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Ovarian cancer is a malignant tumor originating from the ovary, characterized by its high mortality rate and propensity for recurrence. In some patients, especially those with recurrent cancer, conventional treatments such as surgical resection or standard chemotherapy yield suboptimal results. Consequently, there is an urgent need for novel anti-cancer therapeutic strategies. Ferroptosis is a distinct form of cell death separate from apoptosis. Ferroptosis inducers have demonstrated promising potential in the treatment of ovarian cancer, with evidence indicating their ability to enhance ovarian cancer cell sensitivity to cisplatin. However, resistance of cancer cells to ferroptosis still remains an inevitable challenge. Here, we analyzed genome-scale CRISPR-Cas9 loss-of function screens and identified PAX8 as a ferroptosis resistance protein in ovarian cancer. We identified PAX8 as a susceptibility gene in GPX4-dependent ovarian cancer. Depletion of PAX8 rendered GPX4-dependent ovarian cancer cells significantly more sensitive to GPX4 inhibitors. Additionally, we found that PAX8 inhibited ferroptosis in ovarian cancer cells. Combined treatment with a PAX8 inhibitor and RSL3 suppressed ovarian cancer cell growth, induced ferroptosis, and was validated in a xenograft mouse model. Further exploration of the molecular mechanisms underlying PAX8 inhibition of ferroptosis mutations revealed upregulation of glutamate-cysteine ligase catalytic subunit (GCLC) expression. GCLC mediated the ferroptosis resistance induced by PAX8 in ovarian cancer. In conclusion, our study underscores the pivotal role of PAX8 as a therapeutic target in GPX4-dependent ovarian cancer. The combination of PAX8 inhibitors such as losartan and captopril with ferroptosis inducers represents a promising new approach for ovarian cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Ferroptosis 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. Methods This 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. Results Our 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. Conclusions Together, 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.

Background: Ferroptosis 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. Methods: This 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. Results: Our 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. Conclusions: Together, 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. [ABSTRACT FROM AUTHOR]

The parenteral nutrition (PN) received by premature newborns is contaminated with peroxides that induce global DNA hypermethylation via oxidative stress. Exposure to peroxides could be an important factor in the induction of chronic diseases such as those observed in adults who were born preterm. As endogenous H2O2 is a major regulator of glucose–lipid metabolism, our hypothesis was that early exposure to PN induces permanent epigenetic changes in H2O2 metabolism. Three-day-old guinea pigs were fed orally (ON), PN or glutathione-enriched PN (PN+GSSG). GSSG promotes endogenous peroxide detoxification. After 4 days, half the animals were sacrificed, and the other half were fed ON until 16 weeks of age. The liver was harvested. DNA methylation and mRNA levels were determined for the SOD2, GPx1, GCLC, GSase, Nrf2 and Keap1 genes. PN induced GPx1 hypermethylation and decreased GPx1, GCLC and GSase mRNA. These findings were not observed in PN+GSSG. PN+GSSG induced Nrf2 hypomethylation and increased Nrf2 and SOD2 mRNA. These observations were independent of age. In conclusion, in neonatal guinea pigs, PN induces epigenetic changes, affecting the expression of H2O2 metabolism genes. These changes persist for at least 15 weeks after PN. This disruption may signify a permanent reduction in the capacity to detoxify peroxides. [ABSTRACT FROM AUTHOR]

Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Yinzheng Li,1 Shulin Ma,1 Zheng Wang,1 Mengxia Shi,1 Rui Zeng,1– 4,* Ying Yao1,5,* 1Department of Nephrology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China; 2Key Laboratory of Organ Transplantation, Ministry of Education, Wuhan, 430030, People’s Republic of China; 3NHC Key Laboratory of Organ Transplantation, Wuhan, 430030, People’s Republic of China; 4Key Laboratory of Organ Transplantation, Chinese Academy of Medical Sciences, Wuhan, 430030, People’s Republic of China; 5Department of Nutrition, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ying Yao; Rui Zeng, Department of Nephrology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China, Tel +86 13720379867 ; +86 15002726366, Email yaoyingkk@126.com; zengrui@tjh.tjmu.edu.cnPurpose: The distal nephron of kidney plays a pivotal role in advancing acute kidney injury (AKI). Understanding the role of distal nephrons in AKI and identifying markers of injured distal nephrons are critical to comprehending the mechanism of renal injury and identifying novel therapeutic targets.Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data from mice with AKI induced by ischemia-reperfusion (IR), unilateral ureteral obstruction (UUO), cisplatin (CP), sodium oxalate (SO) and lipopolysaccharide (LPS). Additionally, we analyzed renal transcriptomics samples for AKI. Subsequently, we validated the effectiveness of targeting the biomarker Gclc in vitro and in vivo through metabolomics and immunofluorescence.Results: The LOH-Inj and DCT-Inj subtypes were identified through scRNA-seq. Compared to normal distal nephrons, the injured distal nephrons exhibited higher levels of ferroptosis, pro-inflammation, and fibrosis. The expression of ferroptosis-related gene Gclc were high in various AKI models. Furthermore, Gclc was exclusively expressed in the distal nephron and upregulated in the injury subtype. To confirm our findings, we suppressed GCLC expression in the kidneys, resulting to aggravated IR-induced AKI. Inhibition of Gclc promoted damage to primarily renal tubular epithelial cells by promoting inflammatory infiltration, inhibiting glutathione metabolism and exacerbating oxidative stress.Conclusion: Our research findings suggest that Gclc is a potential marker for injured distal nephron.Keywords: Gclc, distal nephron, acute kidney injury, single-cell sequencing

Abstract Background Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis. Methods The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe2+/Fe3+, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis. Results Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation. Conclusions Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.

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. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis. Methods: The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe2+/Fe3+, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis. Results: Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation. Conclusions: Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC. [ABSTRACT FROM AUTHOR]

Ferroptosis is a regulated cell death induced by iron-dependent lipid peroxidation. The heme-responsive transcription factor BTB and CNC homology 1 (BACH1) promotes ferroptosis by repressing the transcription of genes involved in glutathione (GSH) synthesis and intracellular labile iron metabolism, which are key regulatory pathways in ferroptosis. We found that BACH1 re-expression in Bach1 −/− immortalized mouse embryonic fibroblasts (iMEFs) can induce ferroptosis upon 2-mercaptoethanol removal, without any ferroptosis inducers. In these iMEFs, GSH synthesis was reduced, and intracellular labile iron levels were increased upon BACH1 re-expression. We used this system to investigate whether the major ferroptosis regulators glutathione peroxidase 4 (Gpx4) and apoptosis-inducing factor mitochondria-associated 2 (Aifm2), the gene for ferroptosis suppressor protein 1, are target genes of BACH1. Neither Gpx4 nor Aifm2 was regulated by BACH1 in the iMEFs. However, we found that BACH1 represses AIFM2 transcription in human pancreatic cancer cells. These results suggest that the ferroptosis regulators targeted by BACH1 may vary across different cell types and animal species. Furthermore, we confirmed that the ferroptosis induced by BACH1 re-expression exhibited a propagating effect. BACH1 re-expression represents a new strategy for inducing ferroptosis after GPX4 or system Xc− suppression and is expected to contribute to future ferroptosis research. [ABSTRACT FROM AUTHOR]

The Kelch-like ECH-associated protein 1–nuclear factor erythroid 2-related factor 2 (KEAP1–NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress. [ABSTRACT FROM AUTHOR]

The aim of this pilot study was to investigate whether single nucleotide polymorphisms (SNP) in the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC) are associated with the risk and clinical features of psoriasis. A total of 944 unrelated individuals, including 474 patients with a diagnosis of psoriasis and 470 healthy controls, were recruited for the study. Six common SNPs in the GCLC gene were genotyped using the MassArray-4 system. Polymorphisms rs648595 (OR = 0.56, 95% CI 0.35–0.90; Pperm = 0.017) and rs2397147 (OR = 0.54, 95% CI 0.30–0.98; Pperm = 0.05) were associated with susceptibility to psoriasis in males. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDR-adjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The joint effects of SNPs with tobacco smoking (rs648595 and rs17883901) and alcohol abuse (rs648595 and rs542914) on psoriasis risk were observed (Pperm ≤ 0.05). We also found multiple sex-independent associations between GCLC gene polymorphisms and various clinical features such as earlier disease onset, the psoriatic triad, and specific localizations of skin lesions. The present study is the first to show that polymorphisms of the GCLC gene are significantly associated with the risk of psoriasis and related to its clinical features. [ABSTRACT FROM AUTHOR]

Microcystin-LR (MCLR) is an aquatic toxin, which could lead to the development of hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) are considered important regulatory elements in the occurrence and development of cancer. However, the roles and mechanisms of lncRNAs during the process of HCC, induced by MCLR, remain elusive. Here, we identified a novel lncRNA, namely lnc-GCLC-1 (lncGCLC), which is in close proximity to the chromosome location of glutamate–cysteine ligase catalytic subunit (GCLC). We then investigated the role of lncGCLC in MCLR-induced malignant transformation of WRL68, a human hepatic cell line. During MCLR-induced cell transformation, the expression of lncGCLC and GCLC decreased continuously, accompanied with a consistently high expression of miR-122-5p. Knockdown of lncGCLC promoted cell proliferation, migration and invasion, but reduced cell apoptosis. A xenograft nude mouse model demonstrated that knockdown of lncGCLC promoted tumor growth. Furthermore, knockdown of lncGCLC significantly upregulated miR-122-5p expression, suppressed GCLC expression and GSH levels, and enhanced oxidative DNA damages. More importantly, the expression of lncGCLC in human HCC tissues was significantly downregulated in the high-microcystin exposure group, and positively associated with GCLC level in HCC tissues. Together, these findings suggest that lncGCLC plays an anti-oncogenic role in MCLR-induced malignant transformation by regulating GCLC expression. [ABSTRACT FROM AUTHOR]

To determine the expression and function of glutamate-cysteine ligase catalytic (GCLC) and glutamate-cysteine ligase catalytic modifier (GCLM) in gastric adenocarcinoma. Bioinformatics was used to analyze the expression of GCLC and GCLM. We download and analyzed the expression of gastric adenocarcinoma patients from TCGA database. Moreover, the method of immunochemistry was used to verify the expression of GCLC and GCLM in gastric adenocarcinoma. At first, the expression of GCLC and GCLM in gastric adenocarcinoma tissues were both significantly higher compared with normal tissues analyzed via TCGA database. Then, gastric adenocarcinoma tissues were collected and performed with immunochemistry. The gastric adenocarcinoma with positive staining for GCLC and GCLM was 77% and 80%, respectively, which was significantly higher compared with adjacent normal tissues (9% and 11%, respectively). The disordered expression of GCLC and GCLM in gastric adenocarcinoma suggested that these factors may induce tumorigenesis and may be a novel target for diagnosis and treatment of gastric adenocarcinoma. [ABSTRACT FROM AUTHOR]

Ferroptosis, a newly discovered iron-dependent type of cell death, has been found to play a crucial role in the depression of tumorigenesis. However, the prognostic value of ferroptosis-related genes (FRGs) in lung adenocarcinoma (LUAD) remains to be further elucidated. Differential expression analysis and univariate Cox regression analysis were utilized in this study to search for FRGs that were associated with the prognosis of LUAD patients. The influences of candidate markers on LUAD cell proliferation, migration, and ferroptosis were evaluated by CCK8, colony formation, and functional experimental assays in association with ferroptosis. To predict the prognosis of LUAD patients, we constructed a predictive signature comprised of six FRGs. We discovered a critical gene (GCLC) after intersecting the prognostic analysis results of all aspects, and its high expression was associated with a bad prognosis in LUAD. Correlation research revealed that GCLC was related to a variety of clinical information from LUAD patients. At the same time, in the experimental verification, we found that GCLC expression was upregulated in LUAD cell lines, and silencing GCLC accelerated ferroptosis and decreased LUAD cell proliferation and invasion. Taken together, this study established a novel ferroptosis-related gene signature and discovered a crucial gene, GCLC, that might be a new prognostic biomarker of LUAD patients, as well as provide a potential therapeutic target for LUAD patients. [ABSTRACT FROM AUTHOR]

The MYH9 (Myosin heavy chain 9), an architecture component of the actomyosin cytoskeleton, has been reported to be dysregulated in several types of cancers. However, how this molecule contributes to cancer development is still obscure. This study deciphered the molecular function of MYH9 in head and neck cancer (HNC). Cellular methods included clonogenic survival, wound-healing migration, and Matrigel invasion assays. Molecular techniques included RT-qPCR, western blot, luciferase reporter assays, and flow cytometry. Clinical association studies were undertaken by TCGA data mining, Spearman correlation, and Kaplan-Meier survival analysis. We found that MYH9 was overexpressed in tumors and associated with poor prognosis in HNC patients. MYH9 promoted cell motility along with the modulation of the extracellular matrix (fibronectin, ITGA6, fascin, vimentin, MMPs). Also, MYH9 contributed to radioresistance and was related to the expression of anti-apoptotic and DNA repairing molecules (XIAP, MCL1, BCL2L1, ATM, RAD50, and NBN). Mechanically, MYH9 suppressed cellular ROS levels, which were achieved by activating the pan-MAPK signaling molecules (Erk, p38, and JNK), the induction of Nrf2 transcriptional activity, and the up-regulation of antioxidant enzymes (GCLC, GCLM, GPX2). The antioxidant enzyme GCLC was further demonstrated to facilitate cell invasion and radioresistance in HNC cells. Thus, MYH9 exerts malignant functions in HNC by regulating cellular ROS levels via activating the MAPK-Nrf2-GCLC signaling pathway. As MYH9 contributes to radioresistance and metastasis, this molecule may serve as a prognostic biomarker for clinical application. Furthermore, an in vivo study is emergent to support the therapeutic potential of targeting MYH9 to better manage refractory cancers. [ABSTRACT FROM AUTHOR]

Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen–glucose deprivation (OGD)-induced human umbilical endothelial cells' (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the levels of the glutamate–cysteine ligase catalytic subunit (GCLc) and the glutathione synthetase (GSS) proteins, a key enzyme for de novo GSH synthesis, were determined using enzyme-linked immunoassay (ELISA). We demonstrated that the SDX treatment in OGD conditions significantly elevated the intracellular GSH, enhanced the GSH:GSSG ratio, shifting the redox potential to a more pro-reducing status. Furthermore, SDX increased the levels of both GCLc and GSS. The results show that SDX protects the human endothelial cells against ischemic stress by affecting the GSH levels and cellular redox state. These changes suggest that the reduction in the ischemia-induced vascular endothelial cell injury through repressing apoptosis and oxidative stress associated with SDX treatment may be due to an increase in GSH synthesis and modulation of the GSH redox system. [ABSTRACT FROM AUTHOR]

Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The aim of this pilot study was to investigate whether single nucleotide polymorphisms (SNP) in the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC) are associated with the risk and clinical features of psoriasis. A total of 944 unrelated individuals, including 474 patients with a diagnosis of psoriasis and 470 healthy controls, were recruited for the study. Six common SNPs in the GCLC gene were genotyped using the MassArray-4 system. Polymorphisms rs648595 (OR = 0.56, 95% CI 0.35–0.90; Pperm = 0.017) and rs2397147 (OR = 0.54, 95% CI 0.30–0.98; Pperm = 0.05) were associated with susceptibility to psoriasis in males. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDR-adjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The joint effects of SNPs with tobacco smoking (rs648595 and rs17883901) and alcohol abuse (rs648595 and rs542914) on psoriasis risk were observed (Pperm ≤ 0.05). We also found multiple sex-independent associations between GCLC gene polymorphisms and various clinical features such as earlier disease onset, the psoriatic triad, and specific localizations of skin lesions. The present study is the first to show that polymorphisms of the GCLC gene are significantly associated with the risk of psoriasis and related to its clinical features.

Carnosic acid (CA) is a natural phenolic compound with several biomedical actions. This work was performed to study the use of CA-loaded polymeric nanoparticles to improve the antitumor activity of breast cancer cells (MCF-7) and colon cancer cells (Caco-2). CA was encapsulated in bovine serum albumin (BSA), chitosan (CH), and cellulose (CL) nanoparticles. The CA-loaded BSA nanoparticles (CA-BSA-NPs) revealed the most promising formula as it showed good loading capacity and the best release rate profile as the drug reached 80% after 10 h. The physicochemical characterization of the CA-BSA-NPs and empty carrier (BSA-NPs) was performed by the particle size distribution analysis, transmission electron microscopy (TEM), and zeta potential. The antitumor activity of the CA-BSA-NPs was evaluated by measuring cell viability, apoptosis rate, and gene expression of GCLC, COX-2, and BCL-2 in MCF-7 and Caco-2. The cytotoxicity assay (MTT) showed elevated antitumor activity of CA-BSA-NPs against MCF-7 and Caco-2 compared to free CA and BSA-NPs. Moreover, apoptosis test data showed an arrest of the Caco-2 cells at G2/M (10.84%) and the MCF-7 cells at G2/M (4.73%) in the CA-BSA-NPs treatment. RT-PCR-based gene expression analysis showed an upregulation of the GCLC gene and downregulation of the BCL-2 and COX-2 genes in cells treated with CA-BSA-NPs compared to untreated cells. In conclusion, CA-BSA-NPs has been introduced as a promising formula for treating breast and colorectal cancer. [ABSTRACT FROM AUTHOR]

The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections. [Display omitted] • GSH-regulated IL-22, but not IL-17 from Th17 cells, is vital for gut barrier integrity • GCLC expression in IBD patients correlates with gut integrity gene expression • Gclc controls mROS and mitochondrial ATP linked to PI3K/mTOR-driven IL-22 translation • ROS scavenging or T cell's IL-22 saves mutant mice from infection-induced lethality Bonetti et al. show that glutathione's (GSH) control of mitochondrial reactive oxygen species (ROS) in Th17 cells is critical to maintain intestinal barrier integrity during bacterial gastrointestinal infections. They highlight the connection between mitochondrial function and intestinal barrier integrity and identify a Th17 cell-intrinsic GSH/mitochondrial-IL-22 signaling axis as crucial for host protection. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) regulate the tumorigenesis of non-small-cell lung cancer (NSCLC). CircPDSS1 (hsa_circ_0017998) has been newly discovered, and its role in NSCLC remains elusive. We aimed to investigate the functional roles and downstream targets of circPDSS1 in NSCLC cells. Cellular viabilities were measured through the Cell Counting Kit-8 (CCK-8) assay, whereas cell death was assessed through flow cytometry. The lactate dehydrogenase activity, malondialdehyde levels, ferrous iron, and reactive oxygen species were measured using commercial assay kits. The interaction between circPDSSA/ microRNA-137 (miR-137) and miR-137/solute carrier family 7 member 11 (SLC7A11) was assayed through a dual luciferase activity assay. Finally, the mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blots, respectively. CircPDSS1 expression was upregulated in NSCLC cells, compared with healthy lung cells. CircPDSS1 silencing suppressed the viability of NSCLC cells. Additionally, circPDSS1 knockdown induced ferroptosis rather than other types of cell death in NSCLC cells. Mechanically, circPDSS1 functions as a "sponge" to inversely control miR-137 expression, which directly targets SLC7A11. Moreover, circPDSS1 silencing causes the downregulation of glutathione peroxidase 4 (GPX4) and glutamate-cysteine ligase catalytic subunit (GCLC). Targeting the circPDSS1/miR-137/SLC7A11/GPX4/GCLC axis may be a promising strategy to kill NSCLC cells. • Knockdown of circPDSS1 induces ferroptosis in NSCLC cells.CircPDSS1 functions as a "sponge" to inversely control the miR-137 expression, which directly targets the SLC7A11. • Silencing of circPDSS1 also leads to the downregulation of GPX4 and GCLC. [ABSTRACT FROM AUTHOR]

The purpose of this pilot study was to explore whether polymorphisms in genes encoding the catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase, a rate-limiting enzyme in glutathione synthesis, play a role in the development of ischemic stroke (IS) and the extent of brain damage. A total of 1288 unrelated Russians, including 600 IS patients and 688 age- and sex-matched healthy subjects, were enrolled for the study. Nine common single nucleotide polymorphisms (SNPs) of the GCLC and GCLM genes were genotyped using the MassArray-4 system. SNP rs2301022 of GCLM was strongly associated with a decreased risk of ischemic stroke regardless of sex and age (OR = 0.39, 95%CI 0.24–0.62, p < 0.0001). Two common haplotypes of GCLM possessed protective effects against ischemic stroke risk (p < 0.01), but exclusively in nonsmoker patients. Infarct size was increased by polymorphisms rs636933 and rs761142 of GCLC. The mbmdr method enabled identifying epistatic interactions of GCLC and GCLM gene polymorphisms with known IS susceptibility genes that, along with environmental risk factors, jointly contribute to the disease risk and brain infarct size. Understanding the impact of genes and environmental factors on glutathione metabolism will allow the development of effective strategies for the treatment of ischemic stroke and disease prevention. [ABSTRACT FROM AUTHOR]

Microcystin-LR (MCLR) is an aquatic toxin, which could lead to the development of hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) are considered important regulatory elements in the occurrence and development of cancer. However, the roles and mechanisms of lncRNAs during the process of HCC, induced by MCLR, remain elusive. Here, we identified a novel lncRNA, namely lnc-GCLC-1 (lncGCLC), which is in close proximity to the chromosome location of glutamate–cysteine ligase catalytic subunit (GCLC). We then investigated the role of lncGCLC in MCLR-induced malignant transformation of WRL68, a human hepatic cell line. During MCLR-induced cell transformation, the expression of lncGCLC and GCLC decreased continuously, accompanied with a consistently high expression of miR-122-5p. Knockdown of lncGCLC promoted cell proliferation, migration and invasion, but reduced cell apoptosis. A xenograft nude mouse model demonstrated that knockdown of lncGCLC promoted tumor growth. Furthermore, knockdown of lncGCLC significantly upregulated miR-122-5p expression, suppressed GCLC expression and GSH levels, and enhanced oxidative DNA damages. More importantly, the expression of lncGCLC in human HCC tissues was significantly downregulated in the high-microcystin exposure group, and positively associated with GCLC level in HCC tissues. Together, these findings suggest that lncGCLC plays an anti-oncogenic role in MCLR-induced malignant transformation by regulating GCLC expression.

Ferroptosis, a newly discovered iron-dependent type of cell death, has been found to play a crucial role in the depression of tumorigenesis. However, the prognostic value of ferroptosis-related genes (FRGs) in lung adenocarcinoma (LUAD) remains to be further elucidated. Differential expression analysis and univariate Cox regression analysis were utilized in this study to search for FRGs that were associated with the prognosis of LUAD patients. The influences of candidate markers on LUAD cell proliferation, migration, and ferroptosis were evaluated by CCK8, colony formation, and functional experimental assays in association with ferroptosis. To predict the prognosis of LUAD patients, we constructed a predictive signature comprised of six FRGs. We discovered a critical gene (GCLC) after intersecting the prognostic analysis results of all aspects, and its high expression was associated with a bad prognosis in LUAD. Correlation research revealed that GCLC was related to a variety of clinical information from LUAD patients. At the same time, in the experimental verification, we found that GCLC expression was upregulated in LUAD cell lines, and silencing GCLC accelerated ferroptosis and decreased LUAD cell proliferation and invasion. Taken together, this study established a novel ferroptosis-related gene signature and discovered a crucial gene, GCLC, that might be a new prognostic biomarker of LUAD patients, as well as provide a potential therapeutic target for LUAD patients.

Carnosic acid (CA) is a natural phenolic compound with several biomedical actions. This work was performed to study the use of CA-loaded polymeric nanoparticles to improve the antitumor activity of breast cancer cells (MCF-7) and colon cancer cells (Caco-2). CA was encapsulated in bovine serum albumin (BSA), chitosan (CH), and cellulose (CL) nanoparticles. The CA-loaded BSA nanoparticles (CA-BSA-NPs) revealed the most promising formula as it showed good loading capacity and the best release rate profile as the drug reached 80% after 10 h. The physicochemical characterization of the CA-BSA-NPs and empty carrier (BSA-NPs) was performed by the particle size distribution analysis, transmission electron microscopy (TEM), and zeta potential. The antitumor activity of the CA-BSA-NPs was evaluated by measuring cell viability, apoptosis rate, and gene expression of GCLC, COX-2, and BCL-2 in MCF-7 and Caco-2. The cytotoxicity assay (MTT) showed elevated antitumor activity of CA-BSA-NPs against MCF-7 and Caco-2 compared to free CA and BSA-NPs. Moreover, apoptosis test data showed an arrest of the Caco-2 cells at G2/M (10.84%) and the MCF-7 cells at G2/M (4.73%) in the CA-BSA-NPs treatment. RT-PCR-based gene expression analysis showed an upregulation of the GCLC gene and downregulation of the BCL-2 and COX-2 genes in cells treated with CA-BSA-NPs compared to untreated cells. In conclusion, CA-BSA-NPs has been introduced as a promising formula for treating breast and colorectal cancer.

The purpose of this pilot study was to explore whether polymorphisms in genes encoding the catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase, a rate-limiting enzyme in glutathione synthesis, play a role in the development of ischemic stroke (IS) and the extent of brain damage. A total of 1288 unrelated Russians, including 600 IS patients and 688 age- and sex-matched healthy subjects, were enrolled for the study. Nine common single nucleotide polymorphisms (SNPs) of the GCLC and GCLM genes were genotyped using the MassArray-4 system. SNP rs2301022 of GCLM was strongly associated with a decreased risk of ischemic stroke regardless of sex and age (OR = 0.39, 95%CI 0.24–0.62, p < 0.0001). Two common haplotypes of GCLM possessed protective effects against ischemic stroke risk (p < 0.01), but exclusively in nonsmoker patients. Infarct size was increased by polymorphisms rs636933 and rs761142 of GCLC. The mbmdr method enabled identifying epistatic interactions of GCLC and GCLM gene polymorphisms with known IS susceptibility genes that, along with environmental risk factors, jointly contribute to the disease risk and brain infarct size. Understanding the impact of genes and environmental factors on glutathione metabolism will allow the development of effective strategies for the treatment of ischemic stroke and disease prevention.

Antioxidant enzymes play an important role in the defense against oxidative stress in the lung and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Sequence variation in genes encoding antioxidant enzymes may alter susceptibility to COPD by affecting longitudinal change in lung function in adults. We genotyped 384 sequence variants in 56 candidate genes in 1281 African American and 1794 European American elderly adults in the Health, Aging, and Body Composition study. Single-marker associations and gene-by-smoking interactions with rate of change in FEV₁ and FEV₁/FVC were evaluated using linear mixed-effects models, stratified by race/ethnicity. In European Americans, rs17883901 in GCLC was statistically significantly associated with rate of change in FEV₁/FVC; the recessive genotype (TT) was associated with a 0.9% per year steeper decline (P = 4.50 × 10(-5)). Statistically significant gene-by-smoking interactions were observed for variants in two genes in European Americans: the minor allele of rs2297765 in mGST3 attenuated the accelerated decline in FEV₁/FVC in smokers by 0.45% per year (P = 1.13 × 10(-4)); for participants with greater baseline smoking pack-years, the minor allele of rs2073192 in IDH3B was associated with an accelerated decline in FEV₁/FVC (P = 2.10 × 10(-4)). For both genes, nominally significant interactions (P < 0.01) were observed at the gene level in African Americans (P = 0.007 and 4.60 × 10(-4), respectively). Nominally significant evidence of association was observed for variants in SOD3 and GLRX2 in multiple analyses. This study identifies two novel genes associated with longitudinal lung function phenotypes in both African and European Americans and confirms a prior finding for GCLC. These findings suggest novel mechanisms and molecular targets for future research and advance the understanding of genetic determinants of lung function and COPD risk.

Nuclear factor erythroid-2-related factor 2 (Nrf2), is an inducible transcription factor that improves redox balance through stimulating antioxidant gene expression. In older humans the Nrf2 response to a single bout of acute exercise is blunted compared to young indicating impaired redox signaling. The purpose of this randomized controlled trial was to investigate if the signaling impairment could be reversed with exercise training in older men and women, while also comparing to young. Young (18-28y, n = 21) and older (≥60y, n = 19) men and women were randomized to 8-week aerobic exercise training (ET; 3 d/wk, 45 min/d) or a non-exercise control group (CON). Nrf2 nuclear localization, gene expression for NQO1 , HO1 , and GCLC , and GCLC protein were measured in PBMCs in response to acute exercise trial (AET; 30-min cycling at 70% VO 2 peak pre- and post-intervention at 7 timepoints (Pre, +10 m, +30 m, +1 h, +4 h, +8 h, +24 h). Young had greater Nrf2 signaling response compared to older at pre-intervention (p = 0.05), whereas the older had significantly higher basal Nrf2 levels (p = 0.004). ET decreased basal Nrf2 expression compared to CON (p = 0.032) and improved the Nrf2 signaling response in both young and older (p < 0.05). The degree of restoration in Nrf2 signaling response was related to the degree of change in basal Nrf2 (p = 0.039), which was driven by older adults (p = 0.014). Lower basal nuclear Nrf2 levels were associated with changes seen in AET responses for Nrf2 and GCLC protein, as well as NQO1 and GCLC mRNA. Together these data demonstrate that exercise training improves Nrf2 signaling and downstream gene expression and that lower basal Nrf2 levels are associated with a more dynamic acute response. Our results provide evidence that the impaired Nrf2 signaling in sedentary older adults can be restored to a degree with moderate exercise training, albeit not to the level seen in young. NCT03419988. Image 1 • Older individuals had attenuated Nrf2 activation and downstream gene expression following acute exercise versus young. • Exercise Training (ET) can reverse some of the age-related impairment. • The improvements in signaling effects driven by ET in older individuals comes from decreasing basal levels of Nrf2. • The degree of improvement in Nrf2 activation to ET was greater in young than older individuals. • ET does not completely reverse age-related biological declines in Nrf2-dependent signaling systems. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a specific obstetric disorder that may result in maternal and neonatal morbidity and mortality. Increasing evidence has been indicated that some candidate genes related to oxidative stress, such as glutamate-cysteine ligase, catalytic subunit (GCLC), glutamate-cysteine ligase, modifier subunit (GCLM), involve in the pathogenesis of PE. After the genetic contribution of GCLC rs17883901 polymorphism was analyzed by TaqMan allelic discrimination real-time PCR in 1001 PE patients and 1182 normal pregnant women, a case-control association analysis was performed. Although no statistical difference was found in genetic distribution of rs17883901 in GCLC between PE and control group (χ2 = 2.201, p =.333 by genotypic, χ2 = 0.524, p =.469, OR = 0.932, 95%CI = 0.771–1.128 by allelic), significant differences in the genotypic frequencies were investigated between mild PE group (χ2 = 6.999, p =.030) or late-onset PE group (χ2 = 6.197, p =.045) and control group. Furthermore, when dividing the mild PE patients, the late-onset PE patients and the controls into TT/CT + CC, TT + CT/CC, and TT/CC subgroups, we found statistical differences between mild PE and controls (TT/CT + CC:χ2 = 5.132, p =.023, OR = 2.948, 95%CI = 1.107–7.854; TT/CC:χ2 = 4.564, p =.033, OR = 2.793, 95%CI = 1.046–7.460) as well as late-onset PE and controls (TT/CT + CC:χ2 = 4.043, p =.044, OR = 2.248, 95%CI = 1.000–5.055). This is the first study to indicate GCLC rs17883901 polymorphism may be associated with a risk of mild PE and late-onset PE in Chinese Han women. However, additional well-designed studies with multi-ethnic and large-scale samples should be performed to validate our results. [ABSTRACT FROM AUTHOR]

Some effects of aging in animals are tissue-specific. In D. melanogaster neuronal overexpression of Gclc increases lifespan and improves certain physiological parameters associated with health benefits such as locomotor activity, circadian rhythmicity, and stress resistance. Our previous transcriptomic analyses of Drosophila heads, primarily composed of neuronal tissue, revealed significant changes in expression levels of genes involved in aging-related signaling pathways (Jak-STAT, MAPK, FOXO, Notch, mTOR, TGF-beta), translation, protein processing in endoplasmic reticulum, proteasomal degradation, glycolysis, oxidative phosphorylation, apoptosis, regulation of circadian rhythms, differentiation of neurons, synaptic plasticity, and transmission. Considering that various tissues age differently and age-related gene expression changes are tissue-specific, we investigated the effects of neuronal Gclc overexpression on gene expression levels in the imago thorax, which is primarily composed of muscles. A total of 58 genes were found to be differentially expressed between thoraces of control and Gclc overexpressing flies. The Gclc level demonstrated associations with expression of genes involved in the circadian rhythmicity, the genes in categories related to the muscle system process and the downregulation of genes involved in proteolysis. Most of the functional categories altered by Gclc overexpression related to metabolism including Drug metabolism, Metabolism of xenobiotics by cytochrome P450, Glutathione metabolism, Starch and sucrose metabolism, Citrate cycle (TCA cycle), One carbon pool by folate. Thus, the transcriptomic changes caused by neuron-specific Gclc overexpression in the thorax were less pronounced than in the head and affected pathways also differed from previous results. Although these pathways don't belong to the canonical longevity pathways, we suggest that they could participate in the delay of aging of Gclc overexpressing flies.

Given the participation of oxidative stress in the pathogenesis of diabetic complications, we evaluated, in type 1 diabetes (T1D) individuals, the association between diabetic retinopathy (DR) and functional single nucleotide polymorphisms (SNPs) in regulatory regions of two genes belonging to the antioxidant glutathione (GSH) system: rs17883901 in GCLC and rs713041 in GPX4. A cross-sectional case-control study included 288 individuals (61% women, 34[±11] years old, diabetes duration of 22[±9] years, mean [±SD]) sorted according to DR stages: absence of DR (ADR), non-proliferative DR (NPDR) and proliferative DR (PDR). SNPs were genotyped by real-time PCR using fluorescent labelled probes. Logistic regression models with adjustment for confounding covariates were employed. The presence of at least one T-allele of rs17883901 in GCLC was an independent risk factor for PDR (OR 4.13, 95% CI 1.38–13.66, p = 0.014) in a polytomous regression model (PDR versus ADR). The presence of at least one T-allele of rs713041 in GPX4 conferred protection against PDR (OR 0.30, 95% CI 0.11–0.80, p = 0.017) in female T1D individuals. The functional SNPs rs17883901 and rs713041 modulate the risk for PDR in the studied population of T1D individuals, widening the spectrum of candidate genes for this complication. • Proliferative diabetic retinopathy is not fully explained by glucose control. • Genetics also plays an important role in diabetic retinopathy susceptibility. • Redox imbalance is associated with diabetic microvascular complications. • Glutathione (GSH) is one of the major antioxidants in the body. • Variants in genes from GSH system modulate proliferative diabetic retinopathy risk. [ABSTRACT FROM AUTHOR]

Microcystin-LR (MCLR) is a potent hepatotoxin which could lead to the development of hepatocellular carcinoma. However, the mechanisms of its carcinogenic action remain obscure. The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione and is central to the antioxidant capacity of the cell, but emerging data suggest that the GCLC expression is associated with cancer development. The purpose of this study was to investigate the role and molecular mechanisms of GCLC in MCLR-induced malignant transformation of a human liver cell line WRL68. During MCLR-induced cell transformation, the expression of GCLC and activity of glutamate-cysteine ligase (GCL) decreased continuously, accompanied with consistent low levels of glutathione (GSH) but high levels of oxidative DNA damages. Furthermore, MCLR markedly inhibited protein phosphatase 2 A (PP2 A), and increased the level of GCLC phosphorylation. In contrast, overexpression of GCLC significantly enhanced the levels of GSH, inhibited oxidative DNA damages, and suppressed MCLR-induced cell invasion and migration, as well as tumor growth in nude mice. GCLC overexpression partially attenuated MCLR-induced PP2 A inhibition. Together, the current results suggest that down-regulation of GCLC is involved in MCLR-induced malignant transformation of human liver cells by inducing oxidative stress. [ABSTRACT FROM AUTHOR]

Some effects of aging in animals are tissue-specific. In D. melanogaster neuronal overexpression of Gclc increases lifespan and improves certain physiological parameters associated with health benefits such as locomotor activity, circadian rhythmicity, and stress resistance. Our previous transcriptomic analyses of Drosophila heads, primarily composed of neuronal tissue, revealed significant changes in expression levels of genes involved in aging-related signaling pathways (Jak-STAT, MAPK, FOXO, Notch, mTOR, TGF-beta), translation, protein processing in endoplasmic reticulum, proteasomal degradation, glycolysis, oxidative phosphorylation, apoptosis, regulation of circadian rhythms, differentiation of neurons, synaptic plasticity, and transmission. Considering that various tissues age differently and age-related gene expression changes are tissue-specific, we investigated the effects of neuronal Gclc overexpression on gene expression levels in the imago thorax, which is primarily composed of muscles. A total of 58 genes were found to be differentially expressed between thoraces of control and Gclc overexpressing flies. The Gclc level demonstrated associations with expression of genes involved in the circadian rhythmicity, the genes in categories related to the muscle system process and the downregulation of genes involved in proteolysis. Most of the functional categories altered by Gclc overexpression related to metabolism including Drug metabolism, Metabolism of xenobiotics by cytochrome P450, Glutathione metabolism, Starch and sucrose metabolism, Citrate cycle (TCA cycle), One carbon pool by folate. Thus, the transcriptomic changes caused by neuron-specific Gclc overexpression in the thorax were less pronounced than in the head and affected pathways also differed from previous results. Although these pathways don't belong to the canonical longevity pathways, we suggest that they could participate in the delay of aging of Gclc overexpressing flies. [ABSTRACT FROM AUTHOR]

Boron is abundant in vegetables, nuts, legumes, and fruit and intake is associated with reduced risk of cancer and DNA damage and increased antioxidant status. Blood boric acid (BA) levels are approximately 10 μM BA in men at the mean US boron intake. Treatment of DU-145 human prostate cancer cells with 10 μM BA stimulates phosphorylation of elongation initiation factor 2α (eIF2α) at Ser51 leading to activation of the eIF2α/ATF4 pathway which activates the DNA damage-inducible protein GADD34. In the present study, we used MEF WT and MEF PERK (±) cells to test the hypothesis that BA-activated eIF2α phosphorylation requires protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activates Nrf2 and the antioxidant response element (ARE). BA (10 μM) increased phosphorylation of eIF2α Ser51 in MEF WT cells at 1 h, but not in MEF Perk −/− cells exposed for as long as 6 h. GCN2 kinase-dependent phosphorylation of eIF2α Ser51 was activated in MEF PERK −/− cells by amino acid starvation. Nrf2 phosphorylation is PERK dependent and when activated is translocated from the cytoplasm to the nucleus where it acts as a transcription factor for ARE. DU-145 cells were treated with 10 μM BA and Nrf2 measured by immunofluorescence. Cytoplasmic Nrf2 was translocated to the nucleus at 1.5-2 h in DU-145 and MEF WT cells, but not MEF PERK −/− cells. Real-time PCR was used to measure mRNA levels of three ARE genes (HMOX-1, NQO1, and GCLC). Treatment with 10 μM BA increased the mRNA levels of all three genes at 1-4 h in DU-145 cells and HMOX1 and GCLC in MEF WT cells. These results extend the known boric acid signaling pathway to ARE-regulated genes. The BA signaling pathway can be expressed using the schematic [BA + cADPR → cADPR-BA → [[ER]i Ca2+↓] → 3 pathways: PERK/eIF2αP → pathways ATF4 and Nrf2; and [[ER]i Ca2+↓] → ER stress → ATF6 pathway. This signaling pathway provides a framework that links many of the molecular changes that underpin the biological effects of boron intake. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate–glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine–cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

Introduction Glutathione (GSH) pathways play a key role the metabolism and elimination of the neurotoxicant methylmercury (MeHg). We hypothesized that maternal genetic variation linked to GSH pathways could influence MeHg concentrations in pregnant mothers and children and thereby also affect early life development. Methods The GCLM (rs41303970, C/T), GCLC (rs761142, T/G) and GSTP1 (rs1695, A/G) polymorphisms were genotyped in 1449 mothers in a prospective study of the Seychellois population with a diet rich in fish. Genotypes were analyzed in association with maternal hair and blood Hg, fetal blood Hg (cord blood Hg), as well as children's mental (MDI) and motor development (PDI; MDI and PDI assessed by Bayley Scales of Infant Development at 20 months). We also examined whether genotypes modified the association between Hg exposure and developmental outcomes. Results GCLC rs761142 TT homozygotes showed statistically higher mean maternal hair Hg (4.12 ppm) than G carriers (AG 3.73 and GG 3.52 ppm) ( p  = 0.037). For the combination of GCLC rs761142 and GCLM rs41303970, double homozygotes TT + CC showed higher hair Hg (4.40 ppm) than G + T carriers (3.44 ppm; p  = 0.018). No associations were observed between GSTP1 rs1695 and maternal hair Hg or between any genotypes and maternal blood Hg or cord blood Hg. The maternal GSTP1 rs1695 rare allele (G) was associated with a lower MDI among children (β = −1.48, p  = 0.048). We also observed some interactions: increasing Hg in maternal and cord blood was associated with lower PDI among GCLC rs761142 TT carriers; and increasing Hg in hair was associated with lower MDI among GSTP1 rs1695 GG carriers. Conclusions Maternal genetic variation in genes involved in GSH synthesis is statistically associated with Hg concentrations in maternal hair, but not in maternal or fetal blood. We observed interactions that suggest maternal GSH genetics may modify associations between MeHg exposure and neurodevelopmental outcomes. [ABSTRACT FROM AUTHOR]

How macrophages maintain redox homeostasis in the inflammatory process, in which a large amount of oxidants are produced, remains elusive. In this study, we investigated the temporal changes in the intracellular glutathione (GSH), the master antioxidant, and the expression of glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH biosynthesis, in the inflammatory response of human macrophages (THP1 cells) to lipopolysaccharide. Intracellular GSH concentration was decreased significantly in the early phase (~6 h) of LPS exposure, and then gradually went back to the basal level in the late phase (9–24 h). The expression level of the catalytic subunit of GCL (GCLC) followed a similar pattern of change as GSH: its mRNA and protein levels were reduced in the early phase and then back to basal level in the late phase. In contrast, the expression of the modifier subunit of GCL (GCLM) was significantly increased in the phase of LPS exposure. Activation Nrf2, the transcription factor involved in the induction of both GCLC and GCLM, occurred at as early as 3 h after LPS exposure; whereas the activation of NF-κB occurred at as early as 30 min. Inhibition of NF-κB signaling with SN50 prevented the decrease of GCLC and inhibited Nrf2 activation in response to LPS. These data demonstrate time-dependent changes in the expression of GCL and Nrf2 signaling during the inflammatory response, and that the regulation of GCLC and GCLM might be through different pathways in this process. [ABSTRACT FROM AUTHOR]

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo (nobo), encoding a glutathione S-transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the g-glutamylcysteine synthetase catalytic subunit (Gclc) gene in the fruit fly Drosophila melanogaster. Gclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH. [ABSTRACT FROM AUTHOR]