RESUMEN
Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.
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Ferroptosis , Linfa/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/patología , Animales , Supervivencia Celular , Coenzima A Ligasas/metabolismo , Femenino , Ferroptosis/efectos de los fármacos , Glutatión/metabolismo , Humanos , Hierro/metabolismo , Masculino , Melanoma/sangre , Melanoma/metabolismo , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Ácido Oléico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Análisis de Componente PrincipalRESUMEN
Superoxide dismutase 1 (SOD1) is the principal cytoplasmic superoxide dismutase in humans and plays a major role in redox potential regulation. It catalyses the transformation of the superoxide anion (O2â¢-) into hydrogen peroxide. Heterozygous variants in SOD1 are a common cause of familial amyotrophic lateral sclerosis. In this study we describe the homozygous truncating variant c.335dupG (p.C112Wfs*11) in SOD1 that leads to total absence of enzyme activity. The resulting phenotype is severe and marked by progressive loss of motor abilities, tetraspasticity with predominance in the lower extremities, mild cerebellar atrophy, and hyperekplexia-like symptoms. Heterozygous carriers have a markedly reduced enzyme activity when compared to wild-type controls but show no overt neurologic phenotype. These results are in contrast with the previously proposed theory that a loss of function is the underlying mechanism in SOD1-related motor neuron disease and should be considered before application of previously proposed SOD1 silencing as a treatment option for amyotrophic lateral sclerosis.
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Trastornos Heredodegenerativos del Sistema Nervioso/genética , Superóxido Dismutasa-1/deficiencia , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral , Niño , Preescolar , Mutación del Sistema de Lectura , Humanos , Masculino , Linaje , SíndromeRESUMEN
Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and antioxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GAD67GFP+ interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence (GE), the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized DHE oxidation levels in GE and ameliorated the migration delay caused by prenatal stress. Through convergent redox manipula-tions, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.
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Encéfalo/embriología , Interneuronas/metabolismo , Neurogénesis/fisiología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Estrés Psicológico/complicaciones , Animales , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Interneuronas/efectos de los fármacos , Ratones , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Embarazo , Restricción Física/efectos adversos , Estrés Psicológico/metabolismoRESUMEN
Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.
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Adaptación Fisiológica , Canales de Calcio/metabolismo , Corazón/fisiopatología , Mitocondrias Cardíacas/metabolismo , Estrés Fisiológico , Animales , Presión Sanguínea , Calcio/metabolismo , Estimulación Cardíaca Artificial , Reprogramación Celular , Citosol/efectos de los fármacos , Citosol/metabolismo , Diástole , Electrocardiografía , Genes Dominantes , Glucosa/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , Reperfusión Miocárdica , Miocardio/metabolismo , Miocardio/patología , Consumo de Oxígeno , Prostaglandina-Endoperóxido Sintasas/metabolismo , Retículo Sarcoplasmático/metabolismo , Transcripción GenéticaRESUMEN
Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.
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Antioxidantes/farmacología , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Neutrófilos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Taurina/farmacología , Angiotensina II , Animales , Aorta Abdominal/enzimología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Cloruro de Calcio , Modelos Animales de Enfermedad , Eliminación de Gen , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Neutrófilos/enzimología , Elastasa Pancreática , Peroxidasa/deficiencia , Peroxidasa/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMEN
Hepatic levels of the essential micronutrient, zinc, are diminished by several hepatotoxicants, and the dietary supplementation of zinc has proven protective in those cases. 3,3',4,4',5-Pentachlorobiphenyl (PCB126), a liver toxicant, alters hepatic nutrient homeostasis and lowers hepatic zinc levels. The current study was designed to determine the mitigative potential of dietary zinc in the toxicity associated with PCB126 and the role of zinc in that toxicity. Male Sprague-Dawley rats were divided into three dietary groups and fed diets deficient in zinc (7 ppm Zn), adequate in zinc (30 ppm Zn), and supplemented in zinc (300 ppm). The animals were maintained for 3 weeks on these diets, then given a single IP injection of vehicle or 1 or 5 µmol/kg PCB126. After 2 weeks, the animals were euthanized. Dietary zinc increased the level of ROS, the activity of CuZnSOD, and the expression of metallothionein but decreased the levels of hepatic manganese. PCB126 exposed rats exhibited classic signs of exposure, including hepatomegaly, increased hepatic lipids, increased ROS and CYP induction. Liver histology suggests some mild ameliorative properties of both zinc deficiency and zinc supplementation. Other metrics of toxicity (relative liver and thymus weights, hepatic lipids, and hepatic ROS) did not support this trend. Interestingly, the zinc supplemented high dose PCB126 group had mildly improved histology and less efficacious induction of investigated genes than did the low dose PCB126 group. Overall, decreases in zinc caused by PCB126 likely contribute little to the ongoing toxicity, and the mitigative/preventive capacity of zinc against PCB126 exposure seems limited.
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Dieta , Hígado/efectos de los fármacos , Bifenilos Policlorados/toxicidad , Zinc/farmacología , Animales , Conducta Alimentaria , Expresión Génica , Masculino , Metalotioneína/genética , Estrés Oxidativo , Ratas , Receptores de Hidrocarburo de Aril/metabolismo , Superóxido Dismutasa/metabolismo , Zinc/administración & dosificaciónRESUMEN
Increased levels of reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide have been reported in many cancer cells and they have been implicated in carcinogenesis and tumor progression. Antioxidant enzymes, such as Manganese Superoxide Dismutase (MnSOD or SOD2) and Glutathione Peroxidase-1 (GPx1), act coordinately to neutralize ROS. These enzymes are also thought to contribute to cancer cell resistance to conventional radio-chemo-therapies. Although some relationships have been reported between psychosocial factors and the regulation of antioxidant enzymes, little is known about these relationships in the context of cancer progression. The current study investigated the levels of MnSOD and GPx1in confirmed serous, high-grade tumor tissue from 60 ovarian cancer patients, and explored the relationship between the activity of these enzymes, the levels of tumor norepinephrine (NE), and patient mood as determined via pre-operative questionnaires. MnSOD activity was positively related to depressed mood (p=0.025) and tumor NE (p=0.023). In contrast, GPx1 activity was inversely related to fatigue (p=0.015) and tumor NE (p=0.009), and was positively associated with vigor (p=0.024). These findings suggest that psychological state and adrenergic signaling are linked with antioxidant enzyme activity in ovarian cancer and may have implications for patient treatments and outcomes.
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Glutatión Peroxidasa/metabolismo , Norepinefrina/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/psicología , Superóxido Dismutasa/metabolismo , Afecto , Anciano , Antioxidantes/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Sistemas Neurosecretores/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
Biological processes in the Proterozoic Ocean are often inferred from modern oxygen-deficient environments (MODEs) or from stable isotopes in preserved sediment. To date, few MODE studies have simultaneously quantified carbon fixation genes and attendant stable isotopic signatures. Consequently, how carbon isotope patterns reflect these pathways has not been thoroughly vetted. Addressing this, we profiled planktonic productivity and quantified carbon fixation pathway genes and associated organic carbon isotope values (δ13 CPOC ) of size-fractionated (0.2-2.7 and >2.7 µm) particulate matter from meromictic Fayetteville Green Lake, NY, USA. The high-O2 Calvin-Benson-Bassham (CBB) gene (cbbL) was most abundant in the <2.7 µm size fraction in shallow oxic and deep hypoxic waters, corresponding with cyanobacterial and eukaryote algal populations. The low-O2 CBB gene (cbbM) was most abundant near the lower oxycline boundary in the larger size fraction, coincident with purple sulfur bacteria populations. The reverse citric acid cycle gene (aclB) was equally abundant in both size fractions in the deepest photic zone, coinciding with green sulfur bacteria populations. Methane coenzyme reductase A (mcrA), of anaerobic methane cyclers, was most abundant at the lower oxycline boundary in both size fractions, coinciding with Methanoregula populations. δ13 CPOC values overlapped with the high-O2 CBB fixation range except for two negative excursions near the lower oxycline boundary, likely reflecting assimilation of isotopically-depleted groundwater-derived carbon by autotrophs and sulfate-reducers. Throughout aphotic waters, δ13 CPOC values of the large size fraction became 13 C-enriched, likely reflecting abundant purple sulfur bacterial aggregates. Eukaryote algae- or cyanobacteria-like isotopic signatures corresponded with increases in cbbL, cbbM, and aclB, and enrichment of exopolymer-rich prokaryotic photoautotrophs aggregates. Results suggest that δ13 CPOC values of preserved sediments from areas of the Proterozoic Ocean with sulfidic photic zones may reflect a mixture of alternate carbon-fixing populations exported from the deep photic zone, challenging the paradigm that sedimentary stable carbon isotope values predominantly reflect oxygenic photosynthesis from surface waters.
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Chromatiaceae , Cianobacterias , Carbono/metabolismo , Lagos/microbiología , Isótopos de Carbono/análisis , Cianobacterias/metabolismo , Oxígeno/análisis , Chromatiaceae/metabolismo , Metano , Océanos y MaresRESUMEN
OBJECTIVE: Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity. METHODS: We generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal 13C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI. RESULTS: MPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival. CONCLUSIONS: Our findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.
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Lesión Renal Aguda , Rabdomiólisis , Ratones , Animales , Transportadores de Ácidos Monocarboxílicos/metabolismo , Lesión Renal Aguda/metabolismo , Oxidación-Reducción , Rabdomiólisis/inducido químicamente , Rabdomiólisis/metabolismo , Oxidantes/efectos adversosRESUMEN
Natural Fe(III) oxides typically contain a range of trace elements including P. Although solution phase and adsorbed P (as phosphate) have been shown to impact the bioreduction of Fe(III) oxides and the formation of "biogenic" secondary minerals, little is known about the potential effects of occluded/incorporated phosphate. We have examined the bioreduction of Fe(III) oxides (lepidocrocite (γ-FeOOH) and maghemite (γ-Fe2O3)) containing 0-3 mass% P as "bound" (a term we use to include both adsorbed and occluded/incorporated) phosphate. Kinetic dissolution studies showed congruent release of Fe and P, suggesting that the phosphate in these materials was incorporated within the particles; however, 53% or 86% of the total phosphate associated with the lepidocrocites containing 0.7 or 3 mass% P, respectively, was extracted with 0.1 M NaOH and can be considered to be adsorbed, both to exterior surfaces and within micropores. In the absence of phosphate, lepidocrocite was rapidly reduced to magnetite by Shewanella putrefaciens CN32, and over time the magnetite was partially transformed to ferrous hydroxy carbonate (FHC). The presence of 0.2-0.7 mass% P significantly inhibited the initial reduction of lepidocrocite but ultimately resulted in greater Fe(II) production and the formation of carbonate green rust. The bioreduction of maghemite with and without bound phosphate resulted in solid-state conversion to magnetite, with subsequent formation of FHC. We also examined the potential redox cycling of green rust under alternating Fe(III)-reducing and oxic conditions. Oxidation of biogenic green rust by O2 resulted in conversion to ferric green rust, which was readily reduced back to green rust by S. putrefaciens CN32. These results indicate the potential for cycling of green rust between reduced and oxidized forms under redox dynamics similar to those encountered in environments that alternate between iron-reducing and oxic conditions, and they are consistent with the identification of green rust in soils/sediments with seasonal redox cycling.
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Compuestos Férricos/metabolismo , Fosfatos/metabolismo , Shewanella putrefaciens/metabolismo , Oxidación-ReducciónRESUMEN
Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.
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The release of H(2)O(2) from alveolar macrophages has been linked to the development of pulmonary fibrosis, but little is known about its source or mechanism of production. We found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H(2)O(2) and have high expression of Cu,Zn-superoxide dismutase (SOD). Because Cu,Zn-SOD is found in the mitochondrial intermembrane space (IMS), we hypothesized that mitochondrial Cu,Zn-SOD-mediated H(2)O(2) generation contributed to pulmonary fibrosis. Asbestos-induced translocation of Cu,Zn-SOD to the IMS was unique to macrophages and dependent on functional mitochondrial respiration and the presence of at least one of the conserved cysteines required for disulfide bond formation. These conserved cysteine residues were also necessary for enzyme activation and H(2)O(2) generation. Cu,Zn-SOD-mediated H(2)O(2) generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant in that Cu,Zn-SOD(-/-) mice generated significantly less H(2)O(2) and had less oxidant stress in bronchoalveolar lavage fluid and lung parenchyma. Furthermore, Cu,Zn-SOD(-/-) mice did not develop pulmonary fibrosis, and knockdown of Cu,Zn-SOD in monocytes attenuated collagen I deposition by lung fibroblasts. Our findings demonstrate a novel mechanism for the pathogenesis of pulmonary fibrosis where the antioxidant enzyme Cu,Zn-SOD translocates to the mitochondrial IMS to increase H(2)O(2) generation in alveolar macrophages.
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Peróxido de Hidrógeno/metabolismo , Fibrosis Pulmonar/etiología , Superóxido Dismutasa/metabolismo , Adolescente , Adulto , Animales , Amianto/farmacología , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteínas Mitocondriales , Estrés Oxidativo , Transporte de Proteínas , Regulación hacia Arriba , Adulto JovenRESUMEN
The toxicity of many "inert" ingredients of pesticide formulations, such as safeners, is poorly characterized, despite evidence that humans may be exposed to these chemicals. Analysis of ToxCast data for dichloroacetamide safeners with the ToxPi tool identified benoxacor as the safener with the highest potential for toxicity, especially liver toxicity. Benoxacor was subsequently administered to mice via oral gavage for 3 days at concentrations of 0, 0.5, 5, and 50 mg/kg bodyweight (b.w.). Bodyweight-adjusted liver and testes weights were significantly increased in the 50 mg/kg b.w. group. There were no overt pathologies in either the liver or the intestine. 16S rRNA analysis of the cecal microbiome revealed no effects of benoxacor on α- or ß-diversity; however, changes were observed in the abundance of certain bacteria. RNAseq analysis identified 163 hepatic genes affected by benoxacor exposure. Benoxacor exposure expressed a gene regulation profile similar to dichloroacetic acid and the fungicide sedaxane. Metabolomic analysis identified 9 serum and 15 liver metabolites that were affected by benoxacor exposure, changes that were not significant after correcting for multiple comparisons. The activity of antioxidant enzymes was not altered by benoxacor exposure. In vitro metabolism studies with liver microsomes and cytosol from male mice demonstrated that benoxacor is enantioselectively metabolized by cytochrome P450 enzymes, carboxylesterases, and glutathione S-transferases. These findings suggest that the minor toxic effects of benoxacor may be due to its rapid metabolism to toxic metabolites, such as dichloroacetic acid. This result challenges the assumption that inert ingredients of pesticide formulations are safe.
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Microbioma Gastrointestinal , Herbicidas , Animales , Herbicidas/toxicidad , Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Oxazinas , ARN Ribosómico 16SRESUMEN
Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2 â- and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.
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Ketogenic diets (KD) are high in fat and low in carbohydrates, forcing cells to utilize mitochondrial fatty acid oxidation for energy production. Since cancer cells demonstrate increased mitochondrial oxidative stress relative to normal cells, we hypothesized that a KD may selectively enhance metabolic oxidative stress in head and neck cancer cells, sensitizing them to radiation and platinum-based chemotherapy without causing increased toxicity in surrounding normal tissues. This hypothesis was tested in preclinical murine xenografts and in a phase 1 clinical trial (NCT01975766). In this study, mice bearing human head and neck cancer xenografts (FaDu) were fed either standard mouse chow or KetoCal® KD (90% fat, 8% carbohydrate, 2% protein) and exposed to ionizing radiation. Tumors were harvested from mice to test for glutathione, a biomarker of oxidative stress. In parallel, patients with locally advanced head and neck cancer were enrolled in a phase 1 clinical trial where they consumed KD and received radiation with concurrent platinum-based chemotherapy. Subjects consumed KetoCal KD via percutaneous endoscopic gastrostomy (PEG) tube and were also allowed to orally consume water, sugar-free drinks, and foods approved by a dietitian. Oxidative stress markers including protein carbonyls and total glutathione were assessed in patient blood samples both pre-KD and while consuming the KD. Mice bearing FaDu xenografts that received radiation and KD demonstrated a slight improvement in tumor growth rate and survival compared to mice that received radiation alone; however a variation in responses was seen dependent on the fatty acid composition of the diet. In the phase 1 clinical trial, a total of twelve patients were enrolled in the study. Four patients completed five weeks of the KD as per protocol (with variance in compliance). Eight patients did not tolerate the diet with concurrent radiation and platinum-chemotherapy (5 were patient decision and 3 were removed from study due to toxicity). The median number of days consuming a KD in patients who did not complete the study was 5.5 (range: 2-8 days). Reasons for discontinuation included "stress of diet compliance" (1 patient), grade 2 nausea (3 patients), and grade 3 fatigue (1 patient). Three patients were removed from the trial due to dose-limiting toxicities including: grade 4 hyperuricemia (2 patients) and grade 3 acute pancreatitis (1 patient). Median weight loss was 2.95% for the KD-tolerant group and 7.92% for patients who did not tolerate the diet. In conclusion, the ketogenic diet shows promise as a treatment combined with radiation in preclinical mouse head and neck cancer xenografts. A phase 1 clinical trial evaluating the safety and tolerability of KD demonstrated difficulty with diet compliance when combined with standard-of-care radiation therapy and cisplatin chemotherapy.
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Dieta Cetogénica/métodos , Carcinoma de Células Escamosas de Cabeza y Cuello/dietoterapia , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , 3-Hidroxiacil-CoA Deshidrogenasas/efectos de los fármacos , 3-Hidroxiacil-CoA Deshidrogenasas/efectos de la radiación , Acetil-CoA C-Aciltransferasa/efectos de los fármacos , Acetil-CoA C-Aciltransferasa/efectos de la radiación , Adulto , Anciano , Animales , Isomerasas de Doble Vínculo Carbono-Carbono/efectos de los fármacos , Isomerasas de Doble Vínculo Carbono-Carbono/efectos de la radiación , Quimioradioterapia/efectos adversos , Dieta Cetogénica/efectos adversos , Enoil-CoA Hidratasa/efectos de los fármacos , Enoil-CoA Hidratasa/efectos de la radiación , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Racemasas y Epimerasas/efectos de los fármacos , Racemasas y Epimerasas/efectos de la radiación , Radiación Ionizante , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/efectos de la radiaciónRESUMEN
The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.
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Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutaminasa/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Compuestos de Sulfhidrilo/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Glutamina/metabolismo , Glutatión/metabolismo , Glucólisis , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5-16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.
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Auranofina/administración & dosificación , Butionina Sulfoximina/administración & dosificación , Melanoma/tratamiento farmacológico , Mitocondrias/metabolismo , Compuestos Organofosforados/administración & dosificación , Animales , Auranofina/farmacología , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Sinergismo Farmacológico , Femenino , Humanos , Hidrogeles/química , Melanoma/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Estrés Oxidativo/efectos de los fármacos , Relación Estructura-Actividad , Temperatura , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O2vs. 21% O2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.
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Disulfiram , Neoplasias Pulmonares , Línea Celular Tumoral , Cobre , Disulfiram/farmacología , Humanos , Hipoxia , Neoplasias Pulmonares/tratamiento farmacológico , Oxidación-ReducciónRESUMEN
The role of inflammation in the pathogenesis of abdominal aortic aneurysms (AAA) is well established. The inflammatory process leads to protease-mediated degradation of the extracellular matrix and apoptosis of smooth muscle cells (SMC), which are the predominant matrix synthesizing cells of the vascular wall. These processes act in concert to progressively weaken the aortic wall, resulting in dilatation and aneurysm formation. Oxidative stress is invariably increased in, and contributes importantly to, the pathophysiology of inflammation. Moreover, reactive oxygen species (ROS) play a key role in regulation of matrix metalloproteinases and induction of SMC apoptosis. ROS may also contribute to the pathogenesis of hypertension, a risk factor for AAA. Emerging evidence suggests that ROS and reactive nitrogen species (RNS) are associated with AAA formation in animal models and in humans. Although experimental data are limited, several studies suggest that modulation of ROS production or activity may suppress AAA formation and improve experimental outcome in rodent models. Although a number of enzymes can produce injurious ROS in the vasculature, increasing evidence points toward a role for NADPH oxidase as a source of oxidative stress in the pathogenesis of AAA.
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Aneurisma de la Aorta Abdominal/fisiopatología , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis , Endotelio Vascular/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/fisiopatología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Músculo Liso Vascular/metabolismo , Sensibilidad y EspecificidadRESUMEN
Highly glycolytic cervical cancers largely resist treatment by cisplatin and coadministered pelvic irradiation as the present standard of care. In this study, we investigated the effects of inhibiting glycolysis and thiol redox metabolism to evaluate them as alternate treatment strategies in these cancers. In a panel of multiple cervical cancer cell lines, we evaluated sensitivity to inhibition of glycolysis (2-deoxyglucose, 2-DG) with or without simultaneous inhibition of glutathione and thioredoxin metabolism (BSO/AUR). Intracellular levels of total and oxidized glutathione, thioredoxin reductase activity, and indirect measures of intracellular reactive oxygen species were compared before and after treatment. Highly radioresistant cells were the most sensitive to 2-DG, whereas intermediate radioresistant cells were sensitive to 2-DG plus BSO/AUR. In response to 2-DG/BSO/AUR treatment, we observed increased levels of intracellular oxidized glutathione, redox-sensitive dye oxidation, and decreased glucose utilization via multiple metabolic pathways including the tricarboxylic acid cycle. 2-DG/BSO/AUR treatment delayed the growth of tumors composed of intermediate radioresistant cells and effectively radiosensitized these tumors at clinically relevant radiation doses both in vitro and in vivo Overall, our results support inhibition of glycolysis and intracellular redox metabolism as an effective alternative drug strategy for the treatment of highly glycolytic and radioresistant cervical cancers.Significance: This study suggests a simple metabolic approach to strike at an apparent Achilles' heel in highly glycolytic, radioresistant forms of cervical cancers, possibly with broader applications in cancer therapy. Cancer Res; 78(6); 1392-403. ©2018 AACR.