Expression Of Γ-glutamylcysteine Synthetase Research Articles

Glutathione Biosynthesis via Activation of the Nuclear Factor E2–Related Factor 2 (Nrf2) – Antioxidant-Response Element (ARE) Pathway Is Essential for Neuroprotective Effects of Sulforaphane and 6-(Methylsulfinyl) Hexyl Isothiocyanate

Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress–induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H2O2- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2–related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-l-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H2O2 and paraquat. Moreover, exposure to l-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.

Regulatory T Cells Interfere with Glutathione Metabolism in Dendritic Cells and T Cells

Naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) suppress proliferation of CD4(+)CD25(-) effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.

Fibroblast growth factor 9 upregulates heme oxygenase-1 and γ-glutamylcysteine synthetase expression to protect neurons from 1-methyl-4-phenylpyridinium toxicity

Oxidative stress and lower levels of trophic factors involved in nigrostriatal dopaminergic neurodegeneration are a hallmark of Parkinson disease. Our previous studies found that fibroblast growth factor 9 (FGF9) prevented 1-methyl-4-phenylpyridinium (MPP +)-induced nigral dopaminergic neuron death and was involved in the neuroprotection of the antioxidant melatonin. However, the protective mechanisms mediated by FGF9 remain unclear. Herein, we explored whether FGF9 regulated the cellular antioxidant defense protecting dopaminergic neurons against MPP + intoxication. We found that FGF9 treatment alone induced a decrease in hydrogen peroxide (H 2O 2) level, an increase in glutathione content, and an upregulation of γ-glutamylcysteine synthetase (γ-GCS) and heme oxygenase 1 (HO-1) expression in primary cortical neurons but not in astrocytes. Simultaneous treatment with FGF9 and MPP + prevented MPP +-induced neuron death and H 2O 2 overproduction but did not affect the FGF9-increased γ-GCS and HO-1 protein expression. Inhibition of γ-GCS or HO-1 prevented the inhibitory effect of FGF9 on MPP +-induced H 2O 2 production and death in mesencephalic dopaminergic and cortical neurons. However, in the absence of MPP +, the FGF9-induced H 2O 2 reduction was blocked by HO-1 inhibitors, but not by γ-GCS inhibitors. These results indicate that FGF9 upregulates γ-GCS and HO-1 expression to protect cortical and dopaminergic neurons from MPP +-induced oxidative insult.

Alterations in hepatic metabolism of sulfur‐containing substances by silymarin in mice

Silymarin has been shown to exhibit hepatoprotective effects against chemical‐induced liver injury, but the underlying mechanism of its beneficial action remains unclear. In this study we examined the changes in hepatic metabolism of sulfur‐amino acids induced by silymarin. Male mice were treated with silymarin (100 or 200 mg/kg, po) every 12 hr for a total of 3 doses. Methionine level was increased, but methionine adenosyltransferase (MAT) activity was decreased by silymarin in a dose‐dependent manner. S‐Adenosylmethione or homocysteine was not altered whereas cystathionine, cysteine and glutathione levels were increased significantly by silymarin treatment. Western blotting analysis revealed that silymarin treatment decreased protein expression of betaine‐homocysteine methyl transferase, MATI/III and cysteine dioxygenase, but increased cystathione β‐synthase expression in liver. Expression of γ‐glutamylcysteine synthetase, the rate‐limiting enzyme of glutathione synthesis, was not influenced. The present results show that silymarin induces significant changes in the metabolomics of sulfur‐containing substances in liver, which appears to be associated with the hepatoprotective effects of this substance. Further studies to identify its significance in chemical‐induced liver injury are currently being conducted in this laboratory.Grant Funding Source: KOSEF

Mild thermotolerance induced at 40°C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 degrees C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 degrees C, 3h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H(2)O(2). Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and gamma-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H(2)O(2). This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H(2)O(2)-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.

Hepatocyte growth factor protects hepatocytes against oxidative injury induced by ethanol metabolism

Hepatocyte growth factor (HGF) is involved in many cellular responses, such as mitogenesis and apoptosis protection; however, its effect against oxidative injury induced by ethanol metabolism is not well understood. The aim of this work was to address the mechanism of HGF-induced protection against ethanol-generated oxidative stress damage in the human cell line VL-17A (cytochrome P450 2E1/alcohol dehydrogenase-transfected HepG2 cells). Cells were pretreated with 50 ng/ml HGF for 12 h and then treated with 100 mM ethanol for 0–48 h. Some parameters of oxidative damage were evaluated. We found that ethanol induced peroxide formation (3.3-fold) and oxidative damage as judged by lipid peroxidation (5.4-fold). Damage was prevented by HGF. To address the mechanisms of HGF-induced protection we investigated the cellular antioxidant system. We found that HGF increased the GSH/GSSG ratio, as well as SOD1, catalase, and γ-glutamylcysteine synthetase expression. To explore the signaling pathways involved in this process, VL-17A cells were pretreated with inhibitors against PI3K, Akt, and NF-κB. We found that all treatments decreased the expression of the antioxidant enzymes, thus abrogating the HGF-induced protection against oxidative stress. Our results demonstrate that HGF protects cells from the oxidative damage induced by ethanol metabolism by a mechanism driven by NF-κB and PI3K/Akt signaling.

Dysregulated glutathione metabolism links to impaired insulin action in adipocytes

Oxidative stress plays an important role in obesity-related metabolic diseases. Glutathione peroxidase (GPX) is an antioxidant enzyme downregulated in adipose tissue of obese mice. However, the role of GPX in adipocytes remains elusive. The objective of this study was to clarify the pathophysiological changes in GPX activity and glutathione metabolism and their roles in the pathogenesis of insulin resistance in adipocytes. To achieve this goal, we measured cellular GPX activity, glutathione (GSH) contents, GSH/GSSG ratio, and mRNA expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for de novo GSH synthesis, in adipose tissue of control and ob/ob mice and in 3T3-L1 adipocytes treated with insulin, H(2)O(2), free fatty acid (FFA), or TNFalpha. Furthermore, we investigated the effects of GPX inhibition with a specific GPX inhibitor or RNA interference against GPX, H(2)O(2), and reduced GSH on insulin signaling in 3T3-L1 adipocytes. ob/ob Mice showed not only a decrease in cellular activity of GPXs (GPX1, -4, and -7) but also an increase in gamma-GCS expression, resulting in increased GSH contents in adipose tissue. These alterations in glutathione metabolism were also observed during differentiation of 3T3-L1 cells and their exposure to insulin, FFA, or H(2)O(2). Inhibition of GPX activity, addition of GSH, and H(2)O(2) resulted in impaired insulin signaling in 3T3-L1 adipocytes. These results suggest that decreased GPX activity and increased gamma-GCS expression lead to overaccumulation of GSH, which might be involved in the pathogenesis of insulin resistance in obesity.

Indomethacin overcomes doxorubicin resistance by decreasing intracellular content of glutathione and its conjugates with decreasing expression of γ-glutamylcysteine synthetase via promoter activity in doxorubicin-resistant leukemia cells

Drug resistance continues to be a serious problem in cancer therapy. We investigated whether indomethacin, which inhibits cyclooxygenases, is able to overcome doxorubicin resistance in K562/ADR leukemia cells. Indomethacin at 10 microM increased the cytotoxicity of doxorubicin and vincristine in K562/ADR cells. Intracellular glutathione content was elevated in K562/ADR cells. Indomethacin treatment decreased glutathione content and glutathione-conjugates in K562/ADR cells. Increased expression of gamma-glutamylcysteine synthetase (gamma-GCS) was observed in K562/ADR cells, but this expression was decreased by indomethacin treatment. The activity of the gamma-GCS promoter from K562/ADR cells decreased after indomethacin treatment in MDA231 cells. These data strongly suggest that the cyclooxygenase inhibitor indomethacin increases the cytotoxicity of doxorubicin by decreasing the intracellular contents of glutathione and its conjugates with decreasing expression of gamma-GCS by inhibiting gamma-GCS promoter activity.

Dehydroepiandrosterone augments sensitivity to γ-ray irradiation in human H4 neuroglioma cells through down-regulation of Akt signaling

Dehydroepiandrosterone (DHEA) modulates sensitivity to radiation-induced injury in human neuroglioma cells (H4) through effects on Akt signalling by glutathione (GSH)-dependent redox regulation. Previous treatment of H4 cells with DHEA for 18 h reduced the γ-ray-induced phosphorylation of Akt, activated p21waf1 synthesis and up-regulated phosphorylation of Rb independent of p53. These reactions were followed by a decrease in cell number and an increase in apoptosis and G2/M checkpoint arrest. The suppression of phosphorylation of Akt by DHEA was due to regulation of the dephosphorylation by protein phosphatase 2A (PP2A). DHEA up-regulated the expression of γ-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione (GSH) synthesis, and the levels of GSH to maintain PP2A activity. The results suggested that DHEA increases the sensitivity of cells to γ-ray irradiation by inducing apoptosis and cell cycle arrest through GSH-dependent regulation of the reduced form of PP2A to down-regulate the Akt signalling pathway.

Suppression of Oxidant-Induced Glutathione Synthesis by Erythromycin in Human Bronchial Epithelial Cells

Background: Macrolide antibiotics have anti-inflammatory effects which are utilized for the treatment of chronic inflammatory airway diseases. Recently, their anti-inflammatory effects have been proposed to be beneficial in patients with chronic obstructive pulmonary disease (COPD). Objectives: Since the molecular mechanisms of anti-inflammatory effects are associated with inhibition of activator protein 1 (AP-1) and nuclear factor (NF)-ĸB, and both are reported to be involved in the expression of γ-glutamylcysteine synthetase (γ-GCS), we set out to determine if these drugs influence the oxidant-antioxidant balance in human bronchial epithelial (HBE) cells. Methods: 16HBE cells were preincubated with erythromycin (EM) at different concentrations and times and then exposed to hydrogen peroxide (0.01 mM). Levels of interleukin (IL)-8 and glutathione (GSH), and activity of γ-GCS and γ-GCS heavy subunit (γ-GCS-HS) protein production were assayed. AP-1 and NF-ĸB binding to the 5′-flanking region of IL-8 and γ-GCS-HS genes was assessed by electrophoretic mobility-shift assay. Results: The increase in IL-8 levels and activity of AP-1 induced by H₂O₂ were abrogated by preincubation of the cells with EM (5 µg/ml) for 36 h. We also showed that preincubation with EM for 48 h inhibited H₂O₂-induced GSH levels, γ-GCS activity and expression of γ-GCS-HS, and decreased AP-1 binding to the γ-GCS-HS 5′-flanking region. Conclusions: The confirmation of antioxidants maintaining enzyme suppression by EM raised concerns on whether this drug could disrupt the oxidant/ antioxidant balance during long-term use. These data provide important insights into the treatment of inflammatory lung diseases with macrolide antibiotics.

Clinicopathological and Tissue Indicators of Para-Aminophenol Nephrotoxicity in Sprague–Dawley Rats

A model of para-aminophenol (PAP) nephrotoxicity in Sprague-Dawley rats was utilized to characterize potential indicators of toxicity in the kidney and in biofluids, and to chronicle the progression of acute renal injury. Rats were administered PAP at a low or high dose and examined terminally at 6, 24 and 48 hours (4 animals/group with matching controls). Acute tubular necrosis was observed in the medullary rays (low and high doses) and the outer stripe of outer medulla (high dose only) as early as 6 hours postdosing. Starting at 24 hours, regeneration of the tubular epithelium was evident in both low and high dose studies. Associated with the tubular lesions, we observed elevation of urinary alpha -glutathione S-transferase levels, an indicator of proximal tubular injury. By immunohistochemistry of the kidney, decreased gamma -glutamylcysteine synthetase expression correlated with tubular injury, especially at high dose, whereas elevation of vimentin, osteopontin, and Ki-67 expression was concurrent with tubular regeneration. Clusterin and kidney injury molecule-1 displayed expression patterns characteristic of both renal injury and regeneration. Taken together, this study provided insight into the progression of nephrotoxicity, and allowed the evaluation of potential urinary and tissue protein biomarkers that could complement the early detection of acute tubular injury.

Chronic hypoxia differentially increases glutathione content and γ-glutamyl cysteine synthetase expression in fetal guinea pig organs

Objective: Glutathione is a natural antioxidant in the fetus and adult. We sought to determine whether maternal hypoxia alters glutathione levels in fetal organs as an adaptive response to the reduced oxygenation. Study design: Timed pregnant guinea pigs were housed in either a Plexiglas chamber containing 10.5% O 2 from 46 to 60 days gestation (HPX, n = 6) or in room air, as the normoxic control (NMX, n = 5). Pregnant guinea pigs were anesthetized at near term (∼ 60 days, term = 65 days) and liver, lung and kidney were excised from anesthetized fetuses and stored frozen (− 80 °C) prior to sample processing. Using the hypoxia marker, pimonidazole, we measured a hypoxia-induced increase in stained cells of fetal liver compared to no change in either the lung or kidney. To measure the effect of hypoxia among different organs, total glutathione (GSH) content and protein levels of γ-glutamyl cysteine synthetase (γ-GCS) were measured from the same organs. Results: Maternal hypoxia increased ( P < 0.05) total glutathione levels by 121% in the fetal liver but had no effect in either fetal lung or kidney. Chronic hypoxia increased ( P < 0.05) γ-GCS protein levels in all three fetal organs studied. Conclusion: These results demonstrate that the fetal response to maternal hypoxia may be organ specific. The increase in fetal liver glutathione via upregulation of γ-GCS may be an important adaptive response to prolonged hypoxic stress.

Enhanced biosynthesis of glutathione in the spiral ganglion of the cochlea after in vivo treatment with dexamethasone in mice

Glucocorticoids have been widely used as a therapeutic drug for sudden sensorineural hearing loss. However, very little is known about the mechanism(s) underlying the protective effect of glucocorticoids against hearing loss. As an approach toward elucidating the mechanism(s), we evaluated the effects of dexamethasone (DEX) treatment on the biosynthesis of GSH in the mouse cochlea in vivo. The systemic administration of DEX led to a significant increase in the total GSH level in the cochlea 2 to 24 h later. This DEX-induced increase in GSH occurred selectively in the spiral ganglion, but not significantly in the lateral wall tissues or in the organ of Corti. Furthermore, RT-PCR analysis revealed that DEX treatment resulted in enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), which is the rate-limiting enzyme for de novo GSH synthesis, 1 to 24 h after the treatment. In addition to enhancing GSH biosynthesis, DEX treatment was effective in reducing lipid peroxidation in the cochlea. Taken together, DEX has the ability to facilitate GSH biosynthesis through enhanced expression of γ-GCS in the cochlear spiral ganglion.

Acceleration of Glutathione Efflux and Inhibition of γ-Glutamyltranspeptidase Sensitize Metastatic B16 Melanoma Cells to Endothelium-induced Cytotoxicity

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of gamma-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to approximately 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.

Bleomycin upregulates expression of gamma-glutamylcysteine synthetase in pulmonary artery endothelial cells.

The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS), which are thought to contribute to cellular damage and pulmonary injury. We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit growth arrest and increased cellular GSH content. gamma-Glutamylcysteine synthetase (gamma-GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the gamma-GCS catalytic subunit [gamma-GCS heavy chain (gamma-GCS(h))] is upregulated by bleomycin within 3 h. The promoter for human gamma-GCS(h) contains consensus sites for nuclear factor-kappaB (NF-kappaB) and the antioxidant response element (ARE), both of which are activated in response to oxidative stress. Electrophoretic mobility shift assays show that bleomycin activates the transcription factor NF-kappaB as well as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation by bleomycin is inhibited by the ROS quenching agent N-acetylcysteine (NAC), but not by U-0126, a MEK1/2 inhibitor that blocks bleomycin-induced MAPK activation. In contrast, NF-kappaB activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of gamma-GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH.

Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of γ-glutamylcysteine synthetase

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, γ-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of γ-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of γ-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2.

Reversal of cisplatin resistance in human ovarian cancer cell lines by a c-jun antisense oligodeoxynucleotide (ISIS 10582): evidence for the role of transcription factor overexpression in determining resistant phenotype

Human ovarian cancer cell lines derived from A2780 by stepwise exposure to increasing cisplatin concentrations show progressive resistance to cisplatin. Previous studies have shown increased cellular glutathione and elevated steady-state expression of γ-glutamylcysteine synthetase (γ-GCS) and of the transcription factor c-Jun, all in proportion to the level of resistance in the resistant cells. We hypothesized that c-Jun was an important locus of control of the detoxicating enzymes mediating resistance, and that resistance reversal would be achieved by specific inhibition of this mechanism. A2780 (sensitive) and C30 (resistant) cells were treated with a 20-mer c-jun phosphorothioate antisense oligodeoxynucleotide (ISIS 10582, 1μM), and a decrease in steady-state c-jun mRNA was demonstrated in the resistant cells. The expression of γ-GCS mRNA was down-regulated and the cellular level of glutathione was decreased in C30 cells. No change in γ-GCS expression occurred in A2780 cells. Using the microtetrazolium (MTT) cytotoxicity assay, we determined that the c-jun antisense decreased the ic50 value for cisplatin in C30 cells from 18.2 to 3.7μM, and had a substantially smaller effect in A2780 cells. To determine if c-jun overexpression alone could confer resistance to the sensitive cell line, we transiently transfected A2780 cells with a c-jun expression vector. The transfected cells exhibited a 10.7-fold elevation of glutathione (GSH) content, a 9.2-fold increase in c-Jun protein content, and a 2-fold increase in the ic50 for cisplatin. These data suggest that altered regulation of transcription factor expression contributes to the acquired resistance phenotype in these ovarian cancer cells, and provide a novel potential target for therapeutic intervention.

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Hyperglycemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of this study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of γ-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis), and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of γ-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions, incubation of the vascular smooth muscle cells with α-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the γ-glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.

Alteration of antioxidants during the progression of heart disease in streptozotocin-induced diabetic rats

Involvement of oxidative stress is implicated in the progression of complication of diabetes mellitus. With respect to heart diseases, we have studied role of oxidative stress/antioxidants using rats treated with streptozotocin to induce diabetes (DM). Hemodynamic and echocardiographic measurements showed thickening of the wall and an increase in the internal dimension of the left ventricle (LV) in DM rats at 8th week. Decrease in diastolic posterior wall velocity and rate of LV pressure change, and increase in LV end diastolic pressures also proved cardiac dysfunction. These changes were further developed in DM rats after 12 weeks. Utilizing rat hearts at 8th and 12th weeks, the following estimations were performed. There was a decrease in the activity of Mn-superoxide dismutase (SOD), suggesting abnormal mitochondrial metabolism of reactive oxygen species. The level of glutathione (GSH) decreased concomitant with a decrease in the expression of γ-glutamylcysteine synthetase (γ-GCS). The expression of transforming growth factor-β1 (TGF-β1), known as a growth factor and a suppressor of GSH synthesis, elevated in DM rat hearts. Immunohistochemical estimation showed an increase in type IV collagen in DM hearts. Collectively, it was suggested a linkage between mitochondrial damage to generate reactive oxygen species and inactivation of Mn-SOD and elevation of the expression of TGF-β1 to lead suppression of GSH synthesis and induction of fibrous change for the consequent cardiac dysfunction in DM.

Attenuation of Expression of γ-Glutamylcysteine Synthetase by Ribozyme Transfection Enhance Insulin Secretion by Pancreatic β Cell Line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic β cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in β cell line, MIN6 cells. Anti-γ-glutamylcysteine synthetase (γ-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 μg/106 cells/h in control cells (CC) to 1.48 μg/106 cells/h. This was associated with increased intracellular Ca2+ concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by β cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca2+ concentration.