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American Journal of Physiology.... Jan 2018Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced...
Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor l-buthionine sulfoximine impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Furthermore, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macroscopic and microscopic measures of colonic inflammation. Together, our data highlight the heterogeneous roles of GSH in the myenteric plexus of the ENS and during gastrointestinal inflammation. NEW & NOTEWORTHY Our results show that both enteric glia and neurons express the cellular machinery necessary for glutathione (GSH) synthesis and that glial GSH synthesis is necessary for neuronal survival in isolated enteric nervous system (ENS) preparations. In vivo depletion of GSH with the selective inhibitor l-buthionine sulfoximine is not sufficient to induce inflammation but does alter neuronal neurochemical composition and survival. Together, our data highlight novel heterogeneous roles for GSH in the ENS and during gastrointestinal inflammation.
Topics: Animals; Antioxidants; Buthionine Sulfoximine; Cell Death; Colitis; Colon; Dinitrofluorobenzene; Disease Models, Animal; Enzyme Inhibitors; Glutamate-Cysteine Ligase; Glutathione; In Vitro Techniques; Male; Mice, Inbred C57BL; Myenteric Plexus; Neuroglia; Neurons; Phenotype
PubMed: 28882823
DOI: 10.1152/ajpgi.00165.2017 -
BMC Cancer Jan 2014Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine...
BACKGROUND
Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.
METHODS
HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.
RESULTS
HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.
CONCLUSIONS
The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.
Topics: Apoptosis; Apoptosis Regulatory Proteins; Arsenic Trioxide; Arsenicals; Bcl-2-Like Protein 11; Buthionine Sulfoximine; Drug Combinations; Gene Knockdown Techniques; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Oxidative Stress; Oxides; Phosphorylation; Proto-Oncogene Proteins
PubMed: 24428916
DOI: 10.1186/1471-2407-14-27 -
Theranostics 2022Photodynamic therapy (PDT) is a clinically approved anticancer treatment with a promising therapeutic prospect, however, usually suffers from the unfavorable...
Photodynamic therapy (PDT) is a clinically approved anticancer treatment with a promising therapeutic prospect, however, usually suffers from the unfavorable intracellular environment including cellular hypoxia and excessive glutathione (GSH). Comprehensive and long-term modulation of tumor intracellular environment is crucial for optimizing therapeutic outcomes. However, current strategies do not enable such requirements, mainly limited by flexible networks of intracellular metabolic avenues. A metabolic pre-intervention (MPI) strategy that targets critical pathways of cellular metabolism, ensuring long-term modulation of the intracellular environment. A versatile lipid-coating photosensitive metal-organic framework (MOF) nano-vehicle encapsulating aerobic respiration inhibitor metformin (Met) and GSH biosynthesis inhibitor buthionine sulfoximine (BSO) (termed PBMLR) was developed for comprehensive sustainable hypoxia alleviation and GSH downregulating. Since MPI could effectively circumvent the compensatory accessory pathway, PBMLR, therefore functioned as an efficient singlet oxygen (O) radical generator during the subsequent laser irradiation process and enhanced PDT anti-tumor efficiency. We emphasized the concordance of long-term hypoxia alleviation, persistent GSH depletion, and tumor enrichment of photosensitizers, which is very meaningful for a broad therapeutic time window and the successful enhancement of PDT. Our findings indicate that maintaining the sensitivity of tumor cells via MPI could enhance anti-tumor PDT, and may be applied to other dynamic therapies such as radiodynamic therapy and sonodynamic therapy.
Topics: Buthionine Sulfoximine; Cell Line, Tumor; Glutathione; Humans; Hypoxia; Lipids; Metal-Organic Frameworks; Metformin; Neoplasms; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Singlet Oxygen
PubMed: 36168617
DOI: 10.7150/thno.75837 -
Biochemical and Biophysical Research... Feb 2021Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation. Cancer cells increase cystine uptake for the synthesis of glutathione (GSH), which is...
Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation. Cancer cells increase cystine uptake for the synthesis of glutathione (GSH), which is used by glutathione peroxidase 4 to reduce lipid peroxides. Here, we report that cystine deprivation in glioblastoma cells, but not inhibition of GSH synthesis by l-buthionine sulfoximine (BSO), induces ferroptosis. We found that cystine deprivation decreased the protein levels of ferritin heavy chain FTH1, whereas it was increased by BSO treatment. The lysosome inhibitor bafilomycin A1 or deletion of nuclear receptor coactivator 4 (NCOA4) inhibited cystine deprivation-induced decrease in FTH1 protein levels and cell death. In addition, cystine deprivation induced microtubule-associated protein light chain 3 (LC3)-II protein accumulation, suggesting that cystine deprivation induces ferritinophagy. BSO causes cell death when glioblastoma cells are treated with iron inducers, ferrous ammonium sulfate or hemin. On the other hand, cystine deprivation-induced degradation of FTH1 and cell death required glutamine. This study suggests that ferritinophagy, in addition to GSH depletion, plays an important role in cystine deprivation-induced ferroptosis in glioblastoma cells.
Topics: Antimetabolites, Antineoplastic; Autophagy; Brain Neoplasms; Buthionine Sulfoximine; Cell Death; Cell Line, Tumor; Cystine; Ferritins; Ferroptosis; Glioblastoma; Glutathione; Humans; Iron; Lipid Peroxidation; Oxidoreductases
PubMed: 33421769
DOI: 10.1016/j.bbrc.2020.12.075 -
Neuroscience Mar 1996Astrocyte hypertrophy and swelling occur in a variety of pathophysiological conditions, including diseases associated with hyperammonemia. Ammonia is rapidly...
Astrocyte hypertrophy and swelling occur in a variety of pathophysiological conditions, including diseases associated with hyperammonemia. Ammonia is rapidly incorporated into glutamine by glutamine synthetase localized in astrocytes. We tested the hypotheses that (1) 6 h of hyperammonemia (500-600 microM) is adequate for producing astrocyte enlargement, and (2) astrocyte enlargement is attenuated by inhibition of glutamine synthetase with methionine sulfoximine. Pentobarbital-anesthetized rats received an intravenous infusion of either sodium or ammonium acetate after intraperitoneal pretreatment with vehicle, methionine sulfoximine (0.8 mmol/kg) or buthionine sulfoximine (4 mmol/kg), an analogue that does not inhibit glutamine synthetase. Hyperammonemia produced enlarged cortical astrocytes characterized by (1) decreased electron density of cytoplasmic matrix in perikaryon, processes and perivascular endfeet, (2) increased circumference of nuclear membrane, (3) increased numbers of mitochondria and rough and smooth endoplasmic reticulum in perikarya and large processes, and (4) less compact bundles of intermediate filaments. Pretreatment with methionine sulfoximine, but not buthionine sulfoximine, attenuated the decrease in cytoplasmic density and the increase in nuclear circumference; most perivascular endfeet remained as dense as occurred with sodium acetate infusion. However, increased numbers of organelles in expanded perikarya and large processes occurred after methionine sulfoximine treatment with and without ammonium acetate infusion. In separate groups of rats, hyperammonemia produced an increase in cortical tissue water content which was inhibited by methionine sulfoximine, but not buthionine sulfoximine. We conclude that clinically-relevant levels of hyperammonemia can cause astrocyte enlargement within 6 h in vivo characterized by both watery cytoplasm and increased organelles indicative of a cellular metabolic stress and altered astrocyte function. The watery cytoplasm component of astrocyte enlargement depends on glutamine synthesis rather than on ammonium ions per se, and is possibly caused by the osmotic effect accumulated glutamine.
Topics: Ammonia; Animals; Astrocytes; Body Water; Buthionine Sulfoximine; Capillaries; Cell Nucleus; Cell Size; Endoplasmic Reticulum; Enzyme Inhibitors; Glutamate-Ammonia Ligase; Male; Methionine Sulfoximine; Mitochondria; Rats; Rats, Wistar
PubMed: 9053810
DOI: 10.1016/0306-4522(95)00462-9 -
Free Radical Biology & Medicine May 2019Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are...
Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (HS with AzMC) and polysulfides (HS, where n = 2-7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O) and hypoxia (5% O), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased HS production for 5 days but did not affect HS. The glutathione reductase inhibitor, auranofin, initially decreased HS and HS but after two days HS increased over controls. Inhibition of peroxiredoxins with conoidin A decreased HS and increased HS, whereas the glutathione peroxidase inhibitor, tiopronin, increased HS. Aminoadipic acid, an inhibitor of cystine uptake did not affect either HS or HS. In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with HS and HS, or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS.
Topics: Antioxidants; Buthionine Sulfoximine; Catalase; Glutathione; Glutathione Peroxidase; Glutathione Reductase; HEK293 Cells; Humans; Hydrogen Sulfide; Maleates; Metabolic Networks and Pathways; Reactive Oxygen Species; Sulfides; Sulfur; Superoxide Dismutase
PubMed: 30790656
DOI: 10.1016/j.freeradbiomed.2019.02.011 -
Current Drug Targets 2015Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective...
Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective antioxidant systems like glutathione. Current anticancer therapies focus on the cancer dependence on oncogenes and non-oncogenes. Tumors trigger mechanisms to circumvent the oncogenic stress and to escape cell death. In this context we have studied 2-phenylethinesulfoxamine (PES), which disables the cell protective mechanisms to confront the proteotoxicity of damaged and unfolded proteins. Proteotoxic stress is increased in tumor cells, thus providing an explanation for the anticancer selectivity of PES. In addition, we have found that PES induces a severe oxidative stress and the activation of p53. The reduction of the cell content in glutathione by means of L-buthionine-sulfoximine (BSO) synergizes with PES. In conclusion, we have found that ROS constitutes a central element in a series of positive feed-back loops in the cell. ROS, p53, proteotoxicity, autophagy and mitochondrial dynamics are interconnected with the mechanisms leading to cell death, either apoptotic or necrotic. This network of interactions provides multiple targets for drug discovery and development in cancer.
Topics: Antineoplastic Agents; Buthionine Sulfoximine; Drug Discovery; Gene Expression Regulation, Neoplastic; Humans; Mitochondrial Dynamics; Neoplasms; Oxidative Stress; Reactive Oxygen Species; Sulfonamides; Tumor Suppressor Protein p53
PubMed: 25395102
DOI: 10.2174/1389450115666141114153536 -
Antioxidants & Redox Signaling Jul 2015Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the...
AIMS
Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines.
RESULTS
We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors.
INNOVATION
We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA.
CONCLUSION
In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).
Topics: Antineoplastic Agents; Breast Neoplasms; Buthionine Sulfoximine; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Oxidation-Reduction; Primary Cell Culture; Vorinostat
PubMed: 25897982
DOI: 10.1089/ars.2014.6189 -
Chemico-biological Interactions Mar 2022Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote...
Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclc;Le-Cre (KO) and Gclc;Le-Cre control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclc;Le-Cre (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.
Topics: Acetylcysteine; Animals; Buthionine Sulfoximine; Cell Line; Chemokine CCL7; Cytokines; Down-Regulation; Epithelial Cells; Epithelial-Mesenchymal Transition; Eye; Glutamate-Cysteine Ligase; Immunity, Innate; Lens, Crystalline; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Reactive Oxygen Species; Up-Regulation
PubMed: 35123994
DOI: 10.1016/j.cbi.2022.109804 -
Journal of Applied Genetics Feb 2024Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in...
Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells.
Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.
Topics: Child; Humans; Buthionine Sulfoximine; Glutamate-Cysteine Ligase; Burkitt Lymphoma; Catalytic Domain; Cyclophosphamide; Doxorubicin; Glutathione
PubMed: 37917375
DOI: 10.1007/s13353-023-00797-1