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Chemical Biology & Drug Design May 2021Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and...
Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.
Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and synthesized isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and rationally combined ITC-ARi with GSH synthesis inhibitor buthionine sulfoximine (BSO) to efficiently downregulate AR/AR splice variant and induce ferroptosis in CRPC cells. The representative ITC-ARi 13 is an AR ligand that contains an N-acetyl cysteine-masked ITC moiety and gradually releases parental unconjugated ITC 12b in aqueous solution. The in vitro anti-PCa activities of 13, such as growth inhibition and AR downregulation, are significantly enhanced when combined with BSO. The drug combination caused notable lipid peroxidation and the cell viability was effectively rescued by iron chelator, antioxidants or the inhibitor of heme oxygenase-1, supporting the induction of ferroptosis. 13 and BSO cooperatively downregulate AR and induce ferroptosis likely through increasing the accessibility of 13/12b to cellular targets, escalating free intracellular ferrous iron and attenuating GSH-centered cellular defense and adaptation. Further studies on the combination of ITC-ARi and GSH synthesis inhibitor could result in a new modality against CRPC.
Topics: Androgen Receptor Antagonists; Binding Sites; Buthionine Sulfoximine; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Drug Design; Ferroptosis; Glutathione; Humans; Isothiocyanates; Male; Molecular Docking Simulation; Prostatic Neoplasms, Castration-Resistant; Protein Isoforms; Receptors, Androgen; Transcriptional Activation
PubMed: 33470049
DOI: 10.1111/cbdd.13826 -
PloS One 2013Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of...
BACKGROUND
Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.
MATERIALS AND METHODS
Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.
RESULTS
Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.
CONCLUSION/SIGNIFICANCE
BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.
Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Buthionine Sulfoximine; Cell Line, Tumor; Chlorocebus aethiops; Drug Synergism; Eugenol; Extracellular Signal-Regulated MAP Kinases; Glutathione; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Microscopy, Confocal; NIH 3T3 Cells; Nitric Oxide; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Signal Transduction; U937 Cells; Vero Cells
PubMed: 24040019
DOI: 10.1371/journal.pone.0073672 -
The Journal of Toxicological Sciences 2015In this study, we investigated the in vivo effects of exogenous glutathione and buthionine sulfoximine on arsenic methylation and antioxidant capacity in mice exposed to...
In this study, we investigated the in vivo effects of exogenous glutathione and buthionine sulfoximine on arsenic methylation and antioxidant capacity in mice exposed to arsenic via drinking water. Thirty-six female albino mice were randomly divided into six groups. All groups were given free access to drinking water that contained arsenic continuously except the control group. After ten days, mice were treated with different levels of glutathione or buthionine sulfoximine. The levels of the metabolites of arsenic were determined in the liver and urine. The levels of glutathione and total antioxidant capacity were determined in the whole blood and liver. Our results showed that the increase of arsenic species in the liver as well as the decrease of blood and hepatic glutathione and total antioxidant capacity, were all relieved by exogenous glutathione consistently. We also observed the involvement of glutathione in promoting arsenic methylation and urinary elimination in vivo. Increase of total arsenic in the urine was mainly due to the increase of dimethylated arsenic. Furthermore, administration of glutathione increased the first methylation ratio and secondary methylation ratio in the liver and urine, which resulted in the consequent increase of dimethylated arsenic percent and decrease of inorganic arsenic percent in the urine. Opposite effects appeared with the administration of buthionine sulfoximine, a scavenger of glutathione. Our study indicated that exogenous glutathione not only accelerated the methylation and the excretion of arsenic, but also relieve the arsenic-induced oxidative stress. This provides a potential useful chemopreventive dietary component for human populations being at risk of arsenic exposure.
Topics: Animals; Antioxidants; Arsenic Poisoning; Arsenicals; Buthionine Sulfoximine; Chemoprevention; Female; Glutathione; Liver; Methylation; Mice; Oxidative Stress
PubMed: 26354374
DOI: 10.2131/jts.40.577 -
Biotechnology and Bioengineering Nov 2020Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have...
Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate-cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.
Topics: Animals; Buthionine Sulfoximine; CHO Cells; Cholesterol; Cricetulus; Glutathione; Proteome; Proteomics
PubMed: 32662871
DOI: 10.1002/bit.27495 -
Antimicrobial Agents and Chemotherapy Jan 2005l-Buthionine (S,R)-sulfoximine (BSO) increased the toxicity of nifurtimox and benznidazole toward the epimastigote, trypomastigote, and amastigote forms of Trypanosoma...
l-Buthionine (S,R)-sulfoximine (BSO) increased the toxicity of nifurtimox and benznidazole toward the epimastigote, trypomastigote, and amastigote forms of Trypanosoma cruzi. BSO at 500 muM decreased total glutathione-derived thiols by 70 to 80% in 48 h. In epimastigotes, 500 muM BSO decreased the concentration of nifurtimox needed to inhibit constant growth of the parasites by 50%, from 14.0 to 9.0 muM, and decreased that of benznidazole from 43.6 to 24.1 muM. The survival of epimastigotes or trypomastigotes treated with nifurtimox or benznidazole, as measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction, was significantly decreased by 500 muM BSO. In Vero cells infected with amastigotes, 25 muM BSO was able to potentiate the effect of nifurtimox and benznidazole as measured by the percentage of infected Vero cells multiplied by the average number of intracellular amastigotes (endocytic index). At 0.5 muM nifurtimox, the proportion of Vero cells infected decreased from 27 to 20% and the endocytic index decreased from 2,500 to 980 when 25 muM BSO was added. Similar results were obtained with benznidazole- and BSO-benznidazole-treated cells. This study indicates that potentiation of nifurtimox or benznidazole by BSO could decrease the clinical dose of both drugs and diminish the side effects or the length of therapy.
Topics: Animals; Buthionine Sulfoximine; Chlorocebus aethiops; Drug Synergism; Nifurtimox; Nitroimidazoles; Parasitic Sensitivity Tests; Trypanocidal Agents; Trypanosoma cruzi; Vero Cells
PubMed: 15616285
DOI: 10.1128/AAC.49.1.126-130.2005 -
British Journal of Cancer Nov 1986Two and four treatments of 5 mmol kg-1 of buthionine sulfoximine (BSO) at an interval of 12 h depleted the glutathione (GSH) content in NFSa tumours of C3H/He mice,...
Two and four treatments of 5 mmol kg-1 of buthionine sulfoximine (BSO) at an interval of 12 h depleted the glutathione (GSH) content in NFSa tumours of C3H/He mice, respectively, to 24.0 and 1.78 percent of the untreated controls. BSO pre-treatments every 12 h enhanced the cytotoxicity of cyclophosphamide (CYC) towards artificial lung micrometastases of NFSa tumours giving enhancement ratios (ERs) ranging from 1.75 to 1.83 and from 2.41 to 2.73, for two and four BSO pretreatments respectively. Large ERs were obtained at low CYC doses (high cell survival). Four BSO pre-treatments at an interval of 12 h did not increase the cytotoxicity of CYC to bone marrow stem cells. Our results suggest a clinical applicability of the combination of BSO and CYC.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Buthionine Sulfoximine; Cell Survival; Colony-Forming Units Assay; Cyclophosphamide; Cysteine; Glutathione; Lung Neoplasms; Male; Methionine Sulfoximine; Mice; Mice, Inbred C3H
PubMed: 3801271
DOI: 10.1038/bjc.1986.236 -
Journal of Hepatology Nov 1999Ethanol consumption and pathological conditions such as cirrhosis lead to a reduction of hepatic glutathione. Hepatic methionine adenosyltransferase, the enzyme that...
BACKGROUND/AIMS
Ethanol consumption and pathological conditions such as cirrhosis lead to a reduction of hepatic glutathione. Hepatic methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, the major methylating agent, is regulated in vivo by glutathione levels. We have previously shown that nitric oxide inactivates methionine adenosyltransferase in vivo by S-nitrosylation. In this study, we aimed to investigate the regulation by glutathione of methionine adenosyltransferase S-nitrosylation in rat liver.
METHODS
Rat hepatocytes and whole animals were treated with buthionine sulfoximine, an inhibitor of glutathione synthesis, and methionine adenosyltransferase S-nitrosylation and activity were determined.
RESULTS
In hepatocytes, buthionine sulfoximine led to the S-nitrosylation and inactivation of methionine adenosyltransferase. Restoring glutathione levels in hepatocytes treated with buthionine sulfoximine, by the addition of glutathione monoethyl ester, a permeable derivative of glutathione, led to the denitrosylation and reactivation of methionine adenosyltransferase. In whole animals, buthionine sulfoximine led also to methionine adenosyltransferase S-nitrosylation and inactivation. S-Nitrosylation and inactivation of methionine adenosyltransferase induced by buthionine sulfoximine in whole animals was prevented by glutathione monoethyl ester.
CONCLUSIONS
These results indicate that in vivo hepatic methionine adenosyltransferase exists in two forms in equilibrium, nitrosylated (inactive) and denitrosylated (active), which are regulated by both the cellular levels of nitric oxide and glutathione.
Topics: Animals; Buthionine Sulfoximine; Cells, Cultured; Glutathione; Glutathione Disulfide; Kinetics; Liver; Methionine Adenosyltransferase; Nitric Oxide; Nitroso Compounds; Rats; Rats, Wistar
PubMed: 10580587
DOI: 10.1016/s0168-8278(99)80291-8 -
Pediatric Blood & Cancer Aug 2016Myeloablative therapy for high-risk neuroblastoma commonly includes melphalan. Increased cellular glutathione (GSH) can mediate melphalan resistance. Buthionine...
A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent/Refractory High-Risk Neuroblastoma.
BACKGROUND
Myeloablative therapy for high-risk neuroblastoma commonly includes melphalan. Increased cellular glutathione (GSH) can mediate melphalan resistance. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor, enhances melphalan activity against neuroblastoma cell lines, providing the rationale for a Phase 1 trial of BSO-melphalan.
PROCEDURES
Patients with recurrent/resistant high-risk neuroblastoma received BSO (3 gram/m(2) bolus, then 24 grams/m(2) /day infusion days -4 to -2), with escalating doses of intravenous melphalan (20-125 mg/m(2) ) days -3 and -2, and autologous stem cells day 0 using 3 + 3 dose escalation.
RESULTS
Among 28 patients evaluable for dose escalation, one dose-limiting toxicity occurred at 20 mg/m(2) melphalan (grade 3 aspartate aminotransferase/alanine aminotransferase) and one at 80 mg/m(2) (streptococcal bacteremia, grade 4 hypotension/pulmonary/hypocalcemia) without sequelae. Among 25 patients evaluable for response, there was one partial response (PR) and two mixed responses (MRs) among eight patients with prior melphalan exposure; one PR and three MRs among 16 patients without prior melphalan; one stable disease with unknown melphalan history. Melphalan pharmacokinetics with BSO were similar to reports for melphalan alone. Melphalan Cmax for most patients was below the 10 μM concentration that showed neuroblastoma preclinical activity with BSO.
CONCLUSIONS
BSO (75 gram/m(2) ) with melphalan (125 mg/m(2) ) is tolerable with stem cell support and active in recurrent/refractory neuroblastoma. Further dose escalation is feasible and may increase responses.
Topics: Adolescent; Antimetabolites, Antineoplastic; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Buthionine Sulfoximine; Child; Child, Preschool; Drug Synergism; Female; Glutamate-Cysteine Ligase; Glutathione; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Male; Melphalan; Myeloablative Agonists; Neoplasm Recurrence, Local; Neuroblastoma
PubMed: 27092812
DOI: 10.1002/pbc.25994 -
Toxicology in Vitro : An International... Aug 2021Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay...
Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 μM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 μM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.
Topics: Activation, Metabolic; Aspirin; Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Coculture Techniques; Cytochrome P-450 Enzyme System; Diclofenac; Glutathione; Hepatocytes; High-Throughput Screening Assays; Humans; Microsomes, Liver; Ticlopidine
PubMed: 33823239
DOI: 10.1016/j.tiv.2021.105159 -
Scientific Reports Oct 2022Alzheimer's disease (AD) is a type of dementia that affects memory, thinking and behavior. Symptoms eventually become severe enough to interfere with daily tasks....
Alzheimer's disease (AD) is a type of dementia that affects memory, thinking and behavior. Symptoms eventually become severe enough to interfere with daily tasks. Understanding the etiology and pathogenesis of AD is necessary for the development of strategies for AD prevention and/or treatment, and modeling of this pathology is an important step in achieving this goal. β-amyloid peptide (Aβ) injection is a widely used approach for modeling AD. Nevertheless, it has been reported that the model constructed by injection of Aβ in combination with a prooxidant cocktail (ferrous sulfate, Aβ, and buthionine sulfoximine (BSO) (FAB)) best reflects the natural development of this disease. The relationship between oxidative stress and Aβ deposition and their respective roles in Aβ-induced pathology in different animal models of AD have been thoroughly investigated. In the current paper, we compared the effects of Aβ 1-42 alone with that of Aβ-associated oxidative stress induced by the FAB cocktail on the neurodegeneration of hippocampal cells in vitro. We constructed a FAB-induced AD model using rat primary hippocampal cells and analyzed the contribution of each compound. The study mainly focused on the prooxidant aspects of AD pathogenesis. Moreover, cellular bioenergetics was assessed and routine metabolic tests were performed to determine the usefulness of this model. The data clearly show that aggregated Aβ1-42 alone is significantly less toxic to hippocampal cells. Aggregated Aβ damages neurons, and glial cells proliferate to remove Aβ from the hippocampus. External prooxidant agents (Fe) or inhibition of internal antioxidant defense by BSO has more toxic effects on hippocampal cells than aggregated Aβ alone. Moreover, hippocampal cells fight against Aβ-induced damage more effectively than against oxidative damage. However, the combination of Aβ with external oxidative damage and inhibition of internal antioxidant defense is even more toxic, impairs cellular defense systems, and may mimic the late phase of AD-associated cell damage. Our findings strongly indicate a critical role for the combination of Aβ and oxidative stress in the development of neurodegeneration in vitro.
Topics: Animals; Rats; Amyloid beta-Peptides; Alzheimer Disease; Antioxidants; Buthionine Sulfoximine; Hippocampus; Oxidative Stress; Reactive Oxygen Species; Disease Models, Animal; Peptide Fragments
PubMed: 36284177
DOI: 10.1038/s41598-022-22761-5