Inhibitor Of GSH Synthesis Research Articles

Loss of C/EBPδ enhances IR-induced cell death by promoting oxidative stress and mitochondrial dysfunction

Exposure of cells to ionizing radiation (IR) generates reactive oxygen species (ROS). This results in increased oxidative stress and DNA double strand breaks (DSBs) which are the two underlying mechanisms by which IR causes cell/tissue injury. Cells that are deficient or impaired in the cellular antioxidant response are susceptible to IR-induced apoptosis. The transcription factor CCAAT enhancer binding protein delta (Cebpd, C/EBPδ) has been implicated in the regulation of oxidative stress, DNA damage response, genomic stability and inflammation. We previously reported that Cebpd-deficient mice are sensitive to IR and display intestinal and hematopoietic injury, however the underlying mechanism is not known. In this study, we investigated whether an impaired ability to detoxify IR-induced ROS was the underlying cause of the increased radiosensitivity of Cebpd-deficient cells.We found that Cebpd-knockout (KO) mouse embryonic fibroblasts (MEFs) expressed elevated levels of ROS, both at basal levels and after exposure to gamma radiation which correlated with increased apoptosis, and decreased clonogenic survival. Pre-treatment of wild type (WT) and KO MEFs with polyethylene glycol-conjugated Cu-Zn superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) combination prior to irradiation showed a partial rescue of clonogenic survival, thus demonstrating a role for increased intracellular oxidants in promoting IR-induced cell death. Analysis of mitochondrial bioenergetics revealed that irradiated KO MEFs showed significant reductions in basal, adenosine triphosphate (ATP)-linked, maximal respiration and reserved respiratory capacity and decrease in intracellular ATP levels compared to WT MEFs indicating they display mitochondrial dysfunction. KO MEFs expressed significantly lower levels of the cellular antioxidant glutathione (GSH) and its precursor- cysteine as well as methionine. In addition to its antioxidant function, GSH plays an important role in detoxification of lipid peroxidation products such as 4-hydroxynonenal (4-HNE). The reduced GSH levels observed in KO MEFs correlated with elevated levels of 4-HNE protein adducts in irradiated KO MEFs compared to respective WT MEFs.We further showed that pre-treatment with the GSH precursor, N-acetyl L-cysteine (NAC) prior to irradiation showed a significant reduction of IR-induced cell death and increases in GSH levels, which contributed to the overall increase in clonogenic survival of KO MEFs. In contrast, pre-treatment with the GSH synthesis inhibitor- buthionine sulfoximine (BSO) further reduced the clonogenic survival of irradiated KO MEFs.This study demonstrates a novel role for C/EBPδ in protection from basal as well as IR-induced oxidative stress and mitochondrial dysfunction thus promoting post-radiation survival.

Application of glutathione and dithiothreitol to oil seed rape (Brassica napus L.) roots affects cadmium distribution in roots and inhibits Cd translocation to shoots

ABSTRACTPreviously we have shown that site-specific application of glutathione (GSH) to roots inhibits cadmium (Cd) translocation from roots to shoots. In this study, we investigated the effects of chemical compounds containing sulfur, such as dithiothreitol (DTT), potassium sulfate (K2SO4) and a GSH synthesis inhibitor, buthionine sulfoximine (BSO), on the behavior of Cd in oilseed rape plants (Brassica napus L.). We found that DTT, just like GSH, also inhibited Cd translocation from roots to shoots significantly. However, the inhibitory effects of Cd behaviors differed between GSH and DTT. Cd efflux from root cells was activated by GSH and inactivated by DTT. Positron imaging experiments revealed that there are minor differences in the effects of GSH and DTT on Cd adsorption to the surface of roots. These results provide insights into the function of GSH involved in the inhibitory effects of Cd translocation from roots to shoots.

Abstract A41: Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells

Abstract Pancreatic cancer is a devastating and lethal malignancy, estimated to kill over 40,000 Americans per year. Little has changed in the treatment of this disease over the past half-century, highlighting the need for an improved understanding of the molecular mechanisms driving tumor development and maintenance. The malignant behavior of tumor cells is underwritten by alterations to their cellular metabolism, which is optimized to meet the catabolic needs of a rapidly dividing cell. One of the consequences of this oncogenic growth is increased generation of reactive oxygen species (ROS), a potentially toxic byproduct of many metabolic reactions. Interestingly, oncogenes such as Kras, the dominant driver of pancreatic cancer, can counteract ROS toxicity through activation of an antioxidant program, creating a highly reducing intracellular environment. Cysteine is the rate-limiting precursor for the synthesis of glutathione (GSH), the preeminent antioxidant of the cell, and as such may represent a key metabolite in the maintenance of redox balance in tumor cells. In this study we explore the importance of cysteine metabolism in the viability of pancreatic cancer cells and test the effects of cysteine deprivation and the pharmacological inhibition of cysteine uptake on a variety of pancreatic cancer cell lines. To explore the role of cysteine in pancreatic cancer growth, we cultured a panel of human and murine pancreatic cancer cell lines in the absence of cysteine. We found that cysteine withdrawal rapidly induced a peculiar form of cell death called ferroptosis, which can result from the catastrophic accumulation of certain lipid peroxides. Cystine, the oxidized form of cysteine, is the predominant extracellular source of cysteine in most cells, and it is normally imported into the cell by a specific amino acid transporter known as system xc-. We show that pharmacological inhibition of sytem xc- causes rapid cell death in most pancreatic cancer cells through ferroptosis. We find, however, that some cells do exhibit baseline resistance to cysteine deprivation or system xc- inhibition. We hypothesized that these cells utilize methionine to synthesize cysteine through transsulfuration. We show that these cells can be sensitized to system xc- inhibition when co-treated with propargyglycine (PPG), an irreversible inhibitor of gamma-cystathionase, an enzyme required for transsulfuration. These findings imply that cysteine is an important substrate for appropriate redox homeostasis in these cells. To further explore if GSH synthesis is the key mediator of cysteine-deprivation induced cell death, we treated pancreatic tumor cells with buthionine sulfoxamine (BSO), a potent and specific inhibitor of GSH synthesis. To our surprise, cells treated with BSO do not exhibit any of the ill-effects we see in cysteine-deprived pancreatic cancer cells. Therefore, it is unlikely that the cell death seen in cells deprived of cysteine is due solely to GSH depletion, but possibly due to the activity of other antioxidant pathways that require cysteine. In conclusion, we find that pancreatic cancer cells are highly sensitive to cysteine-deprivation and to treatment with system xc- inhibitors, and that the cell death caused by this stress is an iron-dependent, oxidative form of cell death called ferroptosis. Furthermore, it appears that GSH, the primary antioxidant molecule of the cell, is not solely responsible for mediating this cell death, arguing that cysteine may play other important roles in maintaining redox homeostasis in these cells. Given that previous studies show that global system xc- knockout mice do not exhibit any changes in cellular GSH content, these findings may represent a targetable metabolic dependency in pancreatic cancer cells. Citation Format: Michael A. Badgley, Carmine F. Palermo, Stephen A. Sastra, Brent R. Stockwell, Kenneth P. Olive. Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A41.

Application of Glutathione as Anti-Oxidative and Anti-Aging Drugs.

Glutathione (GSH), an abundant tripeptidyl molecule, plays pivotal roles in protecting cells against oxidative stress-induced cellular damage and in detoxifying xenobiotics and drug metabolism. GSH is now entering a new era of therapeutic applications. Decreased GSH levels are associated with the common features of aging as well as of a wide range of pathological conditions, including neurodegenerative disorders. Notably, GSH depletion and/or alterations in its metabolism appear to be crucial in the onset of Parkinson's disease. Despite the fact that GSH is required for cell survival, the molecular mechanism that links GSH depletion to cell death remains poorly understood. Recently, considerable attention has been focused on a newly defined type of cell death: irondependent cell death, also referred to as "ferroptosis". The iron chelator deferoxamine nearly abolishes ferroptosis induced by inhibiting GSH synthesis or cystine uptake by the xCT transporter. Deferoxamine preferentially abrogates the intralysosomal accumulation of iron and inhibits oxidative stress-induced lysosomal membrane permeabilization and cell death. The use of GSH and a prodrug derived from it can be useful, since the dysfunction of the GSH redox system appears to cause a variety of diseases including neurodegenerative disorders. However, the effectiveness of GSH as a therapeutic agent is limited because of its low bioavailability. We also review trials that have been designed to cope with this difficulty; e.g. the use of precursors such as N-acetyl cysteine and chemical modification such as methylation.

Quantitatively evaluating detoxification of the hepatotoxic microcystin-LR through the glutathione (GSH) pathway in SD rats.

Glutathione (GSH) plays crucial roles in antioxidant defense and detoxification metabolism of microcystin-LR (MC-LR). However, the detoxification process of MC-LR in mammals remains largely unknown. This paper, for the first time, quantitatively analyzes MC-LR and its GSH pathway metabolites (MC-LR-GSH and MC-LR-Cys) in the liver of Sprague-Dawley (SD) rat after MC-LR exposure. Rats received intraperitoneal (i.p.) injection of 0.25 and 0.5 lethal dose 50 (LD50) of MC-LR with or without pretreatment of buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The contents of MC-LR-GSH were relatively low during the experiment; however, the ratio of MC-LR-Cys to MC-LR reached as high as 6.65 in 0.5 LD50 group. These results demonstrated that MC-LR-GSH could be converted to MC-LR-Cys efficiently, and this metabolic rule was in agreement with the data of aquatic animals previously reported. MC-LR contents were much higher in BSO + MC-LR-treated groups than in the single MC-LR-treated groups. Moreover, the ratio of MC-LR-Cys to MC-LR decreased significantly after BSO pretreatment, suggesting that the depletion of GSH induced by BSO reduced the detoxification of MCs. Moreover, MC-LR remarkably induced liver damage, and the effects were more pronounced in BSO pretreatment groups. In conclusion, this study verifies the role of GSH in the detoxification of MC-LR and furthers our understanding of the biochemical mechanism for SD rats to counteract toxic cyanobacteria.

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis.

Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

Micheliolide overcomes KLF4-mediated cisplatin resistance in breast cancer cells by downregulating glutathione.

Micheliolide (MCL) is a promising novel compound with broad-spectrum anticancer activity. However, little is known regarding its action and mechanism in breast cancer. To explore the potential therapeutic application of MCL as a chemosensitivity modulator, this study investigated the effects of MCL on cisplatin sensitivity in breast cancer and the underlying mechanisms. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay and a xenograft tumor model, MCL enhanced the cisplatin sensitivity of the breast cancer cell line MCF-7 both in vitro and in vivo. Treatment of MCF-7 cells with low-dose cisplatin (10 µM) was sufficient to enrich the proportion of ALDH+ cells and upregulate Krüppel-like factor 4 (KLF4) expression. The results obtained from knockdown and overexpression experiments demonstrate that KLF4 is both necessary and sufficient to induce a cisplatin resistance phenotype in breast cancer cells. Furthermore, the glutathione (GSH) content was elevated in MCF-7 cells after overexpression of KLF4. KLF4-mediated resistance to cisplatin was found to be abrogated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis. MCL induced GSH depletion and severe cell death in KLF4-overexpressing MCF-7 cells following exposure to cisplatin. Therefore, these results suggest that MCL-mediated direct depletion of GSH represents a major mechanism in reversing KLF4-induced cisplatin resistance in MCF-7 cells.

Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells.

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. Our study revealed that CA treatment significantly reduced the viability of human colon cancer HCT116, SW480, and HT-29 cells. Treatment with CA induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, activation of caspase-9, and -3, and the cleavage of PARP in HCT116 cells. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of STAT3 in HCT116 cells by blocking the phosphorylation of upstream JAK2 and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, D2, and D3. In STAT3-overexpressed HCT116 cells, CA inhibited cell viability and the expression of cyclin D1 and survivin. Furthermore, CA treatment induced the generation of ROS in these colon cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine abrogated the inhibitory effect of CA on the JAK2-STAT3/Src-STAT3 signaling and rescued cells from CA-induced apoptosis by blocking the induction of p53 and the cleavage of caspase-3 and PARP in HCT116 cells. However, L-buthionine-sulfoximine, a pharmacological inhibitor of GSH synthesis, increased CA-induced ROS production, thereby potentiating apoptotic effect of CA. In conclusion, our study provides the first report that CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases, and inhibition of STAT3 signaling pathway. © 2015 Wiley Periodicals, Inc.

Exogenous nitric oxide-mediated GSH-PC synthesis pathway in tomato under copper stress

Nitric oxide (NO) is a bioactive molecule that is extensively used at various biotic and abiotic stresses. This study investigated the law governing the variation of related enzymatic activity and metabolites in exogenous NO-mediated GSH-PC synthesis pathway in tomato solution culture subjected to copper stress. Results demonstrated that relative to control copper stress was more effective in the activation of γ-ECS and GS in tomato. Moreover, sharp increases in root GSH and PCs were observed, which keep upward as the process continued. Moreover, adding exogenous SNP (NO donor) can further improve γ-ECS and GS activities in tomato roots and facilitate the synthesis of GSH and PCs, thereby enhancing its peroxide removal ability, chelating excessive Cu2+, and reducing its biotoxicity. The GSH-PC metabolism in the tomato leaves lagged behind that in the roots to a certain extent. Although exogenous GSH synthesis inhibitor BSO inhibited γ-ECS activity in tomato roots, as well as GSH and PC syntheses, adding SNP can counteract this effect by lessening the influence to the PCs in leaves. Under copper stress, exogenous NO may stimulate a signaling mechanism and reduce the biotoxicity and oxidative damage caused by excessive Cu2+ through activating or enhancing the enzymatic and non-enzymatic systems in the GSH-PC synthesis pathway.

Effect of the Acidic Dental Resin Monomer 10-methacryloyloxydecyl Dihydrogen Phosphate on Odontoblastic Differentiation of Human Dental Pulp Cells.

Although 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) is frequently used as an acidic resin monomer in dental adhesives, its effect on dental pulp cells (DPCs) has been rarely reported. The purpose of this study was to examine the effects of 10-MDP on the inflammatory response and odontoblastic differentiation of DPCs at minimally toxic concentrations. We found that 10-MDP caused the release of inflammatory cytokines including NO, PGE2, iNOS, COX-2, TNF-α, IL-1β, IL-6 and IL-8 in a concentration-dependent manner. In addition, 10-MDP reduced alkaline phosphatase activity, mineralization nodule formation and mRNA expression of odontoblastic differentiation markers such as dentin sialophosphoprotein, dentin matrix protein-1, osterix and Runx2 in a concentration-dependent manner with low toxicity. In addition, 10-MDP induced activation of nuclear factor-E2-related factor 2 (Nrf2) and its target gene, haeme oxygenase-1 (HO-1). We evaluated whether the effect of 10-MDP was related to the induction of HO-1 and found that treatment with a selective inhibitor of HO-1 reversed the production of 10-MDP-mediated pro-inflammatory cytokines and the inhibition of differentiation markers. Pre-treatment with either a GSH synthesis inhibitor or antioxidants blocked 10-MDP-induced mitogen-activated protein kinases (MAPKs), Nrf2 and NF-κB pathways. Taken together, the results of this study showed that minimally toxic concentrations of 10-MDP promoted an inflammatory response and suppressed odontoblastic differentiation of DPCs by activating Nrf2-mediated HO-1 induction through MAPK and NF-κB signalling.

Effect of antioxidants on the genotoxicity of phenethyl isothiocyanate

Isothiocyanates are plant-derived compounds that may be beneficial in the prevention of certain chronic diseases. Yet, by stimulating the production of reactive oxygen species (ROS), isothiocyanates can be genotoxic. Whether antioxidants influence isothiocyanate-induced genotoxicity is unclear, but this situation was clarified appreciably herein. In HCT116 cells, phenethyl isothiocyanate (PEITC) increased ROS production, which was inhibited by N-acetylcysteine (NAC) and deferoxamine (DFO) but not by ascorbic acid (ASC) and trolox (TRX) that were found to be more potent radical scavengers. Surprisingly, ASC and TRX each intensified the DNA damage that was caused by PEITC, but neither ASC nor TRX by themselves caused any DNA damage. In contrast, NAC and DFO each not only attenuated PEITC-induced DNA damage but also attenuated the antioxidant-intensified, PEITC-induced DNA damage. To determine if the DNA damage could be related to possible changes in the major antioxidant defence system, glutathione (GSH) was investigated. PEITC lowered GSH levels, which was prevented by NAC, whereas ASC, TRX and DFO neither inhibited nor enhanced the GSH-lowering effect of PEITC. The GSH synthesis inhibitor, buthionine sulphoxime, intensified PEITC-induced DNA damage, although by itself buthionine sulphoxime did not directly cause DNA damage. The principal findings suggest that ASC and TRX make PEITC more genotoxic, which might be exploited in killing cancer cells as one approach in killing cancer cells is to extensively damage their DNA so as to initiate apoptosis.

Physiologically based pharmacokinetic modeling for 1-bromopropane in F344 rats using gas uptake inhalation experiments.

1-Bromopropane (1-BP) was introduced into the workplace as an alternative to ozone-depleting solvents and increasingly used in manufacturing industry. The potential exposure to 1-BP and the current reports of adverse effects associated with occupational exposure to high levels of 1-BP have increased the need to understand the mechanism of 1-BP toxicity in animal models as a mean of understanding risk in workers. Physiologically based pharmacokinetic (PBPK) model for 1-BP has been developed to examine 2 metabolic pathway assumptions for gas-uptake inhalation study. Based on previous gas-uptake experiments in the Fischer 344 rat, the PBPK model was developed by simulating the 1-BP concentration in a closed chamber. In the model, we tested the hypothesis that metabolism responsibilities were shared by the p450 CYP2E1 and glutathione (GSH) conjugation. The results showed that 2 metabolic pathways adequately simulated 1-BP closed chamber concentration. Furthermore, the above model was tested by simulating the gas-uptake data of the female rats pretreated with 1-aminobenzotrizole, a general P450 suicide inhibitor, or d,l-buthionine (S,R)-sulfoximine, an inhibitor of GSH synthesis, prior to exposure to 800 ppm 1-BP. The comparative investigation on the metabolic pathway of 1-BP through the PBPK modeling in both sexes provides critical information for understanding the role of p450 and GSH in the metabolism of 1-BP and eventually helps to quantitatively extrapolate current animal studies to human.

Glutathione and Thioredoxin Antioxidant Pathways Synergize to Drive Cancer Initiation and Progression

Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer initiation. Genetic loss of Gclm prevents a tumor's ability to drive malignant transformation. Intriguingly, these findings canbe replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death invitro and invivo, demonstrating theimportance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.

Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-γ-lyase function

Glioblastoma multiforme (GBM) is one of the most common encephalic malignant tumors. Due to a high recurrence rate and a lack of effective treatments, the average survival rate remains low. Temozolomide (TMZ), a class of alkylating agent, is widely used as a first-line therapeutic drug during the adjuvant treatment for GBM patients. However, most patients exhibit a palpable resistance to TMZ treatment. Additionally, the underlying mechanism remains to be clarified. In this study, glutathione (GSH) and reactive oxygen species (ROS) levels were found to be closely associated with the sensitivity of GBM cells to TMZ. We also found that TMZ markedly induced xCT, the subunit of glutamate/cystine transporter system xc- expression, which together with the GSH synthesis was increased while the TMZ-inducible ROS level was decreased in GBM cells. In addition, the cystathionine γ-lyase (CTH) acivity, a key enzyme in the transsulfuration pathway was enhanced by TMZ, which insured a cysteine supply and GSH synthesis in a compensatory manner when xCT was blocked. Thus, the individual inhibition of xCT by siRNA and a pharmacological inhibitor (sulfasalazine) only partially inhibited GSH synthesis and moderately enhanced the GBM cell sensitivity to TMZ. However, the TMZ‑induced cytotoxicity was markedly increased along with a marked decrease in GSH levels as result of co-treatment with erastin, which inhibited cysteine uptake from xCT transporter and suppressed CTH activity, leading to impaired transformation from methionine to cysteine. In conclusion, to GBM therapy with a drug combination of TMZ and erastin may be beneficial.

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly.

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

The role of GSH in microcystin-induced apoptosis in rat liver: Involvement of oxidative stress and NF-κB.

Microcystins (MCs) are potent and specific hepatotoxins produced by cyanobacteria in eutrophic waters, representing a health hazard to animals and humans. The objectives of this study are to determine the relationship between oxidative stress and NF-κB activity in MC-induced apoptosis in rat liver and the role of glutathione (GSH). Sprague-Dawley rats were intraperitoneally (i.p.) injected with microcystin-LR (MC-LR) at 0.25 and 0.5 LD50 with or without pretreatment of buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor. MC-LR induced time-dependent alterations of GSH levels in rat liver. Increased malondialdehyde (MDA) and significant changes of antioxidant enzymes including GSH peroxidase (GPX) and GSH reductase (GR) were also observed, particularly at 24 h post-exposure. The results indicated that acute exposure to MC-LR induced oxidative stress, and GSH depletion (BSO pretreatment) enhanced the level of oxidative stress. Furthermore, the modulation of pro-apoptotic gene p53 and Bax and anti-apoptotic gene Bcl-2 was observed in 0.5 LD50 group at 24 h, and the alteration was more pronounced by BSO injection before MC-LR treatment, suggesting that GSH played a protective role against MC-induced toxicity. Additionally, electrophoretic mobility shift assay (EMSA) showed that NF-κB was induced at 0.25 LD50 but inhibited at 0.5 LD50 . The above results indicated that the possible crosstalk of oxidative stress and NF-κB activity was associated with MC-LR-induced hepatocytes apoptosis in vivo. Our data will provide a new perspective for understanding the mechanisms of MC-induced liver injury.

Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

NG as a novel nitric oxide donor induces apoptosis by increasing reactive oxygen species and inhibiting mitochondrial function in MGC803 cells.

NG, O(2)-(2,4-dinitro-5-{[2-(12-en-28-β-D-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl] amino} phenyl) 1-(N-hydroxyethylmethylamino) diazen-1-ium-1,2-diolate, was identified in our laboratory as a novel nitric oxide-releasing prodrug with antitumor effects. A previous study showed that NG inhibited cell growth, and induced apoptosis in HepG2 cells. In this study, the inhibitory effects of NG on the viability of MGC803 cells were examined using methylthiazolyl tetrazolium biomide (MTT) assay, neutral red assay and trypan blue exclusion test. The results showed that NG had strong cytotoxicity to induce apoptosis, which was characterized by a significant externalization of phosphatidylserine, nuclear morphological changes and enhanced Bax-to-Bcl-2 ratio. Moreover, the release of cytochrome c (Cyt c) from mitochondria and the activation of caspase-9/3 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. NG induced mitochondrial dysfunction in MGC803 cells by altering membrane potential (△Ψm), the inhibition of complexes I, II and IV consequently decreasing ATP level. Furthermore, the treatment of MGC803 cells with NG caused a marked rise in oxidative stress as characterized by accumulation of reactive oxygen species (ROS), excessive malondialdehyde (MDA) production and a reduction in glutathione hormone (GSH) level and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. In addition, pretreatment with N-acetylcysteine (NAC), a GSH synthesis precursor, was partially protective against the NG-induced ROS generation and cell apoptosis. In contrast, pretreatment of MGC803 cells with L-buthionine-S, R-sulfoximine (BSO), a GSH synthesis inhibitor, increased the ROS levels, and aggravated cell apoptosis by NG. These results suggest that NG-induced apoptosis in MGC803 cells is mediated, at least in part, by the increase in ROS production, oxidative stress and mitochondrial dysfunction.