ABSTRACT
Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.
Subject(s)
Escherichia coli , Glutathione , Metabolic Engineering , Escherichia coli/genetics , Escherichia coli/metabolism , Glutathione/metabolism , Glutathione/biosynthesis , Glutathione/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Glutathione Synthase/genetics , Glutathione Synthase/metabolism , Glutamate-Cysteine Ligase/genetics , Glutamate-Cysteine Ligase/metabolism , FermentationABSTRACT
l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E. coli, several genes associated with GSH and l-cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP-binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine-based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole-cell catalytic conditions, the engineered strain GSH17-FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added l-Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high-value metabolites that rely on ATP consumption.
Subject(s)
Adenosine Triphosphate , Adenosine , Escherichia coli , Glutathione , Metabolic Engineering , Glutathione/metabolism , Glutathione/biosynthesis , Adenosine Triphosphate/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , Adenosine/metabolism , Adenosine/geneticsABSTRACT
Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.
Subject(s)
COVID-19 , Energy Metabolism , Ketones , Respiratory Distress Syndrome , SARS-CoV-2 , T-Lymphocytes , 3-Hydroxybutyric Acid/biosynthesis , 3-Hydroxybutyric Acid/metabolism , Amino Acids/biosynthesis , Amino Acids/metabolism , Animals , COVID-19/complications , COVID-19/immunology , COVID-19/pathology , Diet, Ketogenic , Esters/metabolism , Glutathione/biosynthesis , Glutathione/metabolism , Glycolysis , Interferon-gamma/biosynthesis , Ketone Bodies/metabolism , Ketones/metabolism , Mice , Orthomyxoviridae/pathogenicity , Oxidation-Reduction , Oxidative Phosphorylation , Respiratory Distress Syndrome/complications , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/virology , SARS-CoV-2/pathogenicity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
OBJECTIVES: Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury. METHODS: UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting. RESULTS: Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01). CONCLUSIONS: There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.
Subject(s)
Colitis, Ulcerative , Colitis , Glutathione , Animals , Antioxidants , Aspartate Aminotransferases , Colitis/chemically induced , Colitis, Ulcerative/metabolism , Colon/pathology , Glutathione/biosynthesis , Liver/metabolism , Peroxidase/metabolism , Rats , Trinitrobenzenesulfonic AcidABSTRACT
Extracellular glutathione (GSH) and oxidized glutathione (GSSG) can modulate the function of the extracellular calcium sensing receptor (CaSR). The CaSR has a binding pocket in the extracellular domain of CaSR large enough to bind either GSH or GSSG, as well as the naturally occurring oxidized derivative L-cysteine glutathione disulfide (CySSG) and the compound cysteinyl glutathione (CysGSH). Modeling the binding energies (ΔG) of CySSG and CysGSH to CaSR reveals that both cysteine derivatives may have greater affinities for CaSR than either GSH or GSSG. GSH, CySSG, and GSSG are found in circulation in mammals and, among the three, CySSG is more affected by HIV/AIDs and aging than either GSH or GSSG. The beta-carbon linkage of cysteine in CysGSH may model a new class of calcimimetics, exemplified by etelcalcetide. Circulating glutathionergic compounds, particularly CySSG, may mediate calcium-regulatory responses via receptor-binding to CaSR in a variety of organs, including parathyroids, kidneys, and bones. Receptor-mediated actions of glutathionergics may thus complement their roles in redox regulation and detoxification. The glutathionergic binding site(s) on CaSR are suggested to be a target for development of drugs that can be used in treating kidney and other diseases whose mechanisms involve CaSR dysregulation.
Subject(s)
Extracellular Space/metabolism , Glutathione/metabolism , Receptors, Calcium-Sensing/metabolism , Animals , Biomarkers , Calcium/chemistry , Calcium/metabolism , Cysteine/analogs & derivatives , Glutathione/analogs & derivatives , Glutathione/biosynthesis , Glutathione/chemistry , Glutathione Disulfide , Humans , Molecular Structure , Organ Specificity , Oxidation-Reduction , Protein Binding , Receptors, Calcium-Sensing/chemistry , Receptors, Calcium-Sensing/genetics , Structure-Activity RelationshipABSTRACT
OBJECTIVES@#Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury.@*METHODS@#UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting.@*RESULTS@#Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01).@*CONCLUSIONS@#There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.
Subject(s)
Animals , Rats , Antioxidants , Aspartate Aminotransferases , Colitis/chemically induced , Colitis, Ulcerative/metabolism , Colon/pathology , Glutathione/biosynthesis , Liver/metabolism , Peroxidase/metabolism , Trinitrobenzenesulfonic AcidABSTRACT
Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.
Subject(s)
Antiporters/chemistry , Antiporters/metabolism , Cystine/metabolism , Glutamic Acid/metabolism , Glutathione/biosynthesis , Amino Acid Transport System y+/chemistry , Amino Acid Transport System y+/metabolism , Antiporters/genetics , Biochemistry , Cryoelectron Microscopy , Cysteine/metabolism , Fusion Regulatory Protein 1, Heavy Chain/chemistry , Fusion Regulatory Protein 1, Heavy Chain/metabolism , HEK293 Cells , Humans , Neoplasms , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Up-RegulationABSTRACT
Glucose metabolism modulates the islet ß cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.
Subject(s)
Glucose/metabolism , Glutathione/biosynthesis , Pyruvate Carboxylase/metabolism , Adult , Animals , Antioxidants/physiology , Female , Glutathione/metabolism , Humans , Insulin/metabolism , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Middle Aged , Oxidation-Reduction , Oxidative Stress/physiology , Primary Cell CultureABSTRACT
Culex pipiens pallens is an important vector of lymphatic filariasis and epidemic encephalitis. Mosquito control is the main strategy used for the prevention of mosquito-borne diseases. Bacillus thuringiensis israelensis (Bti) is an entomopathogenic bacterium widely used in mosquito control. In this study, we profiled the microbiota and transcriptional response of the larvae of Cx. pipiens pallens exposed to different concentrations of Bti. The results demonstrated that Bti induced a significant effect on both the microbiota and gene expression of Cx. pipiens pallens. Compared to the control group, the predominant bacteria changed from Actinobacteria to Firmicutes, and with increase in the concentration of Bti, the abundance of Actinobacteria was gradually reduced. Similar changes were also detected at the genus level, where Bacillus replaced Microbacterium, becoming the predominant genus in Bti-exposed groups. Furthermore, alpha diversity analysis indicated that Bti exposure changed the diversity of the microbota, possibly because the dysbiosis caused by the Bti infection inhibits some bacteria and provides opportunities to other opportunistic taxa. Pathway analysis revealed significant enhancement for processes associated with sphingolipid metabolism, glutathione metabolism and glycerophospholipid metabolism between all Bti-exposed groups and control group. Additionally, genes associated with the Toll and Imd signaling pathway were found to be notably upregulated. Bti infection significantly changed the bacterial community of larvae of Cx. pipiens pallens.
Subject(s)
Bacillus thuringiensis/pathogenicity , Bacteria/classification , Culex/growth & development , Gene Expression Profiling/methods , Insect Proteins/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA/methods , Animals , Bacteria/genetics , Bacteria/isolation & purification , Culex/microbiology , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Gastrointestinal Microbiome , Gene Expression Regulation , Glutathione/biosynthesis , Glycerophospholipids/biosynthesis , Larva/growth & development , Larva/microbiology , Metabolic Networks and Pathways , Mosquito Control/methods , Phylogeny , Sequence Analysis, RNA , Sphingolipids/biosynthesisABSTRACT
Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.
Subject(s)
Aspartate Aminotransferase, Cytoplasmic/antagonists & inhibitors , Ferroptosis , Pancreatic Neoplasms/metabolism , Animals , Antioxidants/pharmacology , Aspartate Aminotransferase, Cytoplasmic/genetics , Aspartate Aminotransferase, Cytoplasmic/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival/drug effects , Cystine/metabolism , Ferroptosis/drug effects , Glutathione/biosynthesis , Humans , Iron/metabolism , Mice , Mitochondria/metabolism , Pancreatic Neoplasms/pathologyABSTRACT
Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.
Subject(s)
Acidithiobacillus/genetics , Acidithiobacillus/metabolism , Glutathione Synthase/genetics , Iron/metabolism , Salt Tolerance/genetics , Acidithiobacillus/drug effects , Escherichia coli/genetics , Glutathione/biosynthesis , Hydrogen-Ion Concentration , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Sodium Chloride/pharmacologyABSTRACT
Ferroptosis is a form of iron-dependent regulated cell death driven by uncontrolled lipid peroxidation. Mitochondria are double-membrane organelles that have essential roles in energy production, cellular metabolism, and cell death regulation. However, their role in ferroptosis has been unclear and somewhat controversial. In this Perspective, I summarize the diverse metabolic processes in mitochondria that actively drive ferroptosis, discuss recently discovered mitochondria-localized defense systems that detoxify mitochondrial lipid peroxides and protect against ferroptosis, present new evidence for the roles of mitochondria in regulating ferroptosis, and outline outstanding questions on this fascinating topic for future investigations. An in-depth understanding of mitochondria functions in ferroptosis will have important implications for both fundamental cell biology and disease treatment.
Subject(s)
Ferroptosis/genetics , GTP Cyclohydrolase/antagonists & inhibitors , Iron/metabolism , Mitochondria/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amino Acid Transport System y+/antagonists & inhibitors , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Cells, Cultured , Ferroptosis/drug effects , GTP Cyclohydrolase/genetics , GTP Cyclohydrolase/metabolism , Gene Expression Regulation , Glutathione/antagonists & inhibitors , Glutathione/biosynthesis , Humans , Lipid Peroxidation/drug effects , Mitochondria/drug effects , Mitochondria/enzymology , Oxidants/pharmacology , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , S100 Calcium-Binding Protein A4/antagonists & inhibitors , S100 Calcium-Binding Protein A4/genetics , S100 Calcium-Binding Protein A4/metabolism , Signal Transduction , Ubiquinone/antagonists & inhibitors , Ubiquinone/metabolismABSTRACT
Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Phenformin/pharmacology , STAT1 Transcription Factor/metabolism , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Drug Synergism , Electron Transport Complex I/antagonists & inhibitors , Energy Metabolism/drug effects , Female , Glutathione/antagonists & inhibitors , Glutathione/biosynthesis , Humans , Interferon-gamma/administration & dosage , Interferon-gamma/deficiency , Interferon-gamma/metabolism , MCF-7 Cells , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, SCID , NAD(P)H Dehydrogenase (Quinone)/antagonists & inhibitors , NAD(P)H Dehydrogenase (Quinone)/metabolism , Naphthoquinones/administration & dosage , Oxidative Stress/drug effects , Phenformin/administration & dosage , Poly I-C/administration & dosage , Reactive Oxygen Species/metabolism , STAT1 Transcription Factor/agonists , Xenograft Model Antitumor AssaysABSTRACT
Oxidative stress resulting from the depletion of glutathione (GSH) level plays a vital role in generating various degenerative diseases, including type 2 diabetes (T2D). We tested the hypothesis that depleted glutathione levels can be enhanced and the impaired glucose metabolism can be prevented by supplementing Allium hookeri, a herb rich in organosulfur compounds, in a High Fat (HF) diet-induced T2D Male Sprague Dawley rat model. The experimental rats were divided into three groups (n = 6), namely normal diet, high-fat diet, and high-fat diet treated with A.hookeri methanolic leaf extract (250 mg/kg). Consumption of HF diet along with the plant extract resulted in significant reduction of the body weight (7.08%-14.89%) and blood glucose level (6.5%-16.4%) from the 13th week onward. There was a significant decrease in reactive oxygen species, oxidized glutathione (GSSG) levels, and an increase in GSH level in skeletal muscle tissues supplemented with the plant extract. The protein expressions of the signaling molecules such as GCLC and GR involved in GSH synthesis and of GLUT4 in glucose transport were also upregulated in the skeletal muscle tissues of the plant extract-treated group. Results of in vitro studies with muscle cell line (L6) further demonstrated the beneficial effect of the plant extract in increasing glucose uptake and maintaining the GSH/GSSH equilibrium via regulation of protein expression of GCLC/GR/GLUT4 signaling molecules in sodium palmitate (0.75 mM) treated cells. Overall this study suggests that dietary supplementation with Allium hookeri, can restore the glutathione level and regulate the blood glucose level in T2D.
Subject(s)
Allium/chemistry , Diabetes Mellitus, Type 2/metabolism , Glucose/metabolism , Glutathione/biosynthesis , Methanol/chemistry , Plant Extracts/pharmacology , Plant Leaves/chemistry , Animals , Glutathione/metabolism , Glutathione Disulfide/metabolism , Male , Plant Extracts/isolation & purification , Rats , Rats, Sprague-DawleyABSTRACT
Glutathione (GSH), the main redox buffer, has long been recognized as a pivotal modulator of tumor initiation, progression and metastasis. It is also implicated in the resistance of platinum-based chemotherapy and radiation therapy. Therefore, depleting intracellular GSH was considered a potent solution to combating cancer. However, reducing GSH within cancer cells alone always failed to yield desirable therapeutic effects. In this regard, the convergence of GSH-scavenging agents with therapeutic drugs has thus been pursued in clinical practice. Unfortunately, the therapeutic outcomes are still unsatisfactory due to untargeted drug delivery. Advanced nanomedicine of synergistic GSH depletion and cancer treatment has attracted tremendous interest because they promise to deliver superior therapeutic benefits while alleviating life-threatening side effects. In the past five years, the authors and others have demonstrated that numerous nanomedicines, by simultaneously delivering GSH-depleting agents and therapeutic components, boost not only traditional chemotherapy and radiotherapy but also multifarious emerging treatment modalities, including photodynamic therapy, sonodynamic therapy, chemodynamic therapy, ferroptosis, and immunotherapy, to name a few, and achieved decent treatment outcomes in a large number of rodent tumor models. In this review, we summarize the most recent progress in engineering nanomedicine for GSH depletion-enhanced cancer therapies. Biosynthesis of GSH and various types of GSH-consuming strategies will be briefly introduced. The challenges and perspectives of leveraging nanomedicine for GSH consumption-augmented cancer therapies will be discussed at the end.
Subject(s)
Glutathione/biosynthesis , Nanomedicine , Neoplasms/therapy , Drug Resistance, Neoplasm , Ferroptosis/drug effects , Glutathione/chemistry , Glutathione/deficiency , Humans , Neoplasms/drug therapy , Neoplasms/immunology , Oxidants/chemistry , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic use , Reactive Oxygen Species/chemistry , Reactive Oxygen Species/metabolismABSTRACT
Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.
Subject(s)
Antioxidants/pharmacology , Epithelial Cells/drug effects , Lutein/analogs & derivatives , Oxidative Stress/drug effects , Prodrugs/pharmacology , Retinal Pigment Epithelium/drug effects , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Catalase/biosynthesis , Catalase/genetics , Cell Line , Cytochromes c/biosynthesis , Cytochromes c/genetics , Drug Evaluation, Preclinical , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Glutathione/biosynthesis , Glutathione/genetics , Glutathione Peroxidase/biosynthesis , Glutathione Peroxidase/genetics , Humans , Hydrogen Peroxide/toxicity , Lutein/chemistry , Lutein/pharmacology , MAP Kinase Signaling System/drug effects , Macular Degeneration/drug therapy , Molecular Structure , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/cytologyABSTRACT
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.
Subject(s)
Cinnamates/pharmacology , Cytoprotection/drug effects , Glutathione/biosynthesis , Hepatocytes/pathology , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Antioxidant Response Elements/genetics , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Cell Death/drug effects , Cinnamates/chemistry , Glutathione/metabolism , Hep G2 Cells , Hepatocytes/drug effects , Humans , Luciferases/metabolism , NF-E2-Related Factor 2/agonists , Protective Agents/pharmacology , tert-ButylhydroperoxideABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.
When building healthy tissue, the human body must carefully control the growth of new cells to prevent them from becoming cancerous. A core component of this regulation is the protein mTORC1, which responds to various growth-stimulating factors and nutrients, and activates the chemical reactions cells need to grow. Part of this process involves controlling 'nutrient-sensing transcription factors' proteins that regulate the activity of specific genes based on the availability of different nutrients. One of these nutrient-sensing transcription factors, ATF4, has recently been shown to be involved in some of the processes triggered by mTORC1. The role this factor plays in how cells respond to stress such as when specific nutrients are depleted, protein folding is disrupted or toxins are present is well-studied. But how it reacts to the activation of mTORC1 is less clear. To bridge this gap, Torrence et al. studied mouse embryonic cells and human prostate cancer cells grown in the laboratory, to see whether mTORC1 influenced the behavior of ATF4 differently than cellular stress. Cells were treated either with insulin, which activates mTORC1, or an antibiotic that sparks the stress response. The cells were then analyzed using a molecular tool to see which genes were switched on by ATF4 following treatment. This revealed that less than 10% of the genes activated by ATF4 during cellular stress are also activated in response to mTORC1-driven growth. Many of the genes activated in both scenarios were involved in synthesizing and preparing the building blocks that make up proteins. This was consistent with the discovery that ATF4 helps mTORC1 stimulate growth by promoting protein synthesis. Torrence et al. also found that mTORC1's regulation of ATF4 stimulated the synthesis of glutathione, the most abundant antioxidant in cells. The central role mTORC1 plays in controlling cell growth means it is important to understand how it works and how it can lead to uncontrolled growth in human diseases. mTORC1 is activated in many overgrowth syndromes and the majority of human cancers. These new findings could provide insight into how tumors coordinate their drive for growth while adapting to cellular stress, and reveal new drug targets for cancer treatment.
Subject(s)
Activating Transcription Factor 4/metabolism , Glutathione/biosynthesis , Mechanistic Target of Rapamycin Complex 1/drug effects , Activating Transcription Factor 4/genetics , Animals , Cell Line , Cell Line, Tumor , Embryo, Mammalian , Fibroblasts , Humans , Insulin/pharmacology , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Signal TransductionABSTRACT
Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.
Subject(s)
Carcinoma, Pancreatic Ductal/drug therapy , Colonic Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Genes, ras , Glutathione/biosynthesis , Methionine/analogs & derivatives , Mutation , NADPH Oxidases/antagonists & inhibitors , Onium Compounds/pharmacology , Ovarian Neoplasms/drug therapy , Pancreatic Neoplasms/drug therapy , Sulfoxides/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Pancreatic Ductal/enzymology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Death/drug effects , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Female , Genes, p53 , Glutamate-Cysteine Ligase/antagonists & inhibitors , Glutamate-Cysteine Ligase/metabolism , HCT116 Cells , Humans , Methionine/pharmacology , Mice, Nude , Mice, Transgenic , NADPH Oxidases/metabolism , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Oxidative Stress , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
As senescence progresses, the sensitivity of wheat organs to plant hormones during the grain-filling stages cannot be ignored. Especially under water deficit situation, non-leaf organs (spikes) have better photosynthesis and drought-tolerance traits than flag leaves. However, the mechanism of ethylene synthesis in wheat organs under water deficit remains unclear. We have studied the influence of water deficit in wheat flag leaves and spike bracts on photosynthetic parameters and on the expression of key enzymes involved in the ethylene biosynthesis pathway during the late grain-filling stages. More stable chlorophyll content (Chl), maximum PSII quantum yield (Fv/Fm), nonphotochemical quenching (NPQ) and maximal efficiency of PSII photochemistry under light adaptation (Fv'/Fm') were observed in the spike bracts than that in the flag leaves during the late grain-filling stages. In addition, the activity of glutathione reductase (GR), γ-glutamylcysteine synthetase (γ-ECS), 1-aminocyclopropane-1-carboxylic (ACC) acid synthase (ACS), and ACC oxidase (ACO) induced ethylene synthesis and influenced plant growth. Further analysis of genes encoding cysteine-ethylene related proteins (γ-ECS, GR, ACO, ACS1, and ASC2) demonstrated that ear organs and flag leaves exhibited different expression patterns. These findings will facilitate future investigations of the regulatory senescence response mechanisms of cysteine interaction with ethylene in wheat under conditions of drought stress.