-
World Journal of Gastroenterology Sep 2011To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo.
AIM
To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo.
METHODS
Different hepatoma and colon carcinoma cell lines (HepG2, HuH7, Chang liver, LoVo, RKO, SW-48, SW-480) were grown in minimal essencial medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution and maintained in a humidified 37 °C incubator with 5% CO₂. These cells were pretreated with BSO for 24 h and then with AZA for different times. We examined the effects of this combination on some proteins and on cellular death. We also studied the efficacy and the safety of AZA (6 mg/kg per day) and BSO (90 mg/kg per day) in HepG2 tumor growth in vivo using athymic mice. We measured safety by serological markers such as aminotransferases and creatine kinase.
RESULTS
The in vitro studies revealed a new mechanism of action for the AZA plus BSO combination in the cancer cells compared with other thiopurines (6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine and 6-methylthioguanine) in combination with BSO. The cytotoxic effect of AZA plus BSO in HepG2 cells resulted from necroptosis induction in a mitochondrial-dependent manner. From kinetic studies we suggest that glutathione (GSH) depletion stimulates c-Jun amino-terminal kinase and Bax translocation in HepG2 cells with subsequent deregulation of mitochondria (cytochrome c release, loss of membrane potential), and proteolysis activation leading to loss of membrane integrity, release of lactate dehydrogenase and DNA degradation. Some of this biochemical and cellular changes could be reversed by N-acetylcysteine (a GSH replenisher). In vivo studies showed that HepG2 tumor growth was inhibited when AZA was combined with BSO.
CONCLUSION
Our studies suggest that a combination of AZA plus BSO could be useful for localized treatment of hepatocellular carcinoma as in the currently used transarterial chemoembolization method.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Azathioprine; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cattle; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Drug Therapy, Combination; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation
PubMed: 22025878
DOI: 10.3748/wjg.v17.i34.3899 -
Analytical Biochemistry May 1991Buthionine sulfoximine inhibits gamma-glutamylcysteine synthetase, the enzyme catalyzing the first reaction of glutathione (GSH) biosynthesis. GSH synthesis is blocked...
Buthionine sulfoximine inhibits gamma-glutamylcysteine synthetase, the enzyme catalyzing the first reaction of glutathione (GSH) biosynthesis. GSH synthesis is blocked in animals or cultured cells exposed to buthionine sulfoximine, and GSH is substantially depleted in cells or tissues with moderate to high rates of GSH utilization. Studies reported to date have used DL-buthionine (SR)-sulfoximine or L-buthionine (SR)-sulfoximine, mixtures of four and two isomers, respectively. The present report describes a chiral solvent HPLC procedure for the analytical separation of the diastereomers of L-buthionine (SR)-sulfoximine and the separation of those isomers from the unresolved diastereomers of D-buthionine (SR)-sulfoximine. L-buthionine (R)-sulfoximine was isolated preparatively by repeated crystallization of L-buthionine (SR)-sulfoximine from water; L-buthionine (S)-sulfoximine was obtained by crystallization as the trifluoroacetate salt in ethanol/hexane mixtures. The absolute configuration, bond lengths and angles of L-buthionine (R)-sulfoximine were determined by X-ray diffraction. In vitro studies demonstrate that L-buthionine (R)-sulfoximine is a relatively weak inhibitor of rat kidney gamma-glutamylcysteine synthetase; binding is competitive with L-glutamate. L-buthionine (S)-sulfoximine is a tight-binding, mechanism-based inhibitor of the enzyme. Since L-buthionine sulfoximine is initially bound as a transition-state analogue, identification of the inhibitory diastereomer elucidates the steric relationships among ATP, glutamate, and cysteine within the active site. When administered to mice, L-buthionine (S)-sulfoximine (0.2 mmol/kg) was as effective as L-buthionine (SR)-sulfoximine (0.4 mmol/kg) in causing GSH depletion in liver, kidney, and pancreas. L-Buthionine (R)-sulfoximine (0.2 mmol/kg) did not cause significant GSH depletion in liver or pancreas. The L-(R)-diastereomer caused a modest GSH depletion in kidney that is tentatively attributed to interference with gamma-glutamylcyst(e)ine transport.
Topics: Animals; Buthionine Sulfoximine; Chromatography, High Pressure Liquid; Glutamate-Cysteine Ligase; Glutathione; Methionine Sulfoximine; Mice; Organ Specificity; Stereoisomerism; X-Ray Diffraction
PubMed: 1677799
DOI: 10.1016/0003-2697(91)90229-m -
Journal of Nanobiotechnology Aug 2022Abundant glutathione (GSH) is a biological characteristic of lots of tumor cells. A growing number of studies are utilizing GSH depletion as an effective adjuvant...
Abundant glutathione (GSH) is a biological characteristic of lots of tumor cells. A growing number of studies are utilizing GSH depletion as an effective adjuvant therapy for tumor. However, due to the compensatory effect of intracellular GSH biosynthesis, GSH is hard to be completely exhausted and the strategy of GSH depletion remains challenging. Herein, we report an L-buthionine-sulfoximine (BSO)-based hypertoxic self-assembled peptide derivative (NSBSO) with dual functions of GSH depletion and biosynthesis inhibition for selective tumor ferroptosis and pyroptosis. The NSBSO consists of a hydrophobic self-assembled peptide motif and a hydrophilic peptide derivative containing BSO that inhibits the synthesis of GSH. NSBSO was cleaved by GSH and thus experienced a morphological transformation from nanoparticles to nanofibers. NSBSO showed GSH-dependent cytotoxicity and depletion of intracellular GSH. In 4T1 cells with medium GSH level, it depleted intracellular GSH and inactivated GSH peroxidase 4 (GPX4) and thus induced efficient ferroptosis. While in B16 cells with high GSH level, it exhausted GSH and triggered indirect increase of intracellular ROS and activation of Caspase 3 and gasdermin E, resulting in severe pyroptosis. These findings demonstrate that GSH depletion- and biosynthesis inhibition-induced ferroptosis and pyroptosis strategy would provide insights in designing GSH-exhausted medicines.
Topics: Buthionine Sulfoximine; Ferroptosis; Glutathione; Pyroptosis
PubMed: 36045424
DOI: 10.1186/s12951-022-01604-5 -
The Journal of Biological Chemistry Feb 2024Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are...
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
Topics: Animals; Mice; Buthionine Sulfoximine; Disease Models, Animal; Glutamate-Cysteine Ligase; Glutathione; Cell Line, Tumor; Humans
PubMed: 38218225
DOI: 10.1016/j.jbc.2024.105645 -
European Review For Medical and... Jul 2021Cataract which is defined as opacification of eye lens forms approximately 40% of total blindness causes all through the world. Age is the biggest risk factor for...
OBJECTIVE
Cataract which is defined as opacification of eye lens forms approximately 40% of total blindness causes all through the world. Age is the biggest risk factor for cataracts and oxidative stress is known to be one of the most important factors causing cataract formation. Age-related nuclear cataract (ARN) is associated with a loss of glutathione in the center of the lens. Taurine is an important antioxidant in lens tissue. Although, there is a high amount of taurine in lenses in early life, its concentration declines with age. In this study, we aimed to investigate the effects of supplemental taurine in lens tissues in an in vivo oxidative stress model which is induced by glutathione depletion to mimic ARN.
MATERIALS AND METHODS
Glutathione depletion was induced in rabbits subcutaneously with l-Buthionine -(S,R)-sulfoximine (BSO)- a glutathione inhibitor and the rabbits were treated with taurine. Total GSH, reduced GSH, GSH/GSSG ratio and MDA levels were measured.
RESULTS
BSO lowered the reduced GSH and total GSH levels and GSH/GSSG ratio. Taurine reversed these effects. On the other hand, BSO enhanced MDA level which is normalized by taurine.
CONCLUSIONS
These findings suggest that glutathione depletion with BSO may be a useful model to mimic ARN and dietary intake of taurine, may have an important role in decelerating the process of cataract formation.
Topics: Animals; Buthionine Sulfoximine; Cataract; Dietary Supplements; Disease Models, Animal; Female; Glutathione; Humans; Lens, Crystalline; Male; Oxidative Stress; Rabbits; Taurine
PubMed: 34286494
DOI: 10.26355/eurrev_202107_26244 -
Clinical Oral Investigations May 2018The aim of this study was to evaluate the cytotoxicity and the influence of bleaching agents on immunologically cell surface antigens of murine macrophages in vitro.
OBJECTIVES
The aim of this study was to evaluate the cytotoxicity and the influence of bleaching agents on immunologically cell surface antigens of murine macrophages in vitro.
MATERIALS AND METHODS
RAW 264.7 cells were exposed to bleaching gel extracts (40% hydrogen peroxide or 20% carbamide peroxide) and different HO concentrations after 1 and 24-h exposure periods and 1-h exposure and 23-h recovery. Tests were performed with and without N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO). Cell viability was determined by MTT assay. The expression of surface markers CD14, CD40, and CD54 with and without LPS stimulation was detected by flow cytometry, while the production of TNF-α was measured by ELISA. Statistical analysis was performed using the Mann-Whitney U test (α = 0.05).
RESULTS
Extracts of bleaching agents were cytotoxic for cells after a 1-h exposure; cells could not recover after 24 h. This effect can be mitigated by the antioxidant NAC and increased by BSO, an inhibitor of glutathione (GSH) synthesis. LPS stimulated expression of all surface markers and TNF-α production. Exposure to bleaching agent extracts and HO leads to a reduction of TNF-α, CD14, and CD40 expression, while the expression of CD54 was upregulated at non-cytotoxic concentrations. Whereas NAC reduced this effect, it was increased in the presence of BSO.
CONCLUSIONS
Extracts of bleaching agents were irreversibly cytotoxic to macrophages after a 1-h exposure. Only the expression of CD54 was upregulated. The reactions are mediated by the non-enzymatic antioxidant GSH.
CLINICAL RELEVANCE
The addition of an antioxidant can downregulate unfavorable effects of dental bleaching.
Topics: Acetylcysteine; Animals; Antigens, Surface; Bleaching Agents; Buthionine Sulfoximine; Carbamide Peroxide; Cell Survival; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Hydrogen Peroxide; Macrophages; Mice; Peroxides; Tooth Bleaching; Tumor Necrosis Factor-alpha; Urea
PubMed: 29196947
DOI: 10.1007/s00784-017-2273-1 -
Frontiers in Immunology 2021(1→3)-β-D-Glucan (BDG) represents a potent pathogen-associated molecular pattern (PAMP) in triggering the host response to fungal and some bacterial infections....
(1→3)-β-D-Glucan (BDG) represents a potent pathogen-associated molecular pattern (PAMP) in triggering the host response to fungal and some bacterial infections. Monocytes play a key role in recognizing BDG and governing the acute host response to infections. However, the mechanisms regulating monocyte's acute response to BDG are poorly understood. We sought to investigate the response of monocytes to BDG at the epigenetic, transcriptomic, and molecular levels. Response of human monocytes to 1, 4, and 24 hours of BDG exposure was investigated using RNA-seq, ATAC-seq, H3K27ac and H3K4me1 ChIP-seq. We show that pathways including glutathione metabolism, pentose phosphate pathway, and citric acid cycle were upregulated at the epigenetic and transcriptomic levels in response to BDG exposure. Strikingly, unlike bacterial lipopolysaccharides, BDG induced intracellular glutathione synthesis. BDG exposure also induced NADP synthesis, increased NADPH/NADP ratio, and increased expression of genes involved in the pentose phosphate pathway in a GSH-dependent manner. By inhibiting GSH synthesis with L-buthionine sulfoximine (BSO) before BDG exposure we show that the GSH pathway promotes cell survival and regulates monocyte's effector functions including NO production, phagocytosis, and cytokine production. In summary, our work demonstrates that BDG induces glutathione synthesis and metabolism in monocytes, which is a major promoter of the acute functional response of monocytes to infections.
Topics: Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Citric Acid; Epigenesis, Genetic; Gene Expression Profiling; Glutathione; Humans; Immunity, Innate; Monocytes; Nitric Oxide; Pathogen-Associated Molecular Pattern Molecules; Pentose Phosphate Pathway; Phagocytosis; Proteoglycans; Sequence Analysis, RNA
PubMed: 34858388
DOI: 10.3389/fimmu.2021.694152 -
Journal of Nanobiotechnology Aug 2023Emerging ferroptosis-driven therapies based on nanotechnology function either by increasing intracellular iron level or suppressing glutathione peroxidase 4 (GPX4)...
BACKGROUND
Emerging ferroptosis-driven therapies based on nanotechnology function either by increasing intracellular iron level or suppressing glutathione peroxidase 4 (GPX4) activity. Nevertheless, the therapeutic strategy of simultaneous iron delivery and GPX4 inhibition remains challenging and has significant scope for improvement. Moreover, current nanomedicine studies mainly use disulfide-thiol exchange to deplete glutathione (GSH) for GPX4 inactivation, which is unsatisfactory because of the compensatory effect of continuous GSH synthesis.
METHODS
In this study, we design a two-in-one ferroptosis-inducing nanoplatform using iron-based metal-organic framework (MOF) that combines iron supply and GPX4 deactivation by loading the small molecule buthionine sulfoxide amine (BSO) to block de novo GSH biosynthesis, which can achieve sustainable GSH elimination and dual ferroptosis amplification. A coated lipid bilayer (L) can increase the stability of the nanoparticles and a modified tumor-homing peptide comprising arginine-glycine-aspartic acid (RGD/R) can achieve tumor-specific therapies. Moreover, as a decrease in GSH can alleviate resistance of cancer cells to chemotherapy drugs, oxaliplatin (OXA) was also loaded to obtain BSO&OXA@MOF-LR for enhanced cancer chemo-ferrotherapy in vivo.
RESULTS
BSO&OXA@MOF-LR shows a robust tumor suppression effect and significantly improved the survival rate in 4T1 tumor xenograft mice, indicating a combined effect of dual amplified ferroptosis and GSH elimination sensitized apoptosis.
CONCLUSION
BSO&OXA@MOF-LR is proven to be an efficient ferroptosis/apoptosis hybrid anti-cancer agent. This study is of great significance for the clinical development of novel drugs based on ferroptosis and apoptosis for enhanced cancer chemo-ferrotherapy.
Topics: Humans; Mice; Animals; Buthionine Sulfoximine; Oxaliplatin; Metal-Organic Frameworks; Neoplasms; Glutathione
PubMed: 37563614
DOI: 10.1186/s12951-023-01998-w -
Clinical and Experimental Hypertension... Feb 2021Oxidative stress and renal inflammation play a pivotal role in the pathogenesis of hypertension. The redox-sensitive transcription factor, nuclear factor E2-related...
Oxidative stress and renal inflammation play a pivotal role in the pathogenesis of hypertension. The redox-sensitive transcription factor, nuclear factor E2-related factor 2 (Nrf2) is the master regulator of phase II antioxidant enzymes that protects against oxidative stress and inflammation. This study aimed to investigate the effect of Nrf2 inhibition on oxidative stress-associated hypertension and renal dopamine 1 receptor (D1R) dysfunction in mice. Male C57BL/6 J mice were treated with a pro-oxidant, L-buthionine sulfoximine (BSO) (10 mmol/L in drinking water), and ML385 (10 kg body weight/kg body weight/day, intraperitoneally), a novel Nrf2 inhibitor that blocks Nrf2 regulated downstream target genes expression. Mice treated with BSO exhibited oxidative stress, renal functional impairment, inflammation, and elevated blood pressure. Also, BSO treatment increased the activity of phase II antioxidant enzyme, NAD(P)H: quinone oxidoreductase-1 (NQO-1). BSO and ML385 co-treatment exhibited a robust increase in blood pressure, oxidative stress and intensified the renal function deterioration as indicated by a significant increase in serum creatinine, urinary albumin excretion rate, and albumin to creatinine ratio and decreased glomerular filtration rate (GFR). Also, BSO and ML385 co-treatment downregulated NQO-1 and significantly altered the inflammatory cytokines, IL-1β and IL-10 levels. A D1R agonist SKF38393 failed to promote urinary sodium excretion indicating functional impairment in renal D1R. ML385 per se did not affect mean arterial pressure, GFR, and renal D1R function. Taken together, we concluded that the Nrf2 inhibition aggravated oxidative stress and inflammation by diminishing phase II antioxidant defense that deteriorates renal function and contributes to the development of hypertension in mice.
Topics: Animals; Antioxidants; Blood Pressure; Buthionine Sulfoximine; Enzyme Inhibitors; Gene Expression Regulation; Hypertension; Inflammation; Kidney; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species
PubMed: 33070655
DOI: 10.1080/10641963.2020.1836191 -
Asian Pacific Journal of Cancer... 2015Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative...
BACKGROUND
Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration.
MATERIALS AND METHODS
T47D human breast cancer cells were treated with 2-deoxy- D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent- phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays.
RESULTS
Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration.
CONCLUSIONS
Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.
Topics: Actins; Antibiotics, Antineoplastic; Antimetabolites; Antioxidants; Apoptosis; Blotting, Western; Breast Neoplasms; Buthionine Sulfoximine; Cell Adhesion; Cell Movement; Cell Proliferation; Deoxyglucose; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Female; Flow Cytometry; Focal Adhesions; Glutathione; Humans; Lipid Peroxidation; Mitosis; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Tumor Cells, Cultured; Wound Healing
PubMed: 25921122
DOI: 10.7314/apjcp.2015.16.8.3213