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1.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445210

RESUMO

Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCßII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCßII specific inhibitor peptide ßIIV5-3-treated IR. Vehicle or ßIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCßII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 µM) to evaluate the inhibition of GLS1 on neuronal viability. PKCßII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by ßIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCßII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.


Assuntos
Transtornos Cerebrovasculares/enzimologia , Glutaminase/metabolismo , Hipocampo/enzimologia , Mitocôndrias/enzimologia , Proteína Quinase C beta/metabolismo , Traumatismo por Reperfusão/enzimologia , Animais , Transtornos Cerebrovasculares/patologia , Gerbillinae , Hipocampo/patologia , Mitocôndrias/patologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia
2.
Theranostics ; 11(16): 7844-7868, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335968

RESUMO

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.


Assuntos
Glutamina/metabolismo , Neoplasias da Próstata/metabolismo , Tolerância a Radiação/genética , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/metabolismo , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biomed Pharmacother ; 138: 111453, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34187143

RESUMO

Glutamate and dopamine hypotheses are leading theories of the pathophysiology of schizophrenia. Multiple lines of evidence suggest that dopaminergic and glutamatergic dysfunction is an underlying mechanism in schizophrenia. Since currently available antipsychotic drugs have significant untoward side effects, identification of new neuroprotective compounds from the medicinal plants may prove beneficial in neurodegenerative disorders. In our previous investigation we have isolated, characterized and reported a novel bioactive compound viz. 3-(3, 4-dimethoxy phenyl)-1-(4-methoxy phenyl) prop-2-en-1-one from the Celastrus paniculatus (CP) is used for the current clinical intervention of schizophrenia disease. The present study is mainly aimed to evaluate the neuroprotective potential of the above bioactive compound against ketamine-induced schizophrenia with particular reference to glutamate metabolism using in vivo and in silico methods. The decrease in glutamine content and the activity levels of glutamate dehydrogenase, glutamine synthetase, and glutaminase in different regions of the rat brain suggests lowered oxidative deamination and lowered mobilization of glutamate towards glutamine formation during ketamine-induced schizophrenia. Pre-treatment with the plant compound reversed the alterations in glutamate metabolism and restored the normal glutamatergic neurotransmission akin to the reference drug, clozapine. In addition, the compound has shown strong interaction and exhibited the highest binding energies against selected NMDA receptors with the lowest inhibition constant than the reference drug. Recoveries of these parameters during anti-schizophrenic treatment suggest that administration of plant compound might offer neuroprotection by interrupting the pathological cascade of glutamatergic neurotransmission that occurs during schizophrenia.


Assuntos
Antipsicóticos , Celastrus , Flavonoides/uso terapêutico , Ketamina , Fármacos Neuroprotetores/uso terapêutico , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Flavonoides/farmacologia , Glutamato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Masculino , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Ratos Wistar , Esquizofrenia/metabolismo
4.
Mol Cell ; 81(11): 2303-2316.e8, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33991485

RESUMO

Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.


Assuntos
Carcinoma Ductal Pancreático/genética , Glutaminase/genética , Neoplasias Pancreáticas/genética , Succinato-CoA Ligases/genética , Animais , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glutaminase/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , NADP/metabolismo , Estresse Oxidativo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/mortalidade , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Succinato-CoA Ligases/metabolismo , Ácido Succínico/metabolismo , Análise de Sobrevida , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
J Med Chem ; 64(8): 4588-4611, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33792311

RESUMO

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule 13b (LL202) was identified with robust GLS1 inhibitory activity (IC50 = 6 nM) and high GLS1 binding affinity (SPR, Kd = 24 nM; ITC, Kd = 37 nM). The X-ray crystal structure of the 13b-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Glutaminase/antagonistas & inibidores , Compostos Macrocíclicos/química , Sítio Alostérico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glutaminase/metabolismo , Glicólise/efeitos dos fármacos , Meia-Vida , Humanos , Compostos Macrocíclicos/metabolismo , Compostos Macrocíclicos/farmacologia , Compostos Macrocíclicos/uso terapêutico , Camundongos , Camundongos Nus , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade
6.
Curr Opin Chem Biol ; 62: 64-81, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33721588

RESUMO

Targeting glutamine catabolism has been attracting more research attention on the development of successful cancer therapy. Catalytic enzymes such as glutaminase (GLS) in glutaminolysis, a series of biochemical reactions by which glutamine is converted to glutamate and then alpha-ketoglutarate, an intermediate of the tricarboxylic acid (TCA) cycle, can be targeted by small molecule inhibitors, some of which are undergoing early phase clinical trials and exhibiting promising safety profiles. However, resistance to glutaminolysis targeting treatments has been observed, necessitating the development of treatments to combat this resistance. One option is to use synergy drug combinations, which improve tumor chemotherapy's effectiveness and diminish drug resistance and side effects. This review will focus on studies involving the glutaminolysis pathway and diverse combination therapies with therapeutic implications.


Assuntos
Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/química , Ciclo do Ácido Cítrico/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutaminase/antagonistas & inibidores , Glicogênio/química , Glicogênio/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo , Transdução de Sinais
7.
Biochimie ; 185: 96-104, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33746066

RESUMO

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.


Assuntos
Ativação Enzimática , Glutaminase/química , Fígado/enzimologia , Substituição de Aminoácidos , Catálise , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade
8.
Vet Res ; 52(1): 51, 2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33766101

RESUMO

Trichinella spiralis is an important foodborne parasitic nematode distributed worldwide that infects humans and animals. Glutaminase (GLS) is an important gene in the glutamine-dependent acid resistance (AR) system; however, its role in T. spiralis muscle larvae (ML) remains unclear. The present study aimed to characterize T. spiralis GLS (TsGLS) and assess its function in T. spiralis ML AR both in vitro and in vivo using RNA interference. The results indicated that native TsGLS (72 kDa) was recognized by anti-rTsGLS serum at the muscle larvae stage; moreover, an immunofluorescence assay confirmed that TsGLS was located in the epidermis of ML. After silencing the TsGLS gene, the relative expression of TsGLS mRNA and the survival rate of T. spiralis ML were reduced by 60.11% and 16.55%, respectively, compared to those in the PBS and control groups. In vivo AR assays revealed that the worm numbers at 7 and 35 days post-infection (dpi) decreased by 61.64% and 66.71%, respectively, compared to those in the PBS group. The relative expression of TsGLS mRNA in F1 generation T. spiralis ML was reduced by 42.52%, compared to that in the PBS group. To the best of our knowledge, this is the first study to report the presence of the glutamine-dependent AR system in T. spiralis. Our results indicate that TsGLS plays a crucial role in the T. spiralis AR system; thus, it could be used as a potential candidate target molecule for producing vaccines against T. spiralis infection.


Assuntos
Glutaminase/genética , Proteínas de Helminto/genética , Interferência de RNA , Doenças dos Suínos/parasitologia , Trichinella spiralis/fisiologia , Triquinelose/veterinária , Animais , Glutaminase/metabolismo , Proteínas de Helminto/metabolismo , Larva/crescimento & desenvolvimento , Larva/fisiologia , Músculos/parasitologia , Sus scrofa , Suínos , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologia
9.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672789

RESUMO

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.


Assuntos
Glutaminase/genética , Mutação , Fator 2 Relacionado a NF-E2/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Benzenoacetamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Malatos/farmacologia , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sulfetos/farmacologia , Tiadiazóis/farmacologia
10.
Theranostics ; 11(9): 4531-4548, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754076

RESUMO

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) has the ability to counter Th17 responses, but the full mechanisms remain elusive. Herein, we aimed to elucidate this process in view of cellular metabolism, especially glutaminolysis. Methods: MTT, CCK-8, Annexin V-FITC/PI staining or trypan blue exclusion assays were used to analyze cytotoxicity. Flow cytometry and Q-PCR assays were applied to determine Th17 responses. The detection of metabolite levels using commercial kits and rate-limiting enzyme expression using western blotting assays was performed to illustrate the metabolic activity. ChIP assays were used to examine H3K4me3 modifications. Mouse models of dextran sulfate sodium (DSS)-induced colitis and house dust mite (HDM)/lipopolysaccharide (LPS)-induced asthma were established to confirm the mechanisms studied in vitro. Results: The PPARγ agonists rosiglitazone and pioglitazone blocked glutaminolysis but not glycolysis under Th17-skewing conditions, as indicated by the detection of intracellular lactate and α-KG and the fluorescence ratios of BCECF-AM. The PPARγ agonists prevented the utilization of glutamine and thus directly limited Th17 responses even when Foxp3 was deficient. The mechanisms were ascribed to restricted conversion of glutamine to glutamate by reducing the expression of the rate-limiting enzyme GLS1, which was confirmed by GLS1 overexpression. Replenishment of α-KG and 2-HG but not succinate weakened the effects of PPARγ agonists, and α-KG-promoted Th17 responses were dampened by siIDH1/2. Inhibition of KDM5 but not KDM4/6 restrained the inhibitory effect of PPARγ agonists on IL-17A expression, and the H3K4me3 level in the promoter and CNS2 region of the il-17 gene locus down-regulated by PPARγ agonists was rescued by 2-HG and GLS1 overexpression. However, the limitation of PPARγ agonists on the mRNA expression of RORγt was unable to be stopped by 2-HG but was attributed to GSH/ROS signals subsequent to GLS1. The exact role of PPARγ was proved by GW9662 or PPARγ knockout, and the mechanisms for PPARγ-inhibited Th17 responses were further confirmed by GLS1 overexpression in vivo. Conclusion: PPARγ agonists repressed Th17 responses by counteracting GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals, which is beneficial for Th17 cell-related immune dysregulation.


Assuntos
Glutaminase/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , Histonas/metabolismo , PPAR gama/agonistas , Espécies Reativas de Oxigênio/metabolismo , Células Th17/efeitos dos fármacos , Animais , Colite/tratamento farmacológico , Colite/metabolismo , Modelos Animais de Doenças , Feminino , Ácido Glutâmico/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pioglitazona/farmacologia , RNA Mensageiro/metabolismo , Rosiglitazona/farmacologia , Células Th17/metabolismo
11.
J Biomed Sci ; 28(1): 14, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33610185

RESUMO

BACKGROUND: Glutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells. METHODS: Microarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G. RESULTS: MiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression. CONCLUSIONS: Glutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glutaminase/genética , MicroRNAs/metabolismo , Estresse Oxidativo , Linhagem Celular Tumoral , Regulação para Baixo , Glutaminase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Regulação para Cima
12.
J Immunol ; 206(6): 1127-1139, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33558372

RESUMO

T effector cells promote inflammation in asthmatic patients, and both Th2 and Th17 CD4 T cells have been implicated in severe forms of the disease. The metabolic phenotypes and dependencies of these cells, however, remain poorly understood in the regulation of airway inflammation. In this study, we show the bronchoalveolar lavage fluid of asthmatic patients had markers of elevated glucose and glutamine metabolism. Further, peripheral blood T cells of asthmatics had broadly elevated expression of metabolic proteins when analyzed by mass cytometry compared with healthy controls. Therefore, we hypothesized that glucose and glutamine metabolism promote allergic airway inflammation. We tested this hypothesis in two murine models of airway inflammation. T cells from lungs of mice sensitized with Alternaria alternata extract displayed genetic signatures for elevated oxidative and glucose metabolism by single-cell RNA sequencing. This result was most pronounced when protein levels were measured in IL-17-producing cells and was recapitulated when airway inflammation was induced with house dust mite plus LPS, a model that led to abundant IL-4- and IL-17-producing T cells. Importantly, inhibitors of the glucose transporter 1 or glutaminase in vivo attenuated house dust mite + LPS eosinophilia, T cell cytokine production, and airway hyperresponsiveness as well as augmented the immunosuppressive properties of dexamethasone. These data show that T cells induce markers to support metabolism in vivo in airway inflammation and that this correlates with inflammatory cytokine production. Targeting metabolic pathways may provide a new direction to protect from disease and enhance the effectiveness of steroid therapy.


Assuntos
Asma/tratamento farmacológico , Dexametasona/farmacologia , Transportador de Glucose Tipo 1/antagonistas & inibidores , Glutaminase/antagonistas & inibidores , Imunossupressores/farmacologia , Adulto , Alternaria/imunologia , Animais , Asma/sangue , Asma/imunologia , Biomarcadores/análise , Biomarcadores/metabolismo , Glicemia/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Casos e Controles , Células Cultivadas , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Transportador de Glucose Tipo 1/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Voluntários Saudáveis , Humanos , Imunossupressores/uso terapêutico , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Cultura Primária de Células , Pyroglyphidae/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Adulto Jovem
13.
Science ; 371(6526): 265-270, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446552

RESUMO

Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 (GLS1) as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.


Assuntos
Envelhecimento/metabolismo , Senescência Celular/fisiologia , Glutaminase/metabolismo , Tecido Adiposo/enzimologia , Envelhecimento/genética , Amônia/metabolismo , Animais , Sobrevivência Celular , Senescência Celular/genética , Genes Essenciais , Glutaminase/genética , Humanos , Concentração de Íons de Hidrogênio , Pulmão/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pele/enzimologia
14.
Cell Oncol (Dordr) ; 44(2): 385-403, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33464483

RESUMO

PURPOSE: Resistance to androgen-deprivation therapies and progression to so-called castrate-resistant prostate cancer (CRPC) remain challenges in prostate cancer (PCa) management and treatment. Among other alterations, CRPC has been associated with metabolic reprogramming driven by androgens. Here, we investigated the role of androgens in regulating glutaminolysis in PCa cells and determined the relevance of this metabolic route in controlling the survival and growth of androgen-sensitive (LNCaP) and CRPC (DU145 and PC3) cells. METHODS: PCa cells (LNCaP, DU145 and PC3) and 3-month old rats were treated with 5α-dihydrotestosterone (DHT). Alternatively, LNCaP cells were exposed to the glutaminase inhibitor BPTES, alone or in combination with the anti-androgen bicalutamide. Biochemical, Western blot and extracellular flux assays were used to evaluate the viability, proliferation, migration and metabolism of PCa cells in response to DHT treatment or glutaminase inhibition. RESULTS: We found that DHT up-regulated the expression of the glutamine transporter ASCT2 and glutaminase, both in vitro in LNCaP cells and in vivo in rat prostate cells. BPTES diminished the viability and migration of PCa cells, while increasing caspase-3 activity. CRPC cells were found to be more dependent on glutamine and more sensitive to glutaminase inhibition. BPTES and bicalutamide co-treatment had an additive effect on suppressing LNCaP cell viability. Finally, we found that inhibition of glutaminolysis differentially affected glycolysis and lipid metabolism in both androgen-sensitive and CRPC cells. CONCLUSION: Our data reveal glutaminolysis as a central metabolic route controlling PCa cell fate and highlight the relevance of targeting glutaminase for CRPC treatment.


Assuntos
Di-Hidrotestosterona/farmacologia , Glutamina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Androgênios/farmacologia , Anilidas/farmacologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glutaminase/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Ácido Láctico/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Nitrilas/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Ratos , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Compostos de Tosil/farmacologia
15.
Dis Markers ; 2021: 8850990, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33505538

RESUMO

Background: Glutamine metabolism is considered an important metabolic phenotype of proliferating tumor cells. Objective: The objective of this study was to investigate the expression of glutamine metabolism-related and amino acid transporter proteins in adrenal cortical neoplasms (ACNs) and pheochromocytomas (PCCs) in the adrenal gland. Methods: A tissue microarray was constructed for 132 cases of ACN (115 cases of adrenal cortical adenoma and 17 cases of adrenal cortical carcinoma) and 189 cases of PCC. Immunohistochemical staining for glutamine metabolism-related proteins GLS1 and GDH and amino acid transporter proteins SLC1A5, SLC7A5, and SLC7A11 as well as SDHB was performed and compared with clinicopathologic parameters. Results: The expression levels of GLS (p < 0.001), SLC7A5 (p = 0.049), and SDHB (p = 0.007) were higher in ACN than in PCC, whereas the expression levels of SLC1A5 (p < 0.001) and SLC7A11 (p < 0.001) were higher in PCC than in ACN. In ACN, GLS positivity was associated with a higher Fuhrman grade (p = 0.009), and SLC1A5 positivity was associated with SDHB positivity (p = 0.004) and a clear cell proportion < 25% (p = 0.010). SDHB negativity was also associated with tumor cell necrosis (p = 0.007). In PCC, SLC7A11 positivity was associated with nonnorepinephrine type (p = 0.008). In Kaplan-Meier analysis, patients with GLS positivity (p = 0.039) and SDHB negativity (p = 0.005) had significantly shorter overall survival in ACN. In PCC patients with a GAPP score ≥ 3, GLS positivity (p = 0.001) and SDHB positivity (p = 0.001) were associated with shorter disease-free survival, whereas GLS positivity (p = 0.004) was also associated with shorter overall survival. Conclusions: The expression of glutamine metabolism-related and amino acid transporter proteins in ACN and PCC is distinct and associated with prognosis.


Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Biomarcadores Tumorais/genética , Glutamato Desidrogenase/genética , Glutaminase/genética , Feocromocitoma/metabolismo , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Adulto , Idoso , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Biomarcadores Tumorais/metabolismo , Feminino , Glutamato Desidrogenase/metabolismo , Glutaminase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Feocromocitoma/genética , Feocromocitoma/patologia
16.
Brain Behav Immun ; 92: 139-156, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33278560

RESUMO

Neuroinflammation is the inflammatory responses that are involved in the pathogenesis of most neurological disorders. Glutaminase (GLS) is the enzyme that catalyzes the hydrolysis of glutamine to produce glutamate. Besides its well-known role in cellular metabolism and excitatory neurotransmission, GLS has recently been increasingly noticed to be up-regulated in activated microglia under pathological conditions. Furthermore, GLS overexpression induces microglial activation, extracellular vesicle secretion, and neuroinflammatory microenvironment formation, which, are compromised by GLS inhibitors in vitro and in vivo. These results indicate that GLS has more complicated implications in brain disease etiology than what are previously known. In this review, we introduce GLS isoforms, expression patterns in the body and the brain, and expression/activities regulation. Next, we discuss the metabolic and neurotransmission functions of GLS. Afterwards, we summarize recent findings of GLS-mediated microglial activation and pro-inflammatory extracellular vesicle secretion, which, in turns, induces neuroinflammation. Lastly, we provide a comprehensive discussion for the involvement of microglial GLS in the pathogenesis of various neurological disorders, indicating microglial GLS as a promising target to treat these diseases.


Assuntos
Glutaminase , Microglia , Encéfalo/metabolismo , Ácido Glutâmico , Glutaminase/metabolismo , Glutamina , Humanos , Microglia/metabolismo
17.
Endocrinology ; 162(1)2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33206168

RESUMO

The liver plays a critical role in maintaining ammonia homeostasis. Urea cycle defects, liver injury, or failure and glutamine synthetase (GS) deficiency result in hyperammonemia, serious clinical conditions, and lethality. In this study we used a mouse model with a defect in the urea cycle enzyme ornithine transcarbamylase (Otcspf-ash) to test the hypothesis that glucagon receptor inhibition using a monoclonal blocking antibody will reduce the hyperammonemia and associated lethality induced by a high-protein diet, which exacerbates disease. We found reduced expression of glutaminase, which degrades glutamine and increased expression of GS in livers of Otcspf-ash mice treated with the glucagon receptor blocking antibody. The gene expression changes favor ammonia consumption and were accompanied by increased circulating glutamine levels and diminished hyperammonemia. Otcspf-ash mice treated with the glucagon receptor-blocking antibody gained lean and body mass and had increased survival. These data suggest that glucagon receptor inhibition using a monoclonal antibody could reduce the risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle, liver injury, or failure and GS deficiency.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Hiperamonemia/terapia , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Receptores de Glucagon/antagonistas & inibidores , Aminoácidos/sangue , Amônia/sangue , Animais , Peso Corporal , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Masculino , Camundongos , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/mortalidade
18.
Eur J Med Chem ; 210: 112980, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33176943

RESUMO

To develop novel GLS1 inhibitors as effective therapeutic agents for triple-negative breast cancer (TNBC), 25 derivatives were synthesized from the natural inhibitor withangulatin A (IC50 = 18.2 µM). Bioassay optimization identified a novel and selective GLS1 inhibitor 7 (IC50 = 1.08 µM). In MDA-MB-231 cells, 7 diminished cellular glutamate levels by blocking glutaminolysis pathway, further triggering the generation of reactive oxygen species to induce caspase-dependent apoptosis. Molecular docking indicated that 7 interacted with a new reacting site of allosteric binding pocket by forming various interactions in GLS1. The intraperitoneal administration of 7 at a dose of 50 mg/kg exhibited remarkable therapeutic effects and no apparent toxicity in the MDA-MB-231 xenograft model, indicating its potential as a novel GLS1 inhibitor for treatment of TNBC.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Pregnenos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glutaminase/metabolismo , Humanos , Estrutura Molecular , Pregnenos/síntese química , Pregnenos/química , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
19.
Biochem Biophys Res Commun ; 534: 687-693, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33213841

RESUMO

BACKGROUND: Metabolic remodeling in cardiomyocytes is deeply associated with the pathogenesis of heart failure (HF). Glutaminolysis is an anaplerotic pathway that incorporates α-ketoglutarate (αKG) derived from glutamine into the tricarboxylic acid (TCA) cycle. It is well known that cancer cells depend on glutamine for their increased energy demand and proliferation; however, the physiological roles of glutamine metabolism in failing hearts remain unclear. OBJECTIVE: To investigate the regulatory mechanisms and biological effects of glutamine metabolism in oxidative stress-induced failing myocardium. METHODS AND RESULTS: The intracellular levels of glutamine, glutamate, and αKG were significantly decreased by H2O2 stimulation in rat neonatal cardiomyocytes (RNCMs). To better understand the metabolic flux in failing myocardium, we performed a stable isotope tracing study and found that glutaminolysis was upregulated in RNCMs under oxidative stress. Consistent with this, the enzymatic activity of glutaminase (Gls), which converts glutamine to glutamate, was augmented in RNCMs treated with H2O2. These findings suggest that glutamine anaplerosis is enhanced in cardiomyocytes under oxidative stress to compensate for the reduction of αKG. Furthermore, the inhibition of Gls reduced cardiac cell viability, ATP production, and glutathione (GSH) synthesis in RNCMs with H2O2 stimulation. Finally, we evaluated the effects of αKG on failing myocardium and observed that dimethyl α-ketoglutarate (DMKG) suppressed oxidative stress-induced cell death likely due to the enhancement of intracellular ATP and GSH levels. CONCLUSION: Our study demonstrates that under oxidative stress, glutaminolysis is upregulated to compensate for the loss of αKG and its replenishment into the TCA cycle, thereby exerting cardioprotective effects by maintaining ATP and GSH levels. Modulation of glutamine metabolism in failing hearts might provide a new therapeutic strategy for HF.


Assuntos
Glutamina/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Sobrevivência Celular , Células Cultivadas , Ciclo do Ácido Cítrico , Metabolismo Energético , Ácido Glutâmico/metabolismo , Glutaminase/metabolismo , Insuficiência Cardíaca/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas , Miócitos Cardíacos/citologia , Estresse Oxidativo , Ratos
20.
J Sci Food Agric ; 101(4): 1301-1306, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32790072

RESUMO

BACKGROUND: L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells. RESULTS: The results obtained showed that cell permeabilization using osmotic shock with 155 g L-1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L-1 , calcium chloride 30 g L-1 , cell mass 100 g L-1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity. CONCLUSION: The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.


Assuntos
Proteínas de Bactérias/metabolismo , Glutaminase/metabolismo , Pseudomonas/enzimologia , Alginatos/química , Proteínas de Bactérias/química , Biocatálise , Células Imobilizadas/química , Células Imobilizadas/enzimologia , Glutamatos/metabolismo , Glutaminase/química , Pseudomonas/química , Pseudomonas/crescimento & desenvolvimento
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