Glutathione Ethyl Ester Research Articles

Roles of Glutathione and AP-1 in the Enhancement of Vitamin D-Induced Differentiation by Activators of the Nrf2 Signaling Pathway in Acute Myeloid Leukemia Cells.

Active vitamin D derivatives (VDDs)-1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs-are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 μM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 μM) or glutathione ethyl ester (200 μM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 μM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.

Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis

BackgroundPersistent inflammatory response in the brain can lead to tissue damage and neurodegeneration. In Alzheimer's disease (AD), there is an aberrant activation of inflammasomes, molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D (GSDMD), the executor of pyroptosis. However, the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown. We have previously shown that high brain cholesterol levels promote amyloid-β (Aβ) accumulation and oxidative stress. Here, we investigate whether these cholesterol-mediated changes may regulate the inflammasome pathway.MethodsSIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex. After exposure to lipopolysaccharide (LPS) plus muramyl dipeptide or Aβ, activation of the inflammasome pathway was analyzed by immunofluorescence, ELISA and immunoblotting analysis. Fluorescently-labeled Aβ was employed to monitor changes in microglia phagocytosis. Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response.ResultsIn activated microglia, cholesterol enrichment promoted the release of encapsulated IL-1β accompanied by a switch to a more neuroprotective phenotype, with increased phagocytic capacity and release of neurotrophic factors. In contrast, in SH-SY5Y cells, high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aβ peptides, resulting in GSDMD-mediated pyroptosis. Glutathione (GSH) ethyl ester treatment, which recovered the cholesterol-mediated depletion of mitochondrial GSH levels, significantly reduced the Aβ-induced oxidative stress in the neuronal cells, resulting in lower inflammasome activation and cell death. Furthermore, using conditioned media, we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia, lowering its phagocytic activity and, therefore, the ability to degrade extracellular Aβ.ConclusionsChanges in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells. Given the microglia-neuron cross-talk in the brain, cholesterol modulation should be considered a potential therapeutic target for AD treatment, which may help to block the aberrant and chronic inflammation observed during the disease progression.

Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype

IntroductionCirculating extracellular histones acquire relevance as cytotoxic mediators in sepsis. Extracellular histones act as damage-associated molecular patterns (DAMPs), which induce oxidative stress and NLRP3 inflammasome activation. Inflammasome mediates pyroptosis, a programmed cell death mechanism that produces inflammation. Despite evidence for inflammasome activation in immune cells during sepsis, it was unknown whether extracellular histones can produce endothelial inflammasomes activation.MethodsWe used human umbilical vein endothelial cells (HUVEC) to explore the activation of pyroptosis, endothelial function and inflammation by extracellular histones. We evaluated pyroptosis by flow cytometry, caspase-1 activity assay, and gene and protein expression analysis by RT-qPCR and Western blot, respectively. The upstream molecular responses involved in pyroptosis activation by extracellular histones were validated by means of using antioxidant glutathione ethyl ester and NLRP3 inflammasome inhibitors. Finally, using mass spectrometry, we measured circulating histones in blood from critically-ill patients and demonstrated that circulating histone levels correlated with the expression of pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity.ResultsWe found that extracellular histones mediate the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how the hyperacetylation of extracellular histones or the use of antioxidants decreased pyroptosis. In addition, we showed that pyroptosis is a feasible process occurring in septic shock patients.DiscussionCirculating histone levels correlated with the expression of pro-inflammatory and pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. We propose to block histone-mediated pyroptosis as a feasible therapeutic strategy in sepsis.

DNA damage by reactive oxygen species resulting from metabolic activation of 8-epidiosbulbin E acetate in vitro and in vivo

8-Epidiosbulbin E acetate (EEA), a furan-containing diterpenoid lactone, is one of main component of Dioscorea bulbifera L. (DBL). It has been reported that EEA induces severe hepatotoxicity in mice and that its hepatotoxicity is associated with metabolic activation. The present study demonstrated that exposure to EEA (50, 100 or 200 μM) induced DNA damage, including significant DNA fragmentation, increases of tail DNA and olive tail moment, H2AX phosphorylation and PARP-1 activation, in cultured mouse primary hepatocytes. Similar observation was obtained in mice administered EEA at 50, 100 or 200 mg/kg. Pre-treatment with 10 μM ketoconazole (KTC), 200 μM vitamin C (VC), or 200 μM glutathione ethyl ester (GSH-OEt) reversed the over-production of reactive oxygen species (ROS) induced by EEA and attenuated susceptibility of hepatocytes to EEA-induced cytotoxicity and DNA damage in mouse primary hepatocytes. In contrast, pre-treatment with 1.0 mM L-buthionine sulfoximine (BSO) potentiated over-production of ROS, cytotoxicity and DNA damage induced by EEA. In summary, EEA induced DNA damage in cultured primary hepatocytes and the liver of mice. ROS, possibly along with DNA alkylation, participated in the observed DNA damage.

Improved developmental potential of mouse vitrified-warmed oocytes achieved by culturing in recovery medium with glutathione ethyl ester (GSH-OEt).

PurposeThe aim of the present study was to investigate the effect of glutathione ethyl ester (GSH‐OEt) in the recovery medium on the developmental competence of mouse vitrified‐warmed MII oocytes.MethodsVitrified‐warmed oocytes were incubated for 1 h in recovery medium in the presence or absence of 0.5 mM GSH‐OEt. The authors examined the effects of GSH‐OEt, first on the levels of glutathione (GSH) and reactive oxygen species (ROS) in vitrified‐warmed oocytes, and second, on in vitro blastocyst development, division speed to blastocysts, and total cell numbers of blastocysts from vitrified‐warmed oocytes fertilized by Intracytoplasmic sperm injection (ICSI).ResultsAdding GSH‐OEt to the recovery medium significantly (p < 0.05) increased GSH content and decreased ROS levels in vitrified‐warmed oocytes. The blastocyst rate did not differ significantly between the two groups, but the speed of development to blastocysts in the GSH‐OEt (+) group was significantly more rapid. In addition, the total blastocyst cell number was significantly higher in the GSH‐OEt (+) group than in the GSH‐OEt (−) group (92.8 ± 5.1 vs. 71.4 ± 3.5, p < 0.01).ConclusionAdding GSH‐OEt to the recovery medium of vitrified‐warmed mouse oocytes enhances the development potential of oocytes and improves the quality of blastocysts.

The effects of glutathione ethyl ester in in vitro maturation on the developmental ability of oocytes derived from cattle with liver abnormalities

The main objectives of this study was to identify the effects of a relationship of hyper-concentration of Gamma-glutamyltransferase (γ-GTP) in follicle fluid (FF) on the levels of glutathione (GSH)/reactive oxygen species (ROS) in oocytes and subsequent embryo development in cattle with abnormal livers. Furthermore, we investigated the effect of supplementing in vitro maturation medium with glutathione ethyl ester (GSH-OEt) on the subsequent developmental potential of oocytes from such cattle. We used a control group of cattle (with normal livers) and a liver disorder (LD) group, in which the liver was diagnosed as being abnormal. In experiment 1, the LD group was divided to two subgroups according to the concentration of γ-GTP in FF: a low group (≤50 IU/L; the low LD group), and a high group (>50 IU/L: the high LD group). Cumulus oocyte-complexes (COCs) were matured and fertilized in vitro and then cultured to the blastocyst stage. The levels of GSH and ROS in the matured oocytes after IVM were then assessed in each group. On day 7 after fertilization, embryo cleavage and development were assessed. We found that the rate of development to the blastocyst stage was significantly lower in the high LD group than in the control group and the low LD group. The levels of GSH in matured oocytes were significantly lower in the high LD group than in the control group and low LD group. The levels of ROS in matured oocytes was significantly higher in the high LD group than in the control group and the low LD group. In experiment 2, COCs from cattle in the high LD group were matured in m-199 supplemented with 5 mM GSH-OEt, then IVF and IVC was performed for 7 days. The GSH levels were determined in some COCs after IVM. The supplementation of media with GSH-OEt during IVM increased the levels of GSH in mature oocytes and improved the rate of blastocyst development compared with the control group. In conclusion, GSH-OEt supplementation to media during IVM can improve the developmental potential of oocytes in liver-diseased cattle with high γ-GTP concentrations in the FF by increasing intracellular GSH synthesis and scavenging ROS.

Glutathione ethyl ester reverses the deleterious effects of fentanyl on ventilation and arterial blood-gas chemistry while prolonging fentanyl-induced analgesia

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague–Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

Glutathione ethyl ester improved the age-induced decline in the developmental competence of bovine oocytes

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30–50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows.

Glutathione ethyl ester supplementation prevents airway hyper-responsiveness in mice.

BackgroundOxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is considered to be one of the most important antioxidants. Our study systematically investigated the effect of the GSH alternative, glutathione ethyl ester (GSH-EE), on airway hyper-responsiveness (AHR) in mice.MethodsSixty-three male specific pathogen-free mice were used. Asthma was induced using a single dose of ovalbumin (OVA). The normal group (n=15) received vehicle only [Al(OH)3 in saline]. Then, 48 mice were divided into two groups, including a control group who received sodium phosphate buffer (pH =7.4), and the GSH-EE group who received 0.1% GSH-EE. AHR was measured 2, 6, and 12 hours after exposure to nebulized OVA (0.01%). The animals were then sacrificed, and lung tissue and the bronchi-alveolar lavage fluid (BALF) were harvested. Factors involved in the antioxidant response to asthma were then measured in these tissues, including thiol content (from GSH and protein), γ-glutamylcysteine synthetase (γ-GCS) activity and expression, and nuclear factor-erythroid-2-related factor (Nrf2) expression.ResultsThe GSH-EE group showed a significant attenuation of AHR (P<0.01) 2 hours after OVA challenge, and significantly enhanced thiol contents by approximately 45% (P<0.05) at 2 and 6 hours after the last OVA challenge, compared to the control group. γ-GCS activity was also higher in the GSH-EE group compared to the control group at different time points (P<0.01). γ-GCSh and Nrf2 protein expression increased in the GSH-EE group and the control group compared with the normal group, but there was no statistically significant difference (P>0.05) between the GSH-EE group and the control group.ConclusionsGSH-EE supplementation can prevent AHR in asthmatic mice during the early stages. It may function by serving as a precursor for GSH biosynthesis and by protecting sulfhydryl groups from oxidation.

Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming.

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-τ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.

UV radiation in HCT 116 cells influences intracellular H2O2 and glutathione levels, antioxidant expression, and protein glutathionylation.

UV radiation influences cellular levels of hydrogen peroxide (H2O2) and glutathione (GSH) and alters the expression of antioxidant genes in the human colorectal cancer cell line HCT116. In this study, cells were irradiated with UV light of different wavelength (A, B, or C). A surge in H2O2 concentration and total glutathione (level occurred 6 hours later. Consequently, protein glutathionylation increased above control levels. Expression of the antioxidant enzymes: glutathione peroxidase (GPX) and glutathione reductase (GSR), assessed by real-time quantitative PCR, increased by 1.5-2 times after 24 hours post-irradiation, in comparison to the untreated controls. Glutathionylation of proteins was enhanced after UV radiation and the set of biotinylated glutathione ethyl ester (BioGEE) tagged proteins was detected by Western Blot procedure. This specific glutathione analogue is conjugated with antioxidant proteins during glutathionylation especially under oxidized conditions in cells. A pool of glutathionylated proteins in the treated cells showed peculiar characteristics. These proteins exhibited varying molecular weights. For UVA-irradiated cells, 24 hours after the treatment we observed two additional ~60 and ~72 kDa bands of glutathionylated proteins from NADPH oxidases (NOX family). Total glutathione level in the UV-irradiated HCT116 cells was higher than in the control. This correlates with the detection of glutathionylation in UV-irradiated cells in the first and twelfth hour of post-irradiation, and can be defined as a specific antioxidant element activation for cellular protection.

33 Assessment of spindle morphology and reactive oxygen species production after vitrification of bovine oocytes following invitro maturation in the presence of glutathione ethyl ester

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P&amp;lt;0.05). No significant differences in percentages of MII or normal spindle configuration were observed among treatments. Treatment with GSH-OEt before vitrification resulted in similar percentages of abnormal spindle configuration to control oocytes, whereas vitrified oocytes showed significantly higher percentages of abnormal spindle configuration relative with control oocytes (Table 1). When the content of ROS was measured, vitrification resulted in significantly higher levels (15.1E+06±1.2E+06; P&amp;lt;0.05) when compared with other treatments. However, oocytes vitrified after IVM with GSH-OEt (11.8E+06±0.6E+06) showed similar ROS levels to control (10.7E+06±0.2E+06) and GSH-OEt (10.0E+06±0.1E+06) groups. In conclusion, addition of GSH-OEt to IVM before vitrification did not have detrimental effects on spindle morphology and was able to reduce ROS content. Further experiments are warranted to assess whether the addition of GSH-OEt to IVM medium can improve oocyte development competence after vitrification. Table 1.Assessment of spindle morphology after oocyte vitrification (VIT) following IVM in the presence of glutathione ethyl ester (GSH-OEt) Item n MII (%) Spindle configuration (%) Chromosome distribution (%) Normal Abnormal Absent Dispersed Decondensed Absent Control 127 79.4±92 74.1±4.4 16.2±5.6a 9.7±1.1 16.2±3.8 9.7±1.1 0 GSH-OEt 113 76.5±6.5 65.6±5.7 26.2±9.0ab 8.1±4.3 19.5±2.6 13.7±3.0 1.1±1.1 VIT 71 60.0±5.6 54.8±7.8 32.7±10.8b 12.4±1.4 34.4±8.7 10.7±6.4 0 GSH-OEt VIT 53 57.4±10.7 58.5±8.8 31.1±9.3ab 10.4±5.8 34.8±5.6 6.7±6.7 0 a,bDifferent superscripts indicate significant differences (P&amp;lt;0.05). *Percentage referred to the total number of oocytes at MII. This work was supported by MCIU, Spain (AGL2016-79802-P) and Generalitat de Catalunya (García-Martínez, 2017_FI_00451).

Arylquin 1, a potent Par-4 secretagogue, induces lysosomal membrane permeabilization-mediated non-apoptotic cell death in cancer cells.

Arylquin 1, a small-molecule prostate-apoptosis-response-4 (Par-4) secretagogue, targets vimentin to induce Par-4 secretion. Secreted Par-4 binds to its receptor, 78-kDa glucose-regulated protein (GRP78), on the cancer cell surface and induces apoptosis. In the present study, we investigated the molecular mechanisms of arylquin 1 in cancer cell death. Arylquin 1 induces morphological changes (cell body shrinkage and cell detachment) and decreases cell viability in various cancer cells. Arylquin 1-induced cell death is not inhibited by apoptosis inhibitors (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitors (necrostatin-1), and paraptosis inhibitors. Furthermore, arylquin 1 significantly induces reactive oxygen species levels, but antioxidants [N-acetyl-l-cysteine and glutathione ethyl ester] do not inhibit arylquin 1-induced cell death. Furthermore, Par-4 knock-down by small interfering RNA confers no effect on cytotoxicity in arylquin 1-treated cells. Interestingly, arylquin 1 induces lysosomal membrane permeabilization (LMP), and cathepsin inhibitors and overexpression of 70-kDa heat shock protein (HSP70) markedly prevent arylquin 1-induced cell death. Therefore, our results suggest that arylquin 1 induces non-apoptotic cell death in cancer cells through the induction of LMP.

Glutathione-related substances maintain cardiomyocyte contractile function in hypoxic conditions

Severe hypoxia leads to decline in cardiac contractility and induces arrhythmic events in part due to oxidative damage to cardiomyocyte proteins including ion transporters. This results in compromised handling of Ca2+ ions that trigger heart contractile machinery. Here, we demonstrate that thiol-containing compounds such as N-acetylcysteine (NAC), glutathione ethyl ester (et-GSH), oxidized tetraethylglutathione (tet-GSSG), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) are capable of reducing negative effects of hypoxia on isolated rat cardiomyocytes. Preincubation of cardiomyocytes with 0.1 mM GSNO, 0.5 mM et-GSH, GSSG, tet-GSSG or with 10 mM NAC allows cells 5-times longer tolerate the hypoxic conditions and elicit regular Ca2+ transients in response to electric pacing. The shape of Ca2+ transients generated in the presence of GSNO, et-GSH and NAC was similar to that observed in normoxic control cardiomyocytes. The leader compound, GSNO, accelerated by 34% the recovery of normal contractile function of isolated rat heart subjected to ischemia-reperfusion. GSNO increased glutathionylation of Na,K-ATPase alpha-2 subunit, the principal ion-transporter of cardiac myocyte sarcolemma, which prevents irreversible oxidation of Na,K-ATPase and regulates its function to support normal Ca2+ ion handling in hypoxic cardiomyocytes. Altogether, GSNO appears effective cardioprotector in hypoxic conditions worth further studies toward its cardiovascular application.

S-nitrosylation of endothelial nitric oxide synthase impacts erectile function

Neuronal and endothelial nitric oxide synthases (nNOS and eNOS respectively) play major roles in generating the nitric oxide bioactivity necessary for erectile function. S-nitrosylation has been shown to regulate NOS activity. The presence of S-nitrosylated NOS in the penis and the impact of NOS S-nitrosylation/denitrosylation on erectile function were examined. S-nitrosylated forms of NOS were identified by biotin-switch assay followed by western blot analysis. Erectile function in S-nitrosoglutathione reductase deficient (GSNO+/-) and null (GSNO-/-) mice were assessed by continuous cavernous nerve electrical stimulation (CCNES). Glutathione ethyl ester (GSHee) was used to manipulate S-nitrosylated NOS levels. Immunohistological and immunofluorescence analyses were used to identify the location of eNOS and GSNO-R in corporal tissue. eNOS and nNOS were S-nitrosylated in unstimulated penises of the mice. CCNES resulted in a time-dependent increase in eNOS S-nitrosylation with peak eNOS S-nitrosylation observed during detumescence. S-nitrosylated nNOS levels were unchanged. Intracorporal injection of GSHee reduced S-nitrosylated eNOS levels, enhancing time to maximum intracorporal pressure (ICP). eNOS and GSNO-R co-localize to the endothelium of the corpus cavernosum in the mouse and the human. ICP measurements obtained during CCNES demonstrate GSNO-R+/- and GSNO-R-/- animals cannot maintain an elevated ICP. Results suggest eNOS S-nitrosylation/denitrosylation is an important mechanism regulating eNOS activity during erectile function. GSNO-R is a key enzyme involved in the eNOS denitrosylation. The increase in eNOS S-nitrosylation (inactivation) observed with tumescence may begin a cycle leading to detumescence. Clinically this may indicate that alterations in the balance of S-nitrosylation/denitrosylation either directly or indirectly contribute to erectile dysfunction.

N-acetylcysteine ethyl ester as GSH enhancer in human primary endothelial cells: A comparative study with other drugs

Several drugs are currently in use as glutathione (GSH) enhancers in clinical, pre-clinical and experimental research. Here we compare the ability of N-acetylcysteine (NAC), 2-oxothiazolidine-4-carboxylic acid (OTC), glutathione ethyl ester (GSH-EE) and N-acetylcysteine ethyl ester (NACET) to increase the intracellular concentration of GSH using primary human umbilical vein endothelial cells (HUVEC) as in vitro model. Our experiments highlighted that NACET is largely the most efficient molecule in increasing the intracellular levels of GSH, cysteine, and γ-glutamylcysteine. This is because NACET is lipophilic and can freely cross plasma membrane but, inside the cell, it is de-esterified to the more hydrophilic NAC, which, in turn, is trapped into the cell and slowly transformed into cysteine. The higher availability of cysteine is matched by an increase in GSH synthesis, cysteine availability being the rate limiting step for this reaction. Surprisingly, the increase in GSH concentration was not linear but peaked at 0.5 mM NACET and gradually decreased when cells were treated with higher concentrations of NACET. We demonstrated that this puzzling ceiling effect was due to the fact that NAC released from NACET turned out to be a competitive inhibitor of the enzyme glutamate-cysteine ligase, with a Ki value of 3.2 mM. By using a cell culture medium lacking of cysteine and methionine, we could demonstrate that the slight increase in intracellular levels of cysteine and GSH induced by NAC in HUVEC grown in standard medium was due to the reduction of the cystine present in the medium itself there rather than to the action of NAC as Cys pro-drug. This fact may explain why NAC works well as GSH enhancer at very high concentrations in pre-clinical and in vitro studies, whereas it failed in most clinical trials.

Abstract 3963: Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers at 8%. Upregulated mechanisms to curb reactive oxygen species (ROS) accumulation are thought to help the growth of PDACs. Hence, perturbation of oxidative balance might be an effective therapeutic strategy for pancreatic cancer. To achieve this end, we used a human enzyme called Cyst(e)inase that degrades extracellular cysteine (Cys) and cystine (CSSC) - substrates that are necessary for synthesis of the antioxidant glutathione (GSH). Since tumor cells need to supplement intracellular Cys synthesis with extracellular import in order to fulfil their increased demand for GSH and protein synthesis, Cys/CSSC depletion can provide a therapeutic window. We validated this idea in our previous study where Cyst(e)inase treatment markedly inhibited tumor growth in several cancer models in vivo without any toxicity. In this study, we assessed the efficacy of Cyst(e)inase alone as well as in combination with auranofin in reducing pancreatic tumor growth. Cyst(e)inase treatment of cultured pancreatic cancer cells (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Panc1 was very sensitive to Cyst(e)inase in vitro, exhibiting ROS accumulation and activation of stress-related signaling as well as G2-arrest and apoptotic cell death whereas BxPC3 and MIA-PaCa2 showed only a G1-arrest. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway via the eIF2α-ATF4-Sestrin2 axis was observed in all 3 cell lines. In addition, MIA-PaCa2 but not Panc1 exhibited a rewiring of mitochondrial metabolism upon Cyst(e)inase treatment that bypassed succinate dehydrogenase. Combining auranofin and Cyst(e)inase caused synergistic increase in mitochondrial ROS and apoptotic cell death in the resistant cell lines MIA-PaCa2 and BxPC3 with associated activation of oxidative stress signaling. Surprisingly, the thioredoxin reductase system (the putative target of auranofin) was dispensable in producing synergy with Cyst(e)inase. Moreover, auranofin and Cyst(e)inase combination led to inhibition of mitophagy and accumulation of defective mitochondria. Supplementation with the antioxidant glutathione ethyl ester rescued the combination's cytotoxic effects. Biweekly intraperitoneal Cyst(e)inase treatment of nude mice harboring pancreatic cancer xenografts roughly recapitulated the in vitro sensitivity profile with Panc1 showing the most growth inhibition, albeit modest. BxPC3 xenografts were completely resistant to Cyst(e)inase alone but adding auranofin significantly inhibited their growth without causing any systemic toxicity. Collectively, the current data suggest that combining Cyst(e)inase and auranofin, an approved drug for rheumatoid arthritis, might constitute an effective therapy for pancreatic cancer. Citation Format: Sabin Kshattry, George Georgiou, Stefano Tiziani, Achinto Saha, Everett Stone, John DiGiovanni. Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3963.

Preincubation with glutathione ethyl ester improves the developmental competence of vitrified mouse oocytes.

Oocyte vitrification is currently used for human fertility preservation. However, vitrification damage is a problem caused by decreasing ooplasmic levels of glutathione (GSH). The GSH donor glutathione ethyl ester (GSH-OEt) can significantly increase the GSH content in oocytes. However, it is difficult to obtain oocyte from woman. To overcome this, we used mouse oocytes to replace human oocytes as a model of study. Oocytes from B6D2F1 mice were preincubated for 30min with 2.5mmol/L GSH-OEt (GSH-OEt group), without GSH-OEt preincubation before vitrification (control vitrification group) or in nonvitrified oocytes (fresh group). After thawing, oocytes were fertilized for evaluating the developmental competence of embryos in vitro and in vivo. Immunofluorescence, Polscope equipment and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to analyze damage, including mitochondrial distribution, reactive oxygen species (ROS) levels, spindle morphology, and gene expression levels (Bcl-2, BAX, and MnSOD). The rates of fertilization, 3-4 cell, blastocyst formation and expanded blastocysts were significantly higher (p < 0.05) in the GSH-OEt group (90.4%; 91.1%; 88.9% and 63.0%) than in the control (80.0%; 81.4%; 77.7% and 50.5%). Provided embryos overcame the 2-cell block and developed to the blastocyst stage, birth rates of all groups were similar. Vitrification altered mitochondrial distribution, increased ROS levels, and caused abnormal spindle morphology; GSH-OEt preincubation could improve such damage. RT-qPCR showed that the expression of Bcl-2 was lower in the control group compared with the GSH-OEt group; BAX and MnSoD expression levels were higher in the control group than in the GSH-OEt group (p < 0.05). The beneficial effect of GSH-OEt preincubation occurred before the 2-cell stage.

A novel hypobromous acid scavenging activity assay using p-cresol as a spectrofluorometric probe

In this study, a novel spectrofluorometric assay based on $p$-cresol (4-methyl phenol) probe is developed for the measurement of HOBr scavenging activity. It is the first study involving the use of a $p$-cresol probe for the determination of the HOBr scavenging activity of biothiols. While the $p$-cresol probe ($\lambda_{ex}$ = 260 nm, $\lambda_{em}$ = 305 nm) has fluorescence characteristics, its brominated derivatives emerging at the end of the oxidation reaction with HOBr do not show fluorescence. The initial fluorescence intensity of the $p$-cresol probe is decreased in the presence of the brominating agent, HOBr, and this decrease is lower in the presence of HOBr scavenging antioxidants. The scavenging activities of biothiols tested with respect to the developed method decrease in the following order: penicillamine $>$ N-acetyl cysteine $>$ L-glutathione (reduced) $>$ cysteamine $>$ homocysteine $>$ glutathione ethyl ester $>$ cysteine $>$ 1,4-dithiothreitol $>$ lipoic acid $>$ methionine. Penicillamine (IC$_{50}$ = 10.12 $\mu $M) was the most effective HOBr-scavenger among the tested biothiols. The results obtained with the developed method for biothiols and some pharmaceutical samples were statistically compared (using ANOVA) to those found by the reference methods (KI/taurine and UPLC). The advantage of the proposed method over the KI/taurine assay was demonstrated