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Autophagy Jul 2023Ferroptosis is a newly characterized form of programmed cell death, which is driven by the lethal accumulation of lipid peroxides catalyzed by the intracellular...
Ferroptosis is a newly characterized form of programmed cell death, which is driven by the lethal accumulation of lipid peroxides catalyzed by the intracellular bioactive iron. Targeted induction of ferroptotic cell death holds great promise for therapeutic design against other therapy-resistant cancers. To date, multiple post-translational modifications have been elucidated to impinge on the ferroptotic sensitivity. Here we report that the Ser/Thr protein kinase ATM, the major sensor of DNA double-strand break damage, is indispensable for ferroptosis execution. Pharmacological inhibition or genetic ablation of ATM significantly antagonizes ferroptosis. Besides, ATM ablation-induced ferroptotic resistance is largely independent of its downstream target TRP53, as cells defective in both and are still more insensitive to ferroptotic inducers than the single knockout cells. Mechanistically, ATM dominates the intracellular labile free iron by phosphorylating NCOA4, facilitating NCOA4-ferritin interaction and therefore sustaining ferritinophagy, a selective type of macroautophagy/autophagy specifically degrading ferritin for iron recycling. Our results thus uncover a novel regulatory circuit of ferroptosis comprising ATM-NCOA4 in orchestrating ferritinophagy and iron bioavailability. AMPK: AMP-activated protein kinase; ATM: ataxia telangiectasia mutated; BSO: buthionine sulphoximine; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); CQ: chloroquine; DFO: deferoxamine; DFP: deferiprone; Fer: ferrostatin-1; FTH1: ferritin heavy polypeptide 1; GPX4: glutathione peroxidase 4; GSH: glutathione; MEF: mouse embryonic fibroblast; NCOA4: nuclear receptor coactivator 4; PFTα: pifithrin-α; PTGS2: prostaglandin-endoperoxide synthase 2; Slc7a11: solute carrier family 7 member 11; Sul: sulfasalazine; TFRC: transferrin receptor; TRP53: transformation related protein 53.
Topics: Animals; Mice; Ferroptosis; Autophagy; Fibroblasts; Transcription Factors; Ferritins; Iron; Buthionine Sulfoximine
PubMed: 36752571
DOI: 10.1080/15548627.2023.2170960 -
Cell Death & Disease Jul 2018Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE...
Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE and subsequent death of photoreceptors have been observed in age-related macular degeneration (AMD). Although the consequences of GSH depletion have been described previously, questions remain regarding the molecular mechanisms. We herein examined the downstream effects of GSH depletion on stress-induced premature senescence (SIPS) and cell death in human RPE cells. Briefly, cultured ARPE-19 cells were depleted of GSH using: (1) incubation in cystine (Cys)-free culture medium; (2) treatment with buthionine sulphoximine (BSO, 1000 µM) to block de novo GSH synthesis for 24-48 h; or (3) treatment with erastin (10 µM for 12-24 h) to inhibit Cys/glutamate antiporter (system x). These treatments decreased cell viability and increased both soluble and lipid reactive oxygen species (ROS) generation but did not affect mitochondrial ROS or mitochondrial mass. Western blot analysis revealed decreased expression of ferroptotic modulator glutathione peroxidase 4 (GPX4). Increased autophagy was apparent, as reflected by increased LC3 expression, autophagic vacuoles, and autophagic flux. In addition, GSH depletion induced SIPS, as evidenced by increased percentage of the senescence-associated β-galactosidase-positive cells, increased senescence-associated heterochromatin foci (SAHF), as well as cell cycle arrest at the G1 phase. GSH depletion-dependent cell death was prevented by selective ferroptosis inhibitors (8 μM Fer-1 and 600 nM Lip-1), iron chelator DFO (80 μM), as well as autophagic inhibitors Baf-A1 (75 nM) and 3-MA (10 mM). Inhibiting autophagy with Baf-A1 (75 nM) or 3-MA (10 mM) promoted SIPS. In contrast, inducing autophagy with rapamycin (100 nM) attenuated SIPS. Our findings suggest that GSH depletion induces ferroptosis, autophagy, and SIPS. In addition, we found that autophagy is activated in the process of ferroptosis and reduces SIPS, suggesting an essential role of autophagy in ferroptosis and SIPS.
Topics: Apoptosis; Autophagy; Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Cellular Senescence; Glutathione; Glutathione Peroxidase; Humans; Lipid Peroxidation; Oxidative Stress; Phospholipid Hydroperoxide Glutathione Peroxidase; Piperazines; Reactive Oxygen Species; Retinal Pigment Epithelium
PubMed: 29988039
DOI: 10.1038/s41419-018-0794-4 -
Molecules (Basel, Switzerland) Aug 2019l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug...
l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug delivery systems and l-BSO nanoformulations are envisaged as potential chemotherapeutics. The absorption of l-BSO at a low wavelength limits its detection by conventional analytical tools. A simple and sensitive method for l-BSO detection and quantification is now reported. In this study, l-BSO was encapsulated in a folate-targeted generation four polyurea dendrimer (PURE-FA) and its release profile was followed for 24 h at pH 7.4 and 37 °C. The protocol uses in situ l-BSO derivatization, by the formation of a catechol-derived -quinone, followed by visible detection of the derivative at 503 nm. The structure of the studied l-BSO derivative was assessed by NMR spectroscopy.
Topics: Buthionine Sulfoximine; Dendrimers; Magnetic Resonance Spectroscopy; Molecular Structure; Nanoparticles; Polymers
PubMed: 31461931
DOI: 10.3390/molecules24173111 -
PloS One 2017Acquisition of anoikis resistance is a prerequisite for metastasis in hepatocellular carcinoma (HCC). However, little is known about how energy metabolism and...
BACKGROUND & AIMS
Acquisition of anoikis resistance is a prerequisite for metastasis in hepatocellular carcinoma (HCC). However, little is known about how energy metabolism and antioxidant systems are altered in anoikis-resistant (AR) HCC cells. We evaluated anti-tumor effects of a combination treatment of 3-bromopyruvate (3-BP) and buthionine sulfoximine (BSO) in AR HCC cells.
METHODS
We compared glycolysis, reactive oxygen species (ROS) production, and chemoresistance among Huh-BAT, HepG2 HCC cells, and the corresponding AR cells. Expression of hexokinase II, gamma-glutamylcysteine synthetase (rGCS), and epithelial-mesenchymal transition (EMT) markers in AR cells was assessed. Anti-tumor effects of a combination treatment of 3-BP and BSO were evaluated in AR cells and an HCC xenograft mouse model.
RESULTS
AR HCC cells showed significantly higher chemoresistance, glycolysis and lower ROS production than attached cells. Expression of hexokinase II, rGCS, and EMT markers was higher in AR HCC cells than attached cells. A combination treatment of 3-BP/BSO effectively suppressed proliferation of AR HCC cells through apoptosis by blocking glycolysis and enhancing ROS levels. In xenograft mouse models, tumor growth derived from AR HCC cells was significantly suppressed in the group treated with 3-BP/BSO compared to the group treated with 3-BP or sorafenib.
CONCLUSIONS
These results demonstrated that a combination treatment of 3-BP/BSO had a synergistic anti-tumor effect in an AR HCC model. This strategy may be an effective adjuvant therapy for patients with sorafenib-resistant HCC.
Topics: Anoikis; Antineoplastic Agents; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Epithelial-Mesenchymal Transition; Hep G2 Cells; Humans; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Pyruvates; Reactive Oxygen Species; Sorafenib
PubMed: 28362858
DOI: 10.1371/journal.pone.0174271 -
Blood Advances Jan 2024Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid...
Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.
Topics: Humans; Cysteine; Reactive Oxygen Species; Antioxidants; Ferroptosis; Cystathionine; Sulfasalazine; Amino Acids; Glutathione; Buthionine Sulfoximine; Leukemia, Myeloid, Acute
PubMed: 37906522
DOI: 10.1182/bloodadvances.2023010786 -
Redox Biology Nov 2023Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that...
Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.
Topics: Animals; Humans; Zebrafish; Glutathione; Glutamate-Cysteine Ligase; TOR Serine-Threonine Kinases; Mechanistic Target of Rapamycin Complex 1; Seizures; Buthionine Sulfoximine; Mammals
PubMed: 37769522
DOI: 10.1016/j.redox.2023.102895 -
Seminars in Radiation Oncology Jan 2019Dysregulated glucose and redox metabolism are near universal features of cancers. They therefore represent potential selectively toxic metabolic targets. This review... (Review)
Review
Dysregulated glucose and redox metabolism are near universal features of cancers. They therefore represent potential selectively toxic metabolic targets. This review outlines the preclinical and clinical data for targeting glucose and hydroperoxide metabolism in cancer, with a focus on drug strategies that have the most available evidence. In particular, inhibition of glycolysis using 2-deoxyglucose, and inhibition of redox metabolism using the glutathione pathway inhibitor buthionine sulfoximine and the thioredoxin pathway inhibitor auranofin, have shown promise in preclinical studies to increase sensitivity to chemotherapy and radiation by increasing intracellular oxidative stress. Combined inhibition of glycolysis, glutathione, and thioredoxin pathways sensitizes highly glycolytic, radioresistant cancer models in vitro and in vivo. Although the preclinical data support this approach, clinical data are limited to exploratory trials using a single drug in combination with either chemotherapy or radiation. Open research questions include optimizing drug strategies for targeting glycolysis and redox metabolism, determining the appropriate timing for administering this therapy with concurrent chemotherapy and radiation, and identifying biomarkers to determine the cancers that would benefit most from this approach. Given the quality of preclinical evidence, dual targeting of glycolysis and redox metabolism in combination with chemotherapy and radiation should be further evaluated in clinical trials.
Topics: Animals; Auranofin; Buthionine Sulfoximine; Deoxyglucose; Glucose; Glycolysis; Humans; Hydrogen Peroxide; Neoplasms; Oxidation-Reduction; Oxidative Stress; Radiation-Sensitizing Agents
PubMed: 30573182
DOI: 10.1016/j.semradonc.2018.10.007 -
Biochimica Et Biophysica Acta May 1995Glutathione deficiency produced by giving buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase) to animals, leads to biphasic decline in cellular... (Review)
Review
Glutathione deficiency produced by giving buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase) to animals, leads to biphasic decline in cellular glutathione levels associated with sequestration of glutathione in mitochondria. Liver mitochondria lack the enzymes needed for glutathione synthesis. Mitochondrial glutathione arises from the cytosol. Rat liver mitochondria have a multicomponent system (with Kms of approx. 60 microM and 5.4 mM) that underlies their remarkable ability to transport and retain glutathione. Mitochondria produce substantial quantities of reactive oxygen species; this is opposed by reactions involving glutathione. Glutathione deficiency leads to widespread mitochondrial damage which is lethal in newborn rats and guinea pigs, animals that do not synthesize ascorbate. Glutathione esters and ascorbate protect against the lethal and other effects of glutathione deficiency. Ascorbate spares glutathione; it increases mitochondrial glutathione in glutathione-deficient animals. Glutathione esters delay onset of scurvy in ascorbate-deficient guinea pigs; thus, glutathione spares ascorbate. Glutathione and ascorbate function together in protecting mitochondria from oxidative damage.
Topics: Animals; Animals, Newborn; Antimetabolites; Ascorbic Acid; Buthionine Sulfoximine; Cerebral Cortex; Glutathione; Guinea Pigs; Liver; Methionine Sulfoximine; Mice; Mitochondria; Mitochondria, Liver; Models, Biological; Rats
PubMed: 7599223
DOI: 10.1016/0925-4439(95)00007-q -
Scientific Reports Jun 2016Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine...
Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation and poly-ADP ribose polymerase (PARP) cleavage were performed to measure the induction of apoptosis after nifurtimox treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. Tumor cells were then treated with combinations of nifurtimox and BSO and evaluated for viability using MTT assays. All neural tumor cell lines were sensitive to nifurtimox, and IC50 values ranged from approximately 20 to 210 μM. Nifurtimox treatment inhibited ERK phosphorylation and induced apoptosis in tumor cells. Furthermore, the combination of nifurtimox and BSO demonstrated significant synergistic efficacy in all tested cell lines. Additional preclinical and clinical studies of the combination of nifurtimox and BSO in patients with neural tumors are warranted.
Topics: Apoptosis; Buthionine Sulfoximine; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Neuroblastoma; Nifurtimox; Phosphorylation; Signal Transduction
PubMed: 27282514
DOI: 10.1038/srep27458 -
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