Glutamate-cysteine Ligase Catalytic Subunit Research Articles

IRE1α determines ferroptosis sensitivity through regulation of glutathione synthesis

Cellular sensitivity to ferroptosis is primarily regulated by mechanisms mediating lipid hydroperoxide detoxification. We show that inositol-requiring enzyme 1 (IRE1α), an endoplasmic reticulum (ER) resident protein critical for the unfolded protein response (UPR), also determines cellular sensitivity to ferroptosis. Cancer and normal cells depleted of IRE1α gain resistance to ferroptosis, while enhanced IRE1α expression promotes sensitivity to ferroptosis. Mechanistically, IRE1α’s endoribonuclease activity cleaves and down-regulates the mRNA of key glutathione biosynthesis regulators glutamate-cysteine ligase catalytic subunit (GCLC) and solute carrier family 7 member 11 (SLC7A11). This activity of IRE1α is independent of its role in regulating the UPR and is evolutionarily conserved. Genetic deficiency and pharmacological inhibition of IRE1α have similar effects in inhibiting ferroptosis and reducing renal ischemia–reperfusion injury in mice. Our findings reveal a previously unidentified role of IRE1α to regulate ferroptosis and suggests inhibition of IRE1α as a promising therapeutic strategy to mitigate ferroptosis-associated pathological conditions.

Implications of GCLC in prognosis and immunity of lung adenocarcinoma and multi-omics regulation mechanisms

BackgroundFerroptosis is an iron-dependent type of regulated cell death, and has been implicated in lung adenocarcinoma (LUAD). Evidence has proved the key role of glutamate-cysteine ligase catalytic subunit (GCLC) in ferroptosis, but its role in LUAD remains unclear. Herein, we explored the implications of GCLC and relevant genes in LUAD prognosis and immunity as well as underlying molecular mechanisms.MethodsThis work gathered mRNA, miRNA, DNA methylation, somatic mutation and copy-number variation data from TCGA-LUAD. WGCNA was utilized for selecting GCLC-relevant genes, and a GCLC-relevant prognostic signature was built by uni- and multivariate-cox regression analyses. Immune compositions were estimated via CIBERSORT, and two immunotherapy cohorts of solid tumors were analyzed. Multi-omics regulatory mechanisms were finally assessed.ResultsOur results showed that GCLC was overexpressed in LUAD, and potentially resulted in undesirable survival. A prognostic model was generated, which owned accurate and independent performance in prognostication. GCLC, and relevant genes were notably connected with immune compositions and immune checkpoints. High GCLC expression was linked with better responses to anti-PD-L1 and anti-CTLA-4 treatment. Their possible DNA methylation sites were inferred, e.g., hypomethylation in cg19740353 might contribute to GCLC up-regulation. Frequent genetic mutations also affected their expression. Upstream transcription factors (E2F1/3/4, etc.), post-transcriptional regulation of miRNAs (hsa-mir-30c-1, etc.), lncRNAs (C8orf34-AS1, etc.), and IGF2BP1-mediated m6A modification were identified. It was also found NOP58-mediated SUMOylation post-translational modification.ConclusionsTogether, we show that GCLC and relevant genes exert crucial roles in LUAD prognosis and immunity, and their expression can be controlled by complex multi-omics mechanisms.

Solid state fermentation of Diaphragma juglandis fructus with Aspergillus niger alleviates hydrogen peroxide-induced oxidative stress in HepG2 cells via Nrf2 signaling pathway

Diaphragma juglandis fructus (DJF) is a traditional medicine, which is rich in bioactive polyphenols and has been proven to exhibit better antioxidant activity. However, the potential effects of fermentation of DJF on oxidative stress remains unclear. Herein, this study aimed to investigate the impact of solid-state fermentation (SSF) with Aspergillus niger (A. niger) on the polyphenol content and biological activity in DJF. Furthermore, compared with unfermented Diaphragma juglandis fructus (UF-DJF) extracts, ellagic acid (EA), sinapic acid (SA) and (+)-catechin (CA) displayed the highest increase in soluble phenolics (SPs) of fermented Diaphragma juglandis fructus (F-DJF) extracts. Thus, we explored the protective effect of EA, SA, CA and co-existing of EA, SA, CA against hydrogen peroxide (H2O2)-induced oxidative stress in HepG2 cells. Our results demonstrated that the insoluble-bound phenolics (IBPs) could be released, thereby increasing the SPs content and in vitro antioxidant activity after SSF. In addition, we also found that F-DJF extracts had the best inhibitory effect on three digestive enzymes among UF-DJF, before fermented Diaphragma juglandis fructus (BF-DJF) and F-DJF extracts. Moreover, our results revealed that pre-treatment with EA, SA, CA and co-present of EA, SA, CA could protect HepG2 cells from oxidative damage through activating the Nrf2 signaling pathway and up-regulating the expression of heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), cytoplasmic Cu-Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2) and catalase (CAT). Remarkably, the co-presence of EA, SA, CA showed the best protective effect. In conclusion, our research highlights SSF is an effective method for enhancing polyphenol content and biological activity in DJF. Moreover, these findings provide novel insights into the role of F-DJF extracts in prevention chronic diseases associated with oxidative stress and the possibility of their application in functional foods. In addition, fermentation may be a reliable strategy to enhance the medicinal value of DJF.

Binge alcohol induces NRF2-related antioxidant response in the skeletal muscle of female mice

BackgroundChronic alcohol enhances oxidative stress, but the temporal response of antioxidant genes in skeletal muscle following a binge drinking episode remains unknown. MethodsExperiment 1: C57BL/6Hsd female mice received an IP injection of saline (CON; n = 39) or ethanol (ETOH; n = 39) (5 g/kg). Gastrocnemius muscles were collected from baseline (untreated; n = 3), CON (n = 3), and ETOH (n = 3) mice every 4 h for 48 h. Experiment 2: Gastrocnemius muscles were collected from control-fed (CON-FED; n = 17), control-fasted (CON-FAST; n = 18), or alcohol-fed (ETOH-FED; n = 18) mice every 4hrs for 20hrs after saline or ethanol (5 g/kg). ResultsEtOH enhanced Superoxide dismutase 1 (Sod1) and NADPH Oxidase 4 (Nox4) from 24 to 48hr after the binge, while Sod2 and Nox2 were suppressed. Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) increased 12hrs after intoxication. Cytochrome P450 oxidoreductase (Por), Heme oxygenase 1 (Ho1), Peroxiredoxin 6 (Prdx6), Glutamate-cysteine ligase catalytic subunit (Gclc), Glutamate-cysteine ligase modifier subunit (Gclm), and Glutathione-disulfide reductase (Gsr) were increased by ETOH starting 12–16hrs post-binge. Fasting had similar effects on Nrf2 compared to alcohol, but downstream targets of NRF2, including Por, Ho1, Gclc, and Gclm, were differentially altered with fasting and EtOH. ConclusionThese data suggest that acute alcohol intoxication induced markers of oxidative stress and antioxidant signaling through the NRF2 pathway and that there were effects of alcohol independent of a possible decrease in food intake caused by binge intoxication.

Protective Mechanisms of Juncus effusus and Carbonized Juncus effusus against D-Galactosamine-Induced Acute Liver Injury in Mice.

This study investigated the hepatoprotective effects of Juncus effusus (J. effusus) and Carbonized J. effusus against liver injury caused by D-galactosamine (D-GalN) in mice. J. effusus and Carbonized J. effusus were administered by gavage once daily starting seven days before the D-GalN treatment. The results of the study indicated that J. effusus and Carbonized J. effusus suppressed the D-GalN-induced generation of serum alanine transaminase (ALT), aspartate aminotransferase (AST), hepatic malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) was observed. The values of superoxide dismutase (SOD) exhibited an increase. In addition, J. effusus and Carbonized J. effusus promoted the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1) as well as the mRNA expression of Nrf2, HO-1, NQO-1 and Glutamate cysteine ligase catalytic subunit (GCLC). The compressed Carbonized J. effusus demonstrated the optimum impact. These results suggest that J. effusus and Carbonized J. effusus protect against D-GalN-induced acute liver injury through the activation of the Nrf2 pathway.

Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways.

Osteoarthritis (OA) causes joint pain and disability due to the abnormal production of inflammatory cytokines and reactive oxygen species (ROS) in chondrocytes, leading to cell death and cartilage matrix destruction. Selenium (Se) intake can protect cells against oxidative damage. It is still unknown whether Se supplementation is beneficial for OA. This study investigated the effects of Se on sodium iodoacetate (MIA)-imitated OA progress in human chondrocyte cell line (SW1353 cells) and rats. The results showed that 0.3 μM of Se treatment could protect SW1353 cells from MIA-induced damage by the Nrf2 pathway by promoting the gene expression of glutathione-synthesis-related enzymes such as the glutamate-cysteine ligase catalytic subunit, the glutamate-cysteine ligase modifier subunit, and glutathione synthetase. In addition, glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase expressions are also elevated to eliminate excessive ROS production. Moreover, Se could downregulate NF-κB, leading to a decrease in cytokines, matrix proteases, and glycosaminoglycans. In the rats, MIA-induced cartilage loss was lessened after 2 weeks of Se supplementation by oral gavage; meanwhile, glutathione synthesis was increased, and the expressions of pro-inflammatory cytokines were decreased. These results suggest that Se intake is beneficial for OA due to its effects of decreasing cartilage loss by enhancing antioxidant capacity and reducing inflammation.

Hepatoprotective Effect of Annulohypoxylon stygium Melanin on Acute Alcoholic Liver Injury in Mice.

Annulohypoxylon stygium melanin (AsM) has various functional properties such as antioxidant and anti-radiation, but its biological activity in vivo has not been fully investigated. In this study, we researched the effects of AsM on the protection against acute liver injury in mice and its mechanism. The results showed that AsM had no significant effect on body weight in mice but reduced the liver index. It was able to significantly decrease the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the contents of triglyceride (TG) and total cholesterol (TC) in mice. Simultaneously, it raised the levels of superoxide dismutase (SOD), peroxidase (CAT), and glutathione peroxidase (GSH-Px), which obviously exceeded those of the EtOH group. AsM could significantly lower the levels of inflammatory factors, with inhibition rates of 68.30%, 29.0%, and 19.50% for IL-1β, IL-6, and TNF-α, respectively. H&E and Oil red O staining also showed that AsM ameliorated liver damage and lipid accumulation in mice. The protective mechanism of AsM may be associated to the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway, which could activate the downstream antioxidant enzymes heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC). These findings confirmed that AsM had an alleviating effect on alcoholic liver injury and provided new thoughts for the development of natural product.

Cystine deprivation triggers CD36-mediated ferroptosis and dysfunction of tumor infiltrating CD8+ T cells

Cancer cells develop multiple strategies to evade T cell-mediated killing. On one hand, cancer cells may preferentially rely on certain amino acids for rapid growth and metastasis. On the other hand, sufficient nutrient availability and uptake are necessary for mounting an effective T cell anti-tumor response in the tumor microenvironment (TME). Here we demonstrate that tumor cells outcompete T cells for cystine uptake due to high Slc7a11 expression. This competition induces T-cell exhaustion and ferroptosis, characterized by diminished memory formation and cytokine secretion, increased PD-1 and TIM-3 expression, as well as intracellular oxidative stress and lipid-peroxide accumulation. Importantly, either Slc7a11 deletion in tumor cells or intratumoral cystine supplementation improves T cell anti-tumor immunity. Mechanistically, cystine deprivation in T cells disrupts glutathione synthesis, but promotes CD36 mediated lipid uptake due to dysregulated cystine/glutamate exchange. Moreover, enforced expression of glutamate-cysteine ligase catalytic subunit (Gclc) promotes glutathione synthesis and prevents CD36 upregulation, thus boosting T cell anti-tumor immunity. Our findings reveal cystine as an intracellular metabolic checkpoint that orchestrates T-cell survival and differentiation, and highlight Gclc as a potential therapeutic target for enhancing T cell anti-tumor function.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum. In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01). Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry.

Role of Nrf2 in Oxidative Stress, Neuroinflammation and Autophagy in Alzheimer's Disease: Regulation of Nrf2 by Different Signaling Pathways.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder and the leading cause of dementia. AD is characterized by the aggregation of amyloid-ß (Aß) peptide, increased levels of tau protein, and loss of redox homeostasis responsible for mitochondrial dysfunction, oxidative stress, and neuroinflammation. Excessive accumulation of toxic Aß plaques activates microglia, which initiates neuroinflammation and consequently accelerates synaptic damage and neuronal loss. Various proinflammatory cytokines release, microglia proliferation, reactive astrocyte, and oxidative (reactive oxygen species (ROS) production, level of antioxidant enzymes, redox homeostasis, and lipid peroxidation) stress play a major role in AD. Several studies revealed that nuclear factor erythroid 2-related factor 2 (Nrf2) regulates redox homeostasis and works as an anti-inflammatory in various neurodegenerative disorders. D-Glutamate expression of transcription factor Nrf2 and its genes (glutamate-cysteine ligase catalytic subunit (GCLC), Heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase I (NQO1)) has been found in AD. Nrf2-HO-1 enhances the expression of antioxidant genes, inhibits microglia-mediated inflammation, and boosts mitochondrial function, suggesting that modulators of this protein may be useful to manage AD. This review focuses on the role of Nrf2 in AD, with a particular emphasis on the various pathways involved in the positive and negative modulation of Nrf2, namely Phosphoinositide 3-kinase (PI3K), Glycogen synthase kinase-3 (GSK-3), Nuclear factor kappa-B (NF-κB), and p38Mitogen-activated protein kinases (p38MAPK). Also, we have discussed the progress and challenges regarding the Nrf2 activators for AD treatment.

Lemon extract reduces the hepatic oxidative stress and persulfidation levels by upregulating the Nrf2 and Trx1 expression in old rats.

Citrus flavanones are recognized as promising bioactives within the concept of healthy aging. Thus, the present study investigated the effects of a nutritionally relevant dose of lemon extract (LE) on liver redox regulation and persulfidation levels in 24-month-old Wistar rats. LE (40 mg/kg b.m.) was administered orally once daily for 4 weeks. Control groups received either vehicle (sunflower oil) or remained intact. The applied methodology considered qPCR, Western blot, protein persulfidation levels evaluation, histochemistry in line with immunofluorescence, liver biochemical assays (glutathione, total -SH groups and malonaldehyde; MDA), liver enzymes in serum and in silico analysis to explore the potential interaction/binding between the proteins studied in the paper. Our results showed that LE increased glutathione peroxidase (GPx), reductase (GR), glutamate-cysteine ligase catalytic and modifier subunit, respectively, as well as Nrf2 gene expressions, but decreased the expression of superoxide dismutase 2 (SOD2). Upon LE application, protein expression showed upregulation of NRF2, SOD2, GPx, GR, and thioredoxin 1 (Trx1). LE significantly decreased the protein persulfidation levels and concentration of MDA, a marker of oxidative damage in the cell. Histological analysis showed a normal liver histoarchitecture without pathological changes, aligning with the normal serum level of hepatic enzymes. Obtained results showed that LE, by modulating hepatic redox regulators Nrf2 and Trx1, diminishes oxidative stress and alters the persulfidation levels, suggesting a considerable beneficial antioxidant potential of lemon flavanones in the old-aged liver.

Transient gestational hypothyroxinemia accelerates and enhances ulcerative colitis-like disorder in the male offspring.

Gestational hypothyroxinemia (HTX) is a condition that occurs frequently at the beginning of pregnancy, and it correlates with cognitive impairment, autism, and attentional deficit in the offspring. Evidence in animal models suggests that gestational HTX can increase the susceptibility of the offspring to develop strong inflammation in immune-mediated inflammatory diseases. Ulcerative colitis (UC) is a frequent inflammatory bowel disease with unknown causes. Therefore, the intensity of ulcerative colitis-like disorder (UCLD) and the cellular and molecular factors involved in proinflammatory or anti-inflammatory responses were analyzed in the offspring gestated in HTX (HTX-offspring) and compared with the offspring gestated in euthyroidism (Control-offspring). Gestational HTX was induced by the administration of 2-mercapto-1-methylimidazole in drinking water to pregnant mice during E10-E14. The HTX-offspring were induced with UCLD by the acute administration of dextran sodium sulfate (DSS). The score of UCLD symptomatology was registered every day, and colon histopathology, immune cells, and molecular factors involved in the inflammatory or anti-inflammatory response were analyzed on day 6 of DSS treatment. The HTX-offspring displayed earlier UCLD pathological symptoms compared with the Control-offspring. After 6 days of DSS treatment, the HTX-offspring almost doubled the score of the Control-offspring. The histopathological analyses of the colon samples showed signs of inflammation at the distal and medial colon for both the HTX-offspring and Control-offspring. However, significantly more inflammatory features were detected in the proximal colon of the HTX-offspring induced with UCLD compared with the Control-offspring induced with UCLD. Significantly reduced mRNA contents encoding for protective molecules like glutamate-cysteine ligase catalytic subunit (GCLC) and mucin-2 (MUC-2) were found in the colon of the HTX-offspring as compared with the Control-offspring. Higher percentages of Th17 lymphocytes were detected in the colon tissues of the HTX-offspring induced or not with UCLD as compared with the Control-offspring. Gestational HTX accelerates the onset and increases the intensity of UCLD in the offspring. The low expression of MUC-2 and GCLC together with high levels of Th17 Lymphocytes in the colon tissue suggests that the HTX-offspring has molecular and cellular features that favor inflammation and tissue damage. These results are important evidence to be aware of the impact of gestational HTX as a risk factor for UCLD development in offspring.

Rosmarinic Acid Inhibits Ultraviolet B-Mediated Oxidative Damage via the AKT/ERK-NRF2-GSH Pathway In Vitro and In Vivo.

Rosmarinic acid (RA) is a phenolic ester that protects human keratinocytes against oxidative damage induced by ultraviolet B (UVB) exposure, however, the mechanisms underlying its effects remain unclear. This study aimed to elucidate the cell signaling mechanisms that regulate the antioxidant activity of RA and confirm its cyto-protective role. To explore the signaling mechanisms, we used the human keratinocyte cell line HaCaT and SKH1 hairless mouse skin. RA enhanced glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS) expression in HaCaT cells in a dose- and time-dependent manner. Moreover, RA induced nuclear factor erythroid-2-related factor 2 (NRF2) nuclear translocation and activated the signaling kinases protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the ERK inhibitor U0126, and small interfering RNA (siRNA) gene silencing suppressed RA-enhanced GCLC, GSS, and NRF2 expression, respectively. Cell viability tests showed that RA significantly prevented UVB-induced cell viability decrease, whereas the glutathione (GSH) inhibitors buthionine sulfoximine, LY294002, and U0126 significantly reduced this effect. Moreover, RA protected against DNA damage and protein carbonylation, lipid peroxidation, and apoptosis caused by UVB-induced oxidative stress in a concentration-dependent manner in SKH1 hairless mouse skin tissues. These results suggest that RA protects against UVB-induced oxidative damage by activating AKT and ERK signaling to regulate NRF2 signaling and enhance GSH biosynthesis. Thus, RA treatment may be a promising approach to protect the skin from UVB-induced oxidative damage.

Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Suppression of circular RNA serum and glucocorticoid-induced kinase 1 elevates antioxidant molecules and angiogenesis in trophoblast cells to attenuate preeclampsia via microRNA-508-3p to target and restrain PUM homolog 1.

Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Recently, multiple circular RNAs (circRNAs) were considered latent clinical diagnostic markers or therapeutic targets. This study was to explore the impact of circRNA serum and glucocorticoid-induced kinase 1 (SGK1) on PE via influencing the microRNA (miR)-508-3p/PUM homolog 1 (PUM1) axis. Placental tissues of 34 pregnant women with PE and 34 normal pregnant women were collected to measure circRNA SGK1 (circSGK1), miR-508-3p, and PUM1. Human placental trophoblasts HTR-8/SVneo were transfected with plasmids, thereafter to observe proliferation, migration, invasion, and apoptosis, analyze antioxidant molecules Troxerutin (TXN), Glutamate-cysteine ligase catalytic subunit (GCLC), NAD (P) H-quinone oxidoreductase 1 (NQO1), and determine angiogenesis. After the construction of the PE rat model, antioxidant molecules TXN, GCLC, and NQO1, vascular-associated factor vascular endothelial growth factor A (VEGF-A), and histopathological conditions were tested. Examination of the binding of circSGK1 and miR-508-3p with PUM1 was performed. Our data showed that circSGK1 expression was elevated in the placenta of patients with PE. Silenced circSGK1 or elevated miR-508-3p promoted the growth and antioxidant molecules and angiogenesis in trophoblast cells; CircSGK1 combined with miR-508-3p, and miR-508-3p targeted PUM1. In summary, suppression of circSGK1 augments antioxidant molecules and angiogenesis in trophoblast cells to attenuate PE via miR-508-3p to target PUM1.

TMET-17. LEVERAGING METABOLIC VULNERABILITIES INDUCED BY TERT EXPRESSION FOR GLIOBLASTOMA THERAPY IN VIVO

Abstract Telomerase reverse transcriptase (TERT) is essential for telomere maintenance and tumor immortality. Although TERT is an attractive therapeutic target, targeting TERT directly is hampered by toxicity to stem cells that also rely on TERT for proliferation and the lag period before telomere attrition is observed. Targeting metabolic reprogramming associated with TERT is an alternate strategy. Our studies indicate that TERT is essential for redox homeostasis in glioblastoma cells (GBM; GBM6 and U251). Specifically, TERT increases levels of glutathione (GSH) via upregulation of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in de novo GSH synthesis. Inhibiting GCLC using buthionine sulfoximine (BSO) or siRNA reduces synthesis of 13C-GSH from [U-13C]-glutamine and inhibits viability of GBM6 and U251 cells. However, GCLC inhibition causes a compensatory increase in synthesis of glutamate, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine via upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), the enzyme that generates glutamate from glutamine, and carbamoyl phosphate synthetase II (CPSII), the enzyme that converts glutamine to carbamoyl phosphate during pyrimidine biosynthesis. We then examined the therapeutic potential of inhibiting GLS and CPSII in combination with GCLC in GBMs. 6-diazo-5-oxy-L-norleucin (DON), a potent inhibitor of glutamine-utilizing enzymes including GLS and CPSII, synergizes with BSO in inhibiting viability of GBM6 and U251 cells. This effect is specific to TERT-expressing GBM cells because the combination of DON and BSO does not affect viability of normal astrocytes or astrocytomas that use the ALT pathway for telomere maintenance. Importantly, in vivo stable isotope tracing confirmed that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine and induces apoptotic death in mice bearing intracranial GBM6 tumors.Collectively, our studies highlight the therapeutic potential of targeting metabolic vulnerabilities induced by TERT expression in GBMs in vivo.

Composition of Polygonatum zanlanscianense Pamp. Steam and Leaf Phenolic Extract and Its Protective Mechanism on t-BHP-Induced Oxidative Damage of HepG2 Cells.

Plant phenolic compounds have attracted considerable attention because of their health benefits. This study aimed to investigate the composition and antioxidant activity of phenol extracts from Polygonatum zanlanscianense Pamp. steam and leaf (PPP). The FTIR, UPLC-Q-Obtrip-MS, and HPLC-DAD methods were used to analyze the composition of PPP, and 20 phenolic compounds were preliminarily identified. Among them, the contents of hyperin, astragalin, and diosmetin levels were the highest. Treatment with PPP can significantly reduce t-BHP-induced cell damage in HepG2 cells, reactive oxygen species (ROS) accumulation, and malondialdehyde (MDA) content. Meanwhile, the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GSH-Px) activities can be increased. Moreover, PPP enhanced Nrf2 expression, which was consistent with that of heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone oxidoreductase-1 (NQO1), whereas the expression of Keap1, the Nrf2 inhibitor, was decreased. All findings indicate that PPP can serve as a natural bioactive substance for preventing oxidative stress.

Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.

GGT1 Suppresses the Development of Ferroptosis and Autophagy in Mouse Retinal Ganglion Cell Through Targeting GCLC.

Glaucoma is a neurodegenerative disorder characterized with optic nerve injury and the loss of retinal ganglion cells (RGCs). Ferroptosis has been proved to be associated with the degradation of RGCs. The aim of this study is to elucidate the relationship between ferroptosis and glaucoma pathogenesis, and unveil the underlying mechanism. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of RGCs. The accumulation of cellular iron was measured by Iron assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescence probe. The mitochondrial morphology and autophagosomes were analysed by using transmission electron microscopy (TEM). The contents of glutathione (GSH) and malondialdehyde (MDA) were tested by a GSH assay kit and an MDA detection kit, respectively. The expression of autophagy-related proteins was detected by Western blotting. A serious cell damage, aberrant iron homeostasis, and oxidative stress was shown in RGC-5 after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and gamma-Glutamyl transpeptidase 1 (GGT1) knockdown, but these effects were significantly alleviated by overexpression of GGT1 or ferroptosis inhibitors. The TEM and immunofluorescent results indicated that mitochondria impairment and autophagosome accumulation in OGD/R-treated cells was improved after GGT1 overexpression, while the phenomenon in GGT1-silenced cells was aggravated. Furthermore, we found that GGT1 can interact with glutamate cysteine ligase catalytic subunit (GCLC) to inhibit autophagy and ferroptosis in RGC-5 cells. GGT1 represses autophagy in RGC-5 cells by targeting GCLC, which further restrains the development of ferroptosis in cells.

Downregulation of TAZ elicits a mitochondrial redox imbalance and ferroptosis in lung epithelial cells exposed to diesel exhaust particles

Mitochondrial dysfunction was reported to be involved in the development of lung diseases including chronic obstructive pulmonary disease (COPD). However, molecular regulation underlying metabolic disorders in the airway epithelia exposed to air pollution remains unclear. In the present study, lung bronchial epithelial BEAS-2B and alveolar epithelial A549 cells were treated with diesel exhaust particles (DEPs), the primary representative of ambient particle matter. This treatment elicited cell death accompanied by induction of lipid reactive oxygen species (ROS) production and ferroptosis. Lipidomics analyses revealed that DEPs increased glycerophospholipid contents. Accordingly, DEPs upregulated expression of the electron transport chain (ETC) complex and induced mitochondrial ROS production. Mechanistically, DEP exposure downregulated the Hippo transducer transcriptional co-activator with PDZ-binding motif (TAZ), which was further identified to be crucial for the ferroptosis-associated antioxidant system, including glutathione peroxidase 4 (GPX4), the glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione-disulfide reductase (GSR). Moreover, immunohistochemistry confirmed downregulation of GPX4 and upregulation of lipid peroxidation in the bronchial epithelium of COPD patients and Sprague-Dawley rats exposed to air pollution. Finally, proteomics analyses confirmed alterations of ETC-related proteins in bronchoalveolar lavage from COPD patients compared to healthy subjects. Together, our study discovered that involvement of mitochondrial redox dysregulation plays a vital role in pulmonary epithelial cell destruction after exposure to air pollution.