Expression Of GCLC Research Articles

Vitamin E regulates bovine granulosa cell apoptosis via NRF2-mediated defence mechanism by activating PI3K/AKT and ERK1/2 signalling pathways.

High-yield dairy cows are usually subject to high-intensive cell metabolism and produce excessive reactive oxygen species (ROS). Once ROS is beyond the threshold of scavenging ability, it can induce oxidative stress, imperilling the reproductive performance of cows. The study was to investigate the effects of vitamin E (VE) on H2 O2 -induced proliferation and apoptosis of bovine granulosa cells and the underlying molecular mechanism. Granulosa cells were pretreated with VE for 24hr and then treated with H2 O2 for 6hr. The results showed that VE treatment decreased the intracellular ROS levels, increased the MDA content, and improved the antioxidant enzyme activity in a dose-dependent manner. Furthermore, VE treatment promoted the proliferation and inhibited apoptosis in granulosa cells by up-regulation of CCND1 and BCL2 levels and down-regulation of P21, BAX, and CASP3 levels. The cytoprotective effects of VE were attributed to the activation of the NRF2 signalling pathway. Knockdown of the NRF2 impaired the cytoprotective effects of VE on granulosa cells. Besides, the PI3K/AKT and ERK1/2, but not the p38 signalling pathway is involved in the regulation of VE-mediated cell proliferation and apoptosis. The PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited the VE-induced granulosa cell proliferation and promoted apoptosis, whereas the p38 inhibitor SB203580 had the opposite effects. These results were confirmed by proliferation and apoptosis-related gene expression at mRNA and protein levels. The results also showed that the PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited VE-induced NRF2, GCLC, GCLM, and HO-1 expression, whereas the p38 inhibitor SB203580 not. Overall, the results demonstrated that VE-regulated granulosa cell proliferation and apoptosis via NRF2-mediated defence system by activating the PI3K/AKT and ERK1/2 signalling pathway.

Alpinetin exerts anti-inflammatory, anti-oxidative and anti-angiogenic effects through activating the Nrf2 pathway and inhibiting NLRP3 pathway in carbon tetrachloride-induced liver fibrosis

Alpinetin is the major active ingredient of Alpiniakatsumadai Hayata. As a kind of novel plant-derived flavonoid, alpinetin has shown potent hepatoprotective effect against many liver diseases such as non-alcoholic fatty liver and lipopolysaccharide/d-Galactosamine-induced liver injury. However, its roles in liver fibrosis remain to be determined. The aim of the current study was to investigate the effect of alpinetin in mice with carbon tetrachloride (CCl4)-induced liver fibrosis, and to elucidate the underlying mechanisms of action. Alpinetin ameliorated the CCl4-induced liver injury and fibrosis in mice, as shown by decreased collagen deposition and the decreased expression of liver fibrosis marker proteins. Alpinetin suppressed the inflammation and oxidative stress in fibrotic livers of mice, as evidenced by decreased levels of proinflammatory factors, the decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and the increased activities of antioxidant enzymes. In addition, alpinetin attenuated the angiogenesis in fibrotic livers of the test animals. Mechanistically, alpinetin inhibited the CCl4-induced expression of NLRP3, ASC, cleaved caspase-1, mature (cleaved-) IL-1β, and IL-18 in livers of mice. Furthermore, alpinetin resulted in an increased in the nuclear expression and a decrease in the cytoplasmic expression of Nrf2, as well as increased protein expression of downstream target enzymes, GCLC, HO-1, NQO1, and GCLM, thus exerting the antioxidant effect. Overall, these findings suggested that the anti-fibrotic effect of alpinetin can be attributed to the inhibition of NLRP3-mediated anti-inflammatory activities and Nrf2-mediated anti-oxidative activities, in addition to the decrement of hepatic angiogenesis.

Targeting KEAP1/Nrf2, AKT, and PPAR-γ signals as a potential protective mechanism of diosmin against gentamicin-induced nephrotoxicity

AimGentamicin (GM) is an aminoglycoside antibiotic effectively used for severe/life-threatening infections. However, the clinical application of GM is limited by nephrotoxic side effects. Diosmin (DS) is a flavonoid with a wide range of bioactivities. However, its therapeutic potential in GM-induced nephrotoxicity remains unclear. MethodsRats received GM (100 mg/kg, i.p.) for 7 days either separately or in combination with oral DS (50 mg/kg). ResultsGM injection disrupted kidney function along with oxidant/antioxidant imbalance. Also, GM significantly decreased renal nuclear factor erythroid 2-related factor 2 (Nrf2), glutamyl cysteine synthetase (GCLC), heme oxygenase-1 (HO-1), superoxide dismutase3 (SOD-3), protein kinase B (AKT), and p-AKT expressions along with Kelch-like ECH-associated protein 1 (KEAP1) up-regulation. On the contrary, DS administration significantly attenuated GM-induced kidney dysfunction and restored kidney oxidant/antioxidant status. In addition, co-treatment with DS plus GM significantly enhanced Nrf2, GCLC, HO-1, SOD3, AKT, and p-AKT expressions along with KEAP1 down-regulation. Additionally, GM-treated rats exhibited a significant decrease in the expressions of renal peroxisome-proliferator activated receptor-gamma (PPAR-γ) and this reduction was alleviated by DS treatment. Furthermore, histopathological findings demonstrated that DS significantly reduced the GM-induced histological abrasions. Besides, an in-silico study was conducted to confirm our biochemical results. Interestingly, in-silico results strongly supported our biochemical investigation by studying the binding affinity of DS to KEAP1, AKT, and PPAR-γ proteins. SignificanceDS could be a promising protective agent against GM-induced nephrotoxicity through targeting of KEAP1/Nrf2/ARE, AKT, and PPAR-γ signaling pathways.

Paeoniflorin and Plycyrrhetinic Acid Synergistically Alleviate MPP+/MPTP-Induced Oxidative Stress through Nrf2-Dependent Glutathione Biosynthesis Mechanisms.

Recently, combination therapy has proven to be an effective strategy for treating polygenic/multifactorial/complex disorder such as Parkinson's disease (PD). Here, we hypothesized that dual up-regulation of glutamate cysteine ligase (GCL) catalytic subunit (GCLc) and GCL modifier subunit (GCLm) via nuclear factor E2-related factor (Nrf2) contribute to the antioxidant effect of paeoniflorin (PF) synergistically with glycyrrhetinic acid (GA) (henceforth called PF/GA) in the context of MPP+/MPTP neurotoxicity. Expectedly, CompuSyn synergism/antagonism analysis showed that PF/GA exerts synergistic neuroprotection. Moreover, the antioxidant effect of PF was significantly enhanced by the combined administration of GA, although GA alone did not confer the effect. Mechanistically, PF triggered extracellular signal-regulated kinase (ERK1/2) phosphorylation, resulting in Nrf2 nuclear translocation from cytoplasmic pool via de novo synthesis in MPP+-challenged SH-SY5Y cells. Concomitantly, GA activates Akt which in turn induces nuclear accumulation of Nrf2. Especially, PF/GA up-regulated glutamate-cysteine ligase catalytic subunit (Gclc) and glutamate-cysteine ligase modifier subunit (Gclm) are formed via two separate pathways. Furthermore, these results were confirmed through pathway blockade assays using PD98059 (ERK1/2 inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), and shRNA-induced Nrf2 knockdown. Additionally, using a mouse MPTP-induced model of PD, we demonstrated that PF/GA synergistically ameliorates both motor deficits and oxidative stress in the ventral midbrain. In parallel, PF/GA also up-regulated both GCLc and GCLm expression at levels of transcription and translation. Conversely, antiparkinsonism and antioxidant effects of PF/GA were not observed in Nrf2-knockout MPTP-mice. Collectively, these results show that ERK1/2 and Akt activation contribute to the synergistic antioxidant effect of PF/GA. Hence, PF/GA regimen warrants further preclinical and possible clinical study for PD.

N-Phenyl Cinnamamide Derivatives Protect Hepatocytes against Oxidative Stress by Inducing Cellular Glutathione Synthesis via Nuclear Factor (Erythroid-Derived 2)-Like 2 Activation.

Substituted N-phenyl cinnamamide derivatives were designed and synthesized to confirm activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway by the electronic effect on beta-position of Michael acceptor according to introducing the R1 and R2 group. Compounds were screened using the Nrf2/antioxidant response element (ARE)-driven luciferase reporter assay. Compound 1g showed desirable luciferase activity in HepG2 cells without cell toxicity. mRNA and protein expression of Nrf2/ARE target genes such as NAD(P)H quinone oxidoreductase 1, hemeoxygenase-1, and glutamate-cysteine ligase catalytic subunit (GCLC) were upregulated by compound 1g in a concentration-dependent manner. Treatment with 1g resulted in increased endogenous antioxidant glutathione, showing strong correlation with enhanced GCLC expression for synthesis of glutathione. In addition, tert-butyl hydroperoxide (t-BHP)-generated reactive oxygen species were significantly removed by 1g, and the results of a cell survival assay in a t-BHP-induced oxidative cell injury model showed a cytoprotective effect of 1g in a concentration dependent manner. In conclusion, the novel compound 1g can be utilized as an Nrf2/ARE activator in antioxidative therapy.

Prevention of Autoimmune Diabetes in NOD Mice by Dimethyl Fumarate.

Oxidative stress plays critical roles in the pathogenesis of diabetes. This study tested the hypothesis that by protecting β-cells against oxidative stress and inflammation, an Nrf2 activator, dimethyl fumarate (DMF), may prevent or delay the onset of type 1 diabetes in non-obese diabetic (NOD) mice. Firstly, islet isolation was conducted to confirm the antioxidative effects of DMF oral administration on islet cells. Secondly, in a spontaneous diabetes model, DMF (25 mg/kg) was fed to mice once daily starting at the age of 8 weeks up to the age of 22 weeks. In a cyclophosphamide-induced accelerated diabetes model, DMF (25 mg/kg) was fed to mice twice daily for 2 weeks. In the islet isolation study, DMF administration improved the isolation yield, attenuated oxidative stress and enhanced GCLC and NQO1 expression in the islets. In the spontaneous model, DMF significantly reduced the onset of diabetes compared to the control group (25% vs. 54.2%). In the accelerated model, DMF reduced the onset of diabetes from 58.3% to 16.7%. The insulitis score in the islets of the DMF treatment group (1.6 ± 0.32) was significantly lower than in the control group (3.47 ± 0.21). The serum IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-12p70, IFN-γ, TNF-α, MCP-1 and CXCL16 levels in the DMF-treated group were lower than in the control group. In conclusion, DMF may protect islet cells and reduce the incidence of autoimmune diabetes in NOD mice by attenuating insulitis and proinflammatory cytokine production.

Downregulation of CD151 induces oxidative stress and apoptosis in trophoblast cells via inhibiting ERK/Nrf2 signaling pathway in preeclampsia

Preeclampsia (PE) is a pregnancy-related syndrome characterized by new-onset hypertension and proteinuria after gestational 20 weeks. Oxidative stress, resulting from the imbalance between the production of oxidants and antioxidants in placentas, is recognized as a key pathology of PE. To date, the molecules that regulate antioxidants production remain unclear. CD151, a member of tetraspanins, is an important regulator of many physiological functions. However, the function of CD151 in oxidative stress and its association with pregnancy-related complications are currently unknown. In the present study, we have demonstrated that CD151 was a key regulator of antioxidants in placentas. Compared with the placentas of the controls, the placentas of PE patients exhibited decreased CD151 expression accompanying with decreased antioxidant gene expression (HO-1, NQO-1, GCLC and SOD-1). In vitro, overexpression of CD151 in trophoblast cells could enhance HO-1, NQO-1, GCLC and SOD-1 expression but downregulation of CD151 decreased those antioxidant genes expression, which indicates CD151 is the upstream of antioxidants. Importantly, the phenotype of PE (hypertension and proteinuria) was mimicked in the downregulating CD151 induced mouse model. Moreover, the beneficial effect of CD151 in trophoblast cells was hindered when ERK and Nrf2 signaling were blocked. Overall, our results revealed CD151 might be a new target for PE treatment.

G-Rh4 improves pancreatic β-cells dysfunction in vivo and in vitro by increased expression of Nrf2 and its target genes

The aim of this study is to investigate the hypoglycemic mechanism of ginsenoside Rh4 (G-Rh4) in vivo and in vitro models. Our results showed that G-Rh4 markedly improved the symptoms of diabetes, normalized glucose metabolism, and promoted insulin secretion which contributed to attenuate symptoms of hyperglycemia in high-fat diet/streptozocin induced type 2 diabetes mellitus mice. This positive effect was associated with increased expression of Nrf2 by G-Rh4. Further results demonstrated that G-Rh4 promoted Nrf2 nucleus translocation as well as up-regulated the expression of HO-1, NQO1 and GCLC. Furthermore, we also found that G-Rh4 increased insulin secretion by activating the signal pathway of PDX-1, GLUT2 and GCK. More importantly, the protective effects of G-Rh4 on alloxan-induced upregulation of Nrf2 target gene and insulin secretion were abolished by Nrf2 knockdown. Finally, we explored the mechanism of G-Rh4 associated with Nrf2 activation and found that the Akt deficiency inhibited G-Rh4-mediated Nrf2 nuclear translocation. Altogether, we present evidence that G-Rh4 increased expression of Nrf2 and results in increased antioxidant gene, as well as a rise in insulin secretion in vivo and in vitro. Exploiting the Nrf2 pathway may show great potential as a therapeutic strategy to improve pancreatic β-cells dysfunction in the diabetic population.

Development and optimization of halogenated vinyl sulfones as Nrf2 activators for the treatment of Parkinson’s disease

The Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a pivotal role in the cellular defense system against oxidative stress by inducing antioxidant and anti-inflammatory effects. We previously developed Nrf2 activators that potentially protect the death of dopaminergic (DAergic) neuronal cells against oxidative stress in Parkinson's disease (PD). In this study, we designed and synthesized a class of halogenated vinyl sulfones by inserting halogens and pyridine to maximize Nrf2 activation efficacy. Among the synthesized compounds, (E)-3-chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine (9d) significantly exhibited potent Nrf2 activating efficacy (9d: EC50=26nM) at least 10-fold compared with the previous developed compounds (1 and 2). Furthermore, treating with 9d remarkably increased Nrf2 nuclear translocation and Nrf2 protein levels in microglial BV-2cells. 9d was shown to induce the expression of antioxidant response genes HO-1, GCLC, GCLM, and SOD-1 at both the mRNA and protein levels and suppress proinflammatory cytokines and enzymes. Also, 9d remarkably protected DAergic neurons and restored the PD-associated motor dysfunction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model.

Sex-, Age-, and Race/Ethnicity-Dependent Variations in Drug-Processing and NRF2-Regulated Genes in Human Livers.

Individual variations in xenobiotic metabolism affect the sensitivity to diseases. In this study, the impacts of sex, age, and race/ethnicity on drug-processing genes and nuclear factor erythroid 2-related factor 2 (NRF2) genes in human livers were examined via QuantiGene multiplex suspension array (226 samples) and quantitative polymerase chain reaction (qPCR) (247 samples) to profile the expression of nuclear receptors, cytochrome P450s, conjugation enzymes, transporters, bile acid metabolism, and NRF2-regulated genes. Sex differences were found in expression of about half of the genes, but in general the differences were not large. For example, females had higher transcript levels of catalase, glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase 1, and thioredoxin reductase-1 compared with males via qPCR. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit (GCLM) and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of HO-1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, SULT2A1, small heterodimer partner, and bile salt export pump. An examination of 34 diseased and control human liver samples showed that compared with disease-free livers, fibrotic livers had higher NAD(P)H-quinone oxidoreductase 1 (NQO1), GCLC, GCLM, and NRF2; hepatocellular carcinoma had higher transcript levels of NQO1 and KEAP1; and steatotic livers had lower GCLC, GCLM, and HO-1 expression. In summary, in drug-processing gene and NRF2 genes, sex differences were the major findings, and there were no apparent age differences, and race/ethnicity differences occurred for a few genes. These descriptive findings could add to our understanding of the sex-, age-, and race/ethnicity-dependent differences in drug-processing genes as well as NRF2 genes in normal and diseased human livers. SIGNIFICANCE STATEMENT: In human liver drug-processing and nuclear factor erythroid 2-related factor 2 genes, sex differences were the main finding. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit, and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of heme oxygenase 1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, small heterodimer partner, SULT2A1, and bile salt export pump.

MiR-9-5p inhibits mitochondrial damage and oxidative stress in AD cell models by targeting GSK-3β.

This study aims to investigate the effects and underlying mechanisms of overexpression microRNA-9-5p (miR-9-5p) on the Aβ-induced mouse hippocampal neuron cell line HT22. Different concentrations of Aβ25-35 (10, 20, 40, 80, and 160μM) treatment were used to establish AD model in HT22 cells. The CCK-8 assay was used to measure the cell viability. The mRNA expression levels of miR-9-5p and glycogen synthase kinase-3β (GSK-3β) were determined by RT-qPCR. HT22 cell apoptosis was analyzed flow cytometry. MiR-9-5p was down-regulated in Aβ25-35-induced HT22 cells. GSK-3β is a functional target for miR-9-5p. MiR-9-5p overexpression inhibited Aβ25-35-induced mitochondrial dysfunction, cell apoptosis, and oxidative stress by regulating GSK-3β expression in HT22 cells. Furthermore, through targeting GSK-3β, overexpression of miR-9-5p partly activated nuclear factor Nrf2/Keap1 signaling, including part increases of Nrf2, HO-1, SOD-1, GCLC expression and slight decrease of Keap1 expression. Our results showed miR-9-5p may play a powerful role in the pathogenesis of AD.

Glutamate-Cysteine Ligase Catalytic Subunit Attenuated Hepatitis C Virus-Related Liver Fibrosis and Suppressed Endoplasmic Reticulum Stress

The study aimed to clarify the role and molecular mechanism of glutamate-cysteine ligase catalytic subunit (GCLC) in modulating Hepatitis C virus (HCV)-related liver fibrosis. Twenty patients with HCV-related liver fibrosis and 15 healthy controls were enrolled. Differentially expressed plasma mRNAs were detected by digital gene expression profile analysis and validated by qRT-PCR. Hepatic histopathology was observed by H&E and Masson stained liver sections. The mRNA and protein expression of GCLC, endoplasmic reticulum (ER) stress markers, and inflammatory and fibrogenic factors were detected in liver tissues from patients with HCV-related hepatic fibrosis and HCV core protein-expressing LX-2. The GCLC-overexpressing LX-2 were established by transfecting puc19-GCLC plasmid. Then, glutathione and reactive oxygen species (ROS) levels were measured respectively by spectrophotometric diagnostic kit and dihydrodichlorofluorescein diacetate kit. GCLC were dramatically down-regulated in HCV-related fibrotic livers and activated HSCs, which companied with up-regulation of ER stress-related genes, including inositol-requiring 1 (IRE1) and glucose-regulated protein 78 (GRP78). Also, the proinflammatory and profibrogenic gene, including nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNFα), and transforming growth factor 1(TGFβ1), was highly upregulated. Overexpression of GCLC in hepatic stellate cells could suppress α-SMA and collagen I expression, produce hepatic GSH and reduce ROS, and down-regulate IRE1, GRP78, NF-κB, TNF-α, and TGFβ1 expression. GCLC was a negative regulatory factor in the development of HCV-related liver fibrosis and might be a potential therapeutic target for liver fibrosis.

A Systematic Review of the Various Effect of Arsenic on Glutathione Synthesis In Vitro and In Vivo.

Background Arsenic is a toxic metalloid widely present in nature, and arsenic poisoning in drinking water is a serious global public problem. Glutathione is an important reducing agent that inhibits arsenic-induced oxidative stress and participates in arsenic methylation metabolism. Therefore, glutathione plays an important role in regulating arsenic toxicity. In recent years, a large number of studies have shown that arsenic can regulate glutathione synthesis in many ways, but there are many contradictions in the research results. At present, the mechanism of the effect of arsenic on glutathione synthesis has not been elucidated. Objective We will conduct a meta-analysis to illustrate the effects of arsenic on GSH synthesis precursors Glu, Cys, Gly, and rate-limiting enzyme γ-GCS in mammalian models, as well as the regulation of p38/Nrf2 of γ-GCS subunit GCLC, and further explore the molecular mechanism of arsenic affecting glutathione synthesis. Results This meta-analysis included 30 studies in vivo and 58 studies in vitro, among which in vivo studies showed that arsenic exposure could reduce the contents of GSH (SMD = −2.86, 95% CI (-4.45, -1.27)), Glu (SMD = −1.11, 95% CI (-2.20,-0.02)), and Cys (SMD = −1.48, 95% CI (-2.63, -0.33)), with no statistically significant difference in p38/Nrf2, GCLC, and GCLM. In vitro studies showed that arsenic exposure increased intracellular GSH content (SMD = 1.87, 95% CI (0.18, 3.56)) and promoted the expression of p-p38 (SMD = 4.19, 95% CI (2.34, 6.05)), Nrf2 (SMD = 4.60, 95% CI (2.34, 6.86)), and GCLC (SMD = 1.32, 95% CI (0.23, 2.41)); the p38 inhibitor inhibited the expression of Nrf2 (SMD = −1.27, 95% CI (-2.46, -0.09)) and GCLC (SMD = −5.37, 95% CI (-5.37, -2.20)); siNrf2 inhibited the expression of GCLC, and BSO inhibited the synthesis of GSH. There is a dose-dependent relationship between the effects of exposure on GSH in vitro. Conclusions. These indicate the difference between in vivo and in vitro studies of the effect of arsenic on glutathione synthesis. In vivo studies have shown that arsenic exposure can reduce glutamate and cysteine levels and inhibit glutathione synthesis, while in vitro studies have shown that chronic low-dose arsenic exposure can activate the p38/Nrf2 pathway, upregulate GCLC expression, and promote glutathione synthesis.

Protective effect of the NAC and Sal on zinc oxide nanoparticles-induced reproductive and development toxicity in pregnant mice

The growing use of zinc oxide nanoparticles (ZnO NPs) in various applications has raised many concerns about the potential risks to human health. In this research, the protective effects of cellular oxidative stress inhibitor N-Acetyl-cysteine (NAC) and endoplasmic reticulum (ER) stress inhibitor Salubrinal (Sal) on reproductive toxicity induced by ZnO NPs were investigated. The results showed that application of these two kinds of cell stress inhibitors after oral ingestion of ZnO NPs could prevent the weight loss of pregnant mice; reduce zinc content in the uterus, placenta and fetus; reduce abnormal development of the offspring; and decrease fetal abortion. Furthermore, RT-qPCR, Western blot and immunofluorescence assay results indicated that NAC restored the expression of Gclc, reduced the expression of ATF4, JNK and Caspase-12, and decreased the expression of eNOS and IGF-1, in the placenta. Sal decreased the expression of ATF4, JNK and Caspase-12, and increased the expression of eNOS and IGF-1caused by the oral ingestion of ZnO NPs. These results indicated that treatment with NAC and Sal after oral exposure could reduce reproductive and development toxicity caused by ZnO NPs which induced reproductive and development toxicity that was probably caused by the activation of oxide stress and ER stress.

Triptolide suppresses IDH1-mutated malignancy via Nrf2-driven glutathione metabolism.

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, served as a potent Nrf2 inhibitor, which exhibited selective cytotoxicity to patient-derived IDH1-mutated glioma cells in vitro and in vivo. Mechanistically, triptolide compromised the expression of GCLC, GCLM, and SLC7A11, which disrupted glutathione metabolism and established synthetic lethality with reactive oxygen species derived from IDH1 mutant neomorphic activity. Our findings highlight triptolide as a valuable therapeutic approach for IDH1-mutated malignancies by targeting the Nrf2-driven glutathione synthesis pathway.

Neuroprotective effects of Danshensu on rotenone-induced Parkinson\u2019s disease models in vitro and in vivo

BackgroundDanshensu is an active constituent in the extracts of Danshen which is a traditional Chinese medical herb. Rotenone inhibits complex I of the mitochondrial electron transport chain in dopaminergic neurons leading to glutathione (GSH) level reduction and oxidative stress. The aim of this study is to investigate neuroprotective effects of Danshensu on rotenone-induced Parkinson’s disease (PD) in vitro and in vivo.MethodsIn vitro, SH-SY5Y human neuroblastoma cell line was pretreated with Danshensu and challenged with rotenone. Then the reactive oxygen species (ROS) production was assayed. In vivo, male C57BL/6 mice were intragastrically administered with Danshensu (15, 30, or 60 mg/kg), followed by oral administration with rotenone at a dose of 30 mg/kg. Pole and rotarod tests were carried out at 28 d to observe the effects of Danshensu on PD.ResultsDanshensu repressed ROS generation and therefore attenuated the rotenone-induced injury in SH-SY5Y cells. Danshensu improved motor dysfunction induced by rotenone, accompanied with reducing MDA content and increasing GSH level in striatum. Danshensu increased the number of TH positive neurons, the expression of TH and the dopamine contents. The expressions of p-PI3K, p-AKT, Nrf2, hemeoxygenase (HO-1), glutathione cysteine ligase regulatory subunit (GCLC), glutathione cysteine ligase modulatory subunit (GCLM) were significantly increased and the expression of Keap1 was decreased in Danshensu groups.ConclusionsThe neuroprotective effects of Danshensu on rotenone-induced PD are attributed to the anti-oxidative properties by activating PI3K/AKT/Nrf2 pathway and increasing Nrf2-induced expression of HO-1, GCLC, and GCLM, at least in part.

Nuclear Factor Erythroid-Derived 2-Like 2-Induced Reductive Stress Favors Self-Renewal of Breast Cancer Stem-Like Cellsviathe FoxO3a-Bmi-1 Axis

Aims: A subpopulation of cancer cells, termed cancer stem cells (CSCs), has stemness properties, such as self-renewal and differentiation, which drive cancer recurrence and tumor resistance. CSCs possess enhanced protection capabilities to maintain reduced intracellular levels of reactive oxygen species (ROS) compared with nonstem-like cancer cells. This study investigated whether reductive stress could regulate self-renewal activity in breast CSCs. Results: We found that manifestation of stemness in breast cancer stem-like cells was associated with an elevated production of reduced glutathione (GSH) maintained by upregulation of glutamate cysteine ligase catalytic subunit (GCLC) and consequently, lowered ROS levels. This was accompanied by upregulation of phospho-AMP-activated protein kinase, FoxO3a, and Bmi-1. Notably, expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) protein was substantially increased in cells undergoing sphere formation. We noticed that expression of Bmi-1 was inhibited after introduction of Nrf2 short interfering RNA into MCF-7 mammosphere cells. Silencing of Nrf2 expression suppressed the xenograft growth of subcutaneously or orthotopically injected human breast cancer cells. Innovation: Association between Nrf2 and self-renewal signaling in CSCs has been reported, but the underlying molecular mechanism remains largely unresolved. This study demonstrates the Nrf2-mediated signaling pathway in maintenance of reductive stress in breast CSCs. Conclusion: Nrf2 overactivation in breast CSCs upregulates GCLC expression and consequently enhances GSH biosynthesis with concurrent reduction in intracellular ROS accumulation, thereby provoking the reductive stress. The consequent upregulation of nuclear FoxO3a and its binding to the promoter of the gene encoding Bmi-1 account for the self-renewal activity of breast cancer stem-like cells and their growth in a xenograft mouse model.

Aged garlic extract and S-allylcysteine increase the GLUT3 and GCLC expression levels in cerebral ischemia.

During cerebral ischemia, energy restoration through the regulation of glucose transporters and antioxidant defense mechanisms is essential to maintain cell viability. Antioxidant therapy has been considered effective to attenuate brain damage; moreover, the regulation of transcription factors that positively regulate the expression of glucose transporters is associated with this therapy. Recently, it has been reported that the use of antioxidants such as S-allylcysteine (SAC), a component of aged garlic extract (AGE), improves survival in experimental models of cerebral ischemia. The aim of this study was to determine the effect of AGE and SAC on the level of mRNA expression of the main neuronal glucose transporter (GLUT3) and the glutamate cysteine ligase catalytic subunit (GCLC) in rats with transient focal cerebral ischemia. Cerebral ischemia was induced in male Wistar rats by middle cerebral artery occlusion (MCAO) for 2 h. The animals were sacrificed after different reperfusion times (0-48 h). Animals injected with AGE (360 mg/kg, intraperitoneally (i.p.)) and SAC (300 mg/kg, i.p.) at the beginning of reperfusion were sacrificed after 2 h. The mRNA expression level was analyzed in the fronto-parietal cortex using quantitative polymerase chain reaction (qPCR). Two major increases in GLUT3 expression at 1 h and 24 h of reperfusion were found. Both treatments increased GLUT3 and GCLC mRNA levels in control and under ischemic/reperfusion injury animals. This data suggests that SAC and AGE might induce neuroprotection, while controlling reactive oxygen species (ROS) levels, as indicated by the increase in GCLC expression, and regulating the energy content of the cell by increasing glucose transport mediated by GLUT3.

135 FH and FGSH prepared from fish skin gelatin using ginger protease alleviates DSS-induced Colitis via Nrf2 pathway

Abstract Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disease affecting the colon, and its incidence is rising worldwide. Hence, it is important to apply new strategies to cope with the IBD. We previously found that hydrolysate fractions (HF) and fish skin gelatin hydrolysate (FSGH), isolated from fish skin gelatin hydrolysate using ginger protease, could exert antioxidant effects in IPEC-J2 cells. Here, we aimed to evaluate the effects of FH and FGSH in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Mouse were treated with 3% DSS in their drinking water for 7 days to induce acute colitis. FH (200 mg/kg) and FGSH (200 mg/kg) was administered for 7 days before and during DSS treatment via oral gavage once per day. Mouse were sacrificed at day 7 after colitis induction. The results showed that FH and FGSH significantly ameliorated the clinical symptoms of DSS-induced mice colitis, such as weight loss, disease activity index (DAI), colon shortening, spleen hypertrophy and histological scores. For MPO activity, an important indicator of neutrophil infiltration and acute inflammation in DSS-induced mouse colitis, FH and FGSH significantly reduced the level of DSS-induced hyperactivated MPO. Treatment with FH and FGSH also significantly inhibited the secretion proinflammatory cytokines TNF-α, IL-8 and IL-12 in DSS-induced mouse colitis. And FH and FGSH administration significantly increased expression of antioxidant enzymes GCLM, GCLC and GPX4 and maintained tight junction in colon tissues. Furthermore, we found that FH and FGSH significantly increased the level of Nrf2 in colon tissues, it indicated that FH and FGSH may alleviated colitis by Nrf2 pathway. Overall, these results suggested that FH and FGSH could be a potential promising candidate for the treatment of IBD.

Abstract C063: A novel Nrf2 inhibitor suppresses proliferation and enhances the sensitivity of cancer cells to chemotherapy

Abstract The Nrf2-Keap1-ARE pathway, a master defense mechanism against oxidative and electrophilic stresses, has emerged as a target for cancer therapy. Activation of Nrf2 has been a common strategy for cancer prevention, as Nrf2 deficiency enhances susceptibility to carcinogens in a wide variety of preclinical models. However, accumulating evidence suggests a tumor-promoting role for Nrf2 in established tumors. Constitutive activation of Nrf2 because of gain of functionmutations in NFE2L2 or loss of function mutations in its negative regulator Keap1, has been found in many cancers, especially lung cancer, and is associated with chemoresistance and poor survival in human patients. Nrf2 inhibitors, therefore, provide a potential novel strategy for treating cancer. Here, we report a novel Nrf2 inhibitor, MSU225, identified from an existing chemistry library. MSU225 dose-dependently downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase reporter assay without cytotoxicity. MSU225 also decreased the expression of Nrf2 downstream targets, including HO-1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6 in A549 human lung cancer cells, in which Keap1 is mutated and Nrf2 activation is constitutive. Notably, MSU225 induced a striking and significant (p<0.05) decrease of Nrf2 protein in A549 cells. The effect of MSU225 on Nrf2 protein levels can be blocked with the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. Ubiquitination of Nrf2 was also increased after MSU225 treatment. Interestingly, MSU225 also decreased Nrf2 protein levels in Keap1 knockout and βTrCP knockout cells, which are the two major pathways inducing Nrf2 degradation. Nrf2 involvement in cancer cell proliferation is well known. MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in both 2D cell culture and soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to chemotherapies including doxorubicin, carboplatin, 5-fluorouracil and Topotecan. Our results suggest that MSU225 is a novel Nrf2 inhibitor that enhances the degradation of Nrf2 in a Keap1 and βTrCP independent manner. In vivo studies to assess the efficacy of MSU225 for treating experimental lung cancer are ongoing. Citation Format: Di Zhang, Kelly Aldrich, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. A novel Nrf2 inhibitor suppresses proliferation and enhances the sensitivity of cancer cells to chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C063. doi:10.1158/1535-7163.TARG-19-C063