescents and young adults. Despite recent advancements in treatments, the challenges of recurrence and chemoresistance persist. Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation, and emerging evidence suggests significant roles for NRF2 and GPX4 in iron metabolism, regulating tumor cell functions. In osteosarcoma, however, the molecular mechanisms of the NRF2/GPX4 axis and its therapeutic potential remain unknown. In this study, we first determined GPX4 and xCT expression levels and cellular responses to RSL3, a GPX4 inhibitor, and Erastin, an xCT inhibitor in osteosarcoma cells. A total of seven osteosarcoma cell lines, consisting of five human cell lines (HOS, 143B, Saos-2, MG-63, U2OS) and two canine cell lines (COS31 and DOUG), were used. Our results revealed that the IC50 values ranged from 0.02 uM to 1.01 uM in cells treated with RSL3 and from 0.35 uM to 1.41 uM in cells treated with Erastin, respectively. These values were proportional to GPX4 and xCT levels across the cell lines. Then, U2OS, as the most sensitive, and Saos-2, the least sensitive based on their IC50 values, were selected to examine the iron regulatory machinery. In U2OS cells, RSL3 treatment significantly decreased NRF2 and GPX4, while increasing ferroportin-1 and ferritin. GPX4 knockdown also led to comparable changes in NRF2, ferroportin-1, and ferritin expression. NRF2 appears to mediate iron metabolism, ferroportin-1 acts as an iron exporter, and ferritin stores intracellular iron. Therefore, our findings suggest that GPX4-mediated ferroptosis is regulated by iron metabolic pathways, potentially to prevent ferroptotic cell death. We also found that Ferrostatin-1, a lipid peroxidation inhibitor showed no changes in ferroportin-1, ferritin, and NRF2. In Saos-2, RSL3 induced a remarkable reduction in GPX4 expression, akin to U2OS. However, no significant change was found in NRF2, ferroportin-1, and ferritin. Instead, Ferrostatin-1 restored the decreased GPX4 and increased NRF2, ferroportin-1, and ferritin levels. In addition, Z-VAD (a pan-caspase inhibitor), Necrosatin-1s (a RIPK1 inhibitor), and Ferrostatin-1 were capable of preventing Saos-2, COS31, and DOUG cells from RSL3-induced cell death at lethal doses. Such protection was undetected in U2OS. These data suggest that metabolic pathways controlling intracellular iron are crucial for NRF2/GPX4-mediated ferroptosis in osteosarcoma cells. Furthermore, we generated RNA-seq and LC-MS/MS data to identify novel molecular targets governing the metabolic pathways of ferroptosis in osteosarcoma. Our ongoing work includes downstream molecular and functional assays following gene modifications in osteosarcoma cells (overexpression and knockout) and assessing metabolic vulnerabilities by targeting iron-mediated ferroptosis in xenografts. Citation Format: Md Abdullah, Donghee Lee, Rong Li, Jong Hyuk Kim. Metabolic liabilities of iron-dependent ferroptosis mediated through NRF2/GPX4 axis in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4459.

Abstract 1562: HMGA2 regulates GPX4 expression and oxidative stress

Abstract Prostate cancer (PCa) is a leading cause of mortality, primarily due to its ability to metastasize to the bone. The High Mobility Group AT-Hook 2 (HMGA2) plays a crucial role in regulating gene expression and has been implicated in tumorigenesis and the metastatic process. Our recent study revealed that overexpression of the wild-type/full-length HMGA2 isoform in PCa cells promotes cancer progression by triggering epithelial mesenchymal transition (EMT), whereas truncated HMGA2 isoform promotes progression through oxidative stress signaling. We hypothesize that HMGA2 regulates GPX4 expression and oxidative stress in PCa. We studied the expression of HMGA2 and GPX4 in various PCa cell lines including enzalutamide resistant cell line (C4-2B MDVR), LNCaP cells overexpressing HMGA2 isoforms (wild type and truncated HMGA2). Our analysis of HMGA2 and GPX4 expression in diverse PCa cell lines, shows an inverse relationship between HMGA2 and GPX4 levels. Elevated HMGA2 expression coincides with reduced GPX4 expression, leading to heightened oxidative stress and susceptibility to ferroptosis. Moreover, treatment of enzalutamide resistant cell line C4-2B MDVR with ferroptosis inducer RSL3, further suppresses GPX4 expression, resulting in decreased cell proliferation. Our results unveils the intricate relationship between HMGA2, GPX4 regulation and oxidative stress in the context of prostate cancer progression. Exploiting this oxidative stress pathway offers a promising therapeutic approach for cancer treatment, especially for enzalutamide-resistant cancer cells. Acknowledgements: These studies were supported by NIH/NIGMS/RISE SR25GM060414 and NIH/NIMHD 2U54MD007590; 5U54MD013376. Citation Format: Precious Elechi Dike, Taaliah Campbell, Mojisoluwa Awolowo, Valerie Odero-Marah. HMGA2 regulates GPX4 expression and oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1562.

Abstract 6002: Increased ferroptosis sensitivity and epithelial to mesenchymal transition of breast cancer cells overcoming chemotherapeutic mediated apoptotic caspase activation

Abstract The survival of cancer cells post chemotherapeutic treatment can lead to the presence of recurrent tumors and continues to be a barrier to effective cancer treatment. The majority of chemotherapeutics kill cells through the induction of executioner caspases and subsequent apoptotic death, and resistance to apoptosis can lead to the presence of anastatic cancer cells. Executioner caspase release was previously thought to be the point of no return from apoptotic cell death, however, it has been shown that removal of the reagent causing caspase release can lead to recovery of cells from apoptotic signaling, a phenomenon which has been termed ``Anastasis``. This presents a need for a better understanding of the underlying mechanisms behind cancer cell survival and the mechanisms that lead to cell recovery and recurrent tumors. We have hypothesized that when treated with chemotherapeutics, some cells will evade cell death even when executioner caspases have been activated. By identifying surviving anastatic cells and the pathways involved in their evasion of cell death, we hope to propose novel therapeutic strategies to prevent their survival. The novel CasExpress system, developed by the Denise Montell lab group, was used to identify and isolate a population of cells surviving caspase-3 activation as a result of chemotherapeutic treatment in Triple Negative Breast Cancer (TNBC) cell lines. The CasExpress system permanently labels these cells with GFP post caspase-3 activation. Analysis of this population of SUM159 anastatic cells, identified by their GFP expression, demonstrates that these cells have an increased resistance to further chemotherapeutic treatment, a decrease in levels of active caspase-3, an up-regulation of Cancer Stem Cell marker CD44, and a more mesenchymal phenotype. Furthermore, these “anastatic cells” express decreased levels of GPX-4, an enzyme that mitigates ferroptosis via lipid peroxide reduction, and are more sensitive to GPX-4 and xCT inhibitor mediated ferroptosis cell death.To investigate the link between Epithelial to Mesenchymal Transition (EMT) and ferroptosis mediated cell death, we used TGF-b to stimulate EMT in NMe mouse epithelial cells. We found that TGF-b induced EMT is associated with increased sensitivity to ferroptosis mediated by GPX-4 inhibition. Furthermore, the TGF-b induced EMT cells have a marked decrease in the expression of GPX4, similar to what was observed in the TNBC anastatic cells. These results indicate a potential link between EMT and an increased sensitivity to ferroptosis, and provide novel strategies for identifying and targeting chemotherapeutic resistant tumor cells. Citation Format: Rachel Hausman, Wells Brown, Paul McDonald, Shannon Awrey, Gongping Sun, Denise Montell, Shoukat Dedhar. Increased ferroptosis sensitivity and epithelial to mesenchymal transition of breast cancer cells overcoming chemotherapeutic mediated apoptotic caspase activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6002.

Abstract 2550: Investigating glutathione peroxidases as potential prognostic factors in uterine corpus endometrial cancer

Abstract Introduction: Uterine corpus endometrial cancer (UCEC) is one of the deadliest women’s reproductive cancers in the United States, and mortality rates have not improved in the last 30 years due to poor prognostic methodologies. Altered expression patterns of Glutathione Peroxidases (GPX1-8) have been connected to the outcome of many cancers. Still, GPX expression patterns and their use as prognostic biomarkers in UCEC have yet to be explored. This project aimed to evaluate the potential utility of GPX1-8 as prognostic biomarkers for UCEC. Methods: To identify GPX1-8 expression alterations in UCEC tumors, the UCEC tissue samples (n=543) from the Cancer Genome Atlas Database (TCGA) dataset were analyzed using the University of Alabama at Birmingham Cancer Data Analysis portal (UALCAN) software to statistically compare mRNA levels between UCEC tumor samples and non-tumorigenic uterine tissue. To determine the impacts of GPX1-8 expression on overall survival (OS) and relapse-free survival (RFS), the UCEC TCGA dataset was analyzed using the pan-cancer RNA seq platform using Kaplan-Meier Plotter (KM plotter). The patient cohort was split into groups according to high GPX expression status and low GPX expression status using the trichotomization function in KM plotter. Hazard ratio (HR), 95% confidence interval (95% CI), and log-rank P value were calculated using the KM plotter platform. Results: GPX1, GPX2, GPX4, GPX7, and GPX8 were differentially expressed in UCEC tumor tissue compared to non-cancerous uterine tissue. However, unfavorable OS in patients associated with low GPX4 expression in UCEC tumor samples (P=0.0043, HR=0.39, 95% CI=0.2-0.76) only. Unfavorable RFS in UCEC patients only correlated with low GPX1 expression (P=0.019, HR=0.43, 95% CI=0.21-0.89) or high GPX6 expression (P=0.0032, HR=2.37, 95% CI=1.31-4.27) in UCEC tumor samples. Conclusion: Low GPX1 and GPX4 expression and high GPX6 expression may be novel unfavorable UCEC prognostic markers. Citation Format: Alexis Dukesherer, Calli A. Davison-Versagli. Investigating glutathione peroxidases as potential prognostic factors in uterine corpus endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2550.

Abstract 3322: Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling

Abstract Introduction: Pancreatic cancer (PC) is a malignancy with poor prognosis and high mortality with a very low 5-year survival rate. Currently, apoptosis and autophagy are the central mechanisms of programmed cell death (PCD) that maintain cellular homeostasis and regulate cell fate. With the recent emergence of ‘ferroptosis’ as a new type of PCD and its close association with the pathophysiological processes of many diseases including tumors, it is viewed as a potential mechanism to improve systemic treatment of PC. Considering the opportunity to regulate both apoptosis and ferroptosis by a single agent, our group has developed a cancer-targeted small molecule consisting of a sigma-2 ligand (a known apoptotic inducer) delivery moiety linked to an inhibitor of the cystine importer xCT (dm-Erastin, a known ferroptotic inducer) and named it as Sigma-2-Erastin (S2E) or ACXT-3102. The study presented here is aimed at determining the efficacy of ACXT-3102 in PC and defining its underlying mechanism of action. Methods: Using in vitro and in vivo tumor xenograft models of human PC, we determined the effects of ACXT-3102 treatments in regulating PCD of PC cells and defined the signaling pathway involved in mediating the effects. Results: As assessed by TitreGlo assays, we observed dose-dependent inhibition of PC cell (BxPC-3, AsPC-1 &amp; PANC-1) viability. Following the WES-analysis of signaling proteins, we observed that ACXT-3102 induces PC cell death by combinatorial regulation of apoptotic and ferroptotic PCD via lipid ROS-DUSP6-MAPK/ERK-mediated signaling pathway. Interestingly, the results of this study demonstrated a hyperactivation of MAPK/ERK signaling by increased phosphorylation instead of inhibition involved in inducting apoptotic cell death as represented by significant increases in expression of cleaved Capase-3/7 and PARP. In addition, ACXT-3102 treatments deceased GPX4 expression along with an increase in lipid ROS production as key markers of inducing ferroptotic cell death. Results from the murine model of human tumor xenografts using AsPC-1 cells further demonstrated significant decreases in tumor volume and increases in survival proportions in ACXT-3102-treated mice. Conclusions: ACXT-3102, an orally administered conjugate of sigma-2 ligand and Erastin induces PCD of human PC by uniquely regulating apoptosis and ferroptosis together. Therefore, ACXT-3102 offers a novel and improved therapeutic strategy for treating patients of fatal human carcinomas, especially those, which are difficult to diagnose at an early stage. Citation Format: Kumar S. Bishnupuri, Kenneth F. Newcomer, Qingqing Gong, Li Ye, Suwanna Vangveravong, Rony Takchi, Bradley T. Keller, Dirk M. Spitzer, William G. Hawkins. Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3322.