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1.
Food Chem ; 308: 125701, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669946

RESUMO

In this study, we found that glutaminase from Bacillus amyloliquefaciens could increase antioxidant activities of Gln-Cys mixture, demonstrated by the higher superoxide anion and DPPH radical scavenging activities, Fe2+-chelating and reducing power. According to UPLC-Q-TOF-MS/MS results, we identified γ-[Glu](n=1,2,3,4)-Cys in Gln-Cys mixture in the presence of glutaminase. The yields of γ-Glu-Cys (GC) and γ-Glu-γ-Glu-Cys (GGC) were 40.92% and 22.79% respectively, under the established optimum conditions: pH 10, 37 °C, 3 h, 0.1 mol/l Gln: 0.1 mol/l Cys = 1:1, and glutaminase at 0.1% (m/v). The antioxidant properties of GC, GGC, glutathione and Gln-Cys mixture in the presence of glutaminase were further compared and we found GC exhibited the highest superoxide anion and DPPH radical scavenging activities, and Fe2+-chelating and reducing power. Therefore, glutaminase caused the increase of antioxidant activity of Gln-Cys mixture and might be attributed to GC which was synthesized by glutaminase via transpeptidation, and GC has the potential to be used in food and nutraceutical applications as an antioxidant peptide.


Assuntos
Antioxidantes/metabolismo , Bacillus amyloliquefaciens/enzimologia , Dipeptídeos/metabolismo , Glutaminase/metabolismo , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem
2.
Life Sci ; 237: 116893, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606381

RESUMO

AIM: Gastric cancer (GC) is a common human malignancy tumor of digestive tract in worldwide. Physcion 8-O-ß-glucopyranoside (PG) exhibits anti-tumor effects in various cancer cells. This study aimed to explore the biological behavior effects of PG on GC cells, and determine its underlying mechanism. MATERIAL AND METHODS: The effect of PG treatment on the ferroptotic GC cell death was detected by ROS level, intracellular Fe2+ level and malondialdehyde (MDA) generation in vitro. The mRNA expression was detected by RT-qPCR. The interaction between miR-103a-3p and glutaminase 2 (GLS2) were verified by dual-luciferase reporter gene assay. Cell proliferation, invasion and migration were examined by CCK-8 and Transwell assay. Western blot was used to examine the expression of GLS2, SLC1A5 and epithelial-mesenchymal transition (EMT) related proteins. We also evaluated the influence of PG on the tumor growth and metastasis in vivo. RESULTS: PG-induced ferroptosis in GC cells through upregulating ROS level, intracellular Fe2+ level and MDA generation. Besides, PG also significantly enhanced the protein level of GLS2, which was an important transporter of glutamine to glutamate. Importantly, miR-103a-3p directly interacted with GLS2 and suppressed its expression. Mechanistically, PG treatment significantly promoted ferroptosis and anti-tumorigenesis by downregulating inhibitory effect of miR-103a-3p on GLS2 expression. CONCLUSION: Our studies confirmed that PG exerts pro-ferroptosis and anti-tumor effects in vitro and in vivo through regulating miR-103a-3p/GLS2 axis, thereby highlighting its therapeutic potential in GC.


Assuntos
Apoptose/efeitos dos fármacos , Emodina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Glutaminase/metabolismo , Ferro/metabolismo , MicroRNAs/genética , Neoplasias Gástricas/patologia , Animais , Proliferação de Células/efeitos dos fármacos , Emodina/farmacologia , Feminino , Glutaminase/genética , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Food Chem ; 301: 125226, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31357003

RESUMO

The glutenin (Glu) and gliadin (Gli) were modified by protein-glutaminase (PG) to obtain soluble glutenin (PG-Glu) and gliadin (PG-Gli), and PG-Glu or PG-Gli was added to potato starch (PS) according to different amounts (0.5%, 1.0%, and 1.5%, based on dry starch weight, w/w) to explore the effect of modified proteins on the retrogradation behavior and digestibility of PS. The results showed that the long-term retrogradation of PS was accelerated by the addition of PG-Glu or PG-Gli. The addition of PG-Glu or PG-Gli led to an increase in hydrogen bonds within starch molecules and induced a significant increase in resistant starch content. The hydrolysis kinetic parameters, C∞ and K, both decreased with the increasing level of modified protein, indicating the deceleration of hydrolysis rate by the addition of PG-Glu or PG-Gli. In summary, the addition of PG-Glu or PG-Gli could promote the retrogradation of PS and mitigate the digestion of starch.


Assuntos
Digestão , Gliadina/química , Glutaminase/metabolismo , Glutens/química , Solanum tuberosum/química , Amido/química , Amido/metabolismo , Gliadina/metabolismo , Glutens/metabolismo , Hidrólise , Solubilidade
4.
Klin Lab Diagn ; 64(5): 260-264, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31185147

RESUMO

The activity of amino acid metabolism enzymes and the content of free amino acids in the placenta during physiological pregnancy and placental insufficiency (PI) were studied using spectrophotometric methods and ion-exchange chromatography. It was found that in PI placental activity of the studied enzymes: alanine-, cysteine-e, tyrosine-, glutamino- transferase, glutathione synthetase, glutamate dehydrogenase decreases at different periods of gestation. The opposite variations occur for aspartataminotranferase and glutaminase. Similar changes are detected for amino acids synthesized or used in the course of appropriate reactions: aspartic acid, glutamic acid, glutamine, alanine, cysteine, tyrosine, arginine. The correlation between enzyme activity and amino acid content was revealed. Different periods of pregnancy are characterized by varying degrees of change, especially expressed in the second trimester, characterized by the most intense growth and development of the fetus, and its increased needs for trophic material. The revealed changes obviously play a pathogenetic role in the formation and further development of PI.


Assuntos
Aminoácidos/metabolismo , Placenta/enzimologia , Complicações na Gravidez/enzimologia , Aspartato Aminotransferases/metabolismo , Feminino , Glutaminase/metabolismo , Humanos , Oxirredutases/metabolismo , Insuficiência Placentária/enzimologia , Gravidez
5.
Cell Biol Int ; 43(8): 921-930, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31115975

RESUMO

miR-145 has been found to be a participant in cancer metastasis and glucose metabolism in ovarian cancer. However, the role of glutamine metabolism in ovarian cancer remains unclear. In this study, we aim to elucidate the molecular mechanism underlying the regulation of glutamine metabolism by miR-145 in ovarian cancer cells. The messenger RNA (mRNA) levels of miR-145 and glutaminase 1 (GLS1) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of c-myc and GLS1 were detected by western blot analysis. Luciferase reporter assays were used to validate c-myc was a target of miR-145. Glutamine metabolism was analyzed using assay kits. In addition, we performed luciferase reporter assays and chromatin immunoprecipitation assay to validate c-myc transcription activated GLS1 and promoted GLS1 expression. The qRT-PCR demonstrated that the mRNA level of miR-145 and GLS1 was negatively correlated in ovarian cancer tissues and cell lines. Kaplan-Meier survival analysis and the log-rank test showed that patients with high miR-145 expression had significantly increased the overall survival. The overexpression of miR-145 inhibited glutamine consumption, α-ketoglutarate production, and cellular ATP levels. Furthermore, we found miR-145 inhibited glutamine metabolism by targeting c-myc. Moreover, c-myc could promote GLS1 expression by transcription activated. Together, our results revealed that miR-145 inhibited glutamine metabolism through c-myc/GLS1 pathways in ovarian cancer cells, which may improve the current strategy of ovarian cancer diagnosis and therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/metabolismo , Feminino , Humanos , MicroRNAs/genética , Células Tumorais Cultivadas
6.
Breast Cancer Res ; 21(1): 61, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088535

RESUMO

INTRODUCTION: Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. EXPERIMENTAL: MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). RESULTS: Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. CONCLUSION: Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer. The effect of glutaminase inhibition in two breast cancer patient-derived xenograft (PDX) models. 13C HR MAS MRS analysis of tumor tissue from CB-839-treated and untreated models receiving 13C-labeled glutamine ([5-13C] Gln) shows that the glutaminase inhibitor CB-839 is causing an accumulation of glutamine (arrow up) in two PDX models representing luminal-like breast cancer (MAS98.06) and basal-like breast cancer (MAS98.12). In MAS98.06 tumors, CB-839 is in addition causing depletion of proline ([5-13C] Pro), alanine ([1-13C] Ala), and glutamate ([1-13C] Glu), which could explain why CB-839 causes tumor growth inhibition in MAS98.06 tumors, but not in MAS98.12 tumors.


Assuntos
Neoplasias da Mama/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Prolina/metabolismo , Animais , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Biologia Computacional , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Glutaminase/antagonistas & inibidores , Humanos , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética , Metabolômica/métodos , Camundongos , Modelos Biológicos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Biomed Pharmacother ; 116: 108960, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31112871

RESUMO

To uncover the role of microRNA-200a-3p in regulating osteogenic differentiation of MSCs via targeting glutaminase, thus influencing the progression of OP. Serum level of microRNA-200a-3p in OP patients and healthy controls was determined by qRT-PCR. MicroRNA-200a-3p level in MSCs undergoing osteogenic differentiation for different days was examined as well. ALP activity, calcification nodules and relative levels of Bglap, Runx2 and OPN in MSCs overexpressing microRNA-200a-3p undergoing osteogenic differentiation were detected. Relative l-glutaminase uptake in MSCs undergoing osteogenic differentiation for different days was determined. After transfection of si-GLS in MSCs undergoing osteogenic differentiation, l-glutaminase uptake, ALP activity and relative levels of Bglap, Runx2 and OPN were detected. The potential binding relationship between microRNA-200a-3p and GLS was tested by dual-luciferase reporter gene assay. Finally, rescue experiments were conducted to elucidate the role of microRNA-200a-3p/GLS in osteogenic differentiation of MSCs. MicroRNA-200a-3p level was higher in serum of OP patients relative to controls. Its level in MSCs gradually decreased with the prolongation of osteogenic differentiation. Overexpression of microRNA-200a-3p reduced cell viability, ALP activity, number and volume of calcification nodule. The mRNA levels of Bglap, Runx2 and OPN were downregulated by overexpressed microRNA-200a-3p. The cell viability, ALP activity, number and volume of calcification nodule were reduced when microRNA-200a-3p was knocked down. The mRNA levels of Bglap, Runx2 and OPN were upregulated when transfected microRNA-200a-3p inhibitor. l-glutaminase uptake increased with the prolongation of osteogenic differentiation in MSCs. Knockdown of GLS attenuated l-glutaminase uptake and ALP activity, as well as downregulated Bglap, Runx2 and OPN. Besides, GLS was verified to directly bind to microRNA-200a-3p. GLS overexpression reversed the inhibitory effects of overexpressed microRNA-200a-3p on osteogenic differentiation of MSCs. MicroRNA-200a-3p suppresses osteogenic differentiation of MSCs via targeting glutaminase, thereafter accelerating the progression of OP.


Assuntos
Diferenciação Celular/genética , Progressão da Doença , Glutaminase/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteogênese/genética , Osteoporose/genética , Osteoporose/patologia , Sequência de Bases , Regulação para Baixo/genética , Glutamina/metabolismo , Humanos , MicroRNAs/genética , Osteoporose/sangue
8.
Nat Commun ; 10(1): 1296, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899002

RESUMO

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Hipoglicemiantes/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzenoacetamidas/farmacologia , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/antagonistas & inibidores , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Camundongos , Terapia de Alvo Molecular , Fenformin/farmacologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Tiadiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Molecules ; 24(3)2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736411

RESUMO

In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , DNA Ribossômico/genética , Endopeptidases/genética , Fermentação , Glutaminase/biossíntese , Regulação Bacteriana da Expressão Gênica , Glutaminase/metabolismo , Temperatura Ambiente
10.
Oncogene ; 38(24): 4729-4738, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30765862

RESUMO

Cancer cells exhibit metabolic dependence on mitochondrial glutamine metabolism that provides them with the substrates required for rapid proliferation. Despite the extensive efforts to target this glutamine addiction for therapeutic purposes, the adaptive metabolic responses and the mechanisms whereby cells maintain their unlimited growth remain areas of active investigation. Here we report that mitochondrial glutamate-pyruvate transaminase 2 (GPT2) contributes to cell survival and growth by sustaining the tricarboxylic acid (TCA) cycle anaplerosis after the inhibition of glutaminase (GLS), the first enzyme for mitochondrial glutamine metabolism. We found that elevated reactive oxygen species upon GLS inhibition induce GPT2 expression via activating transcription factor 4. Moreover, inhibition of GPT2 synergized with suppression of GLS activity to induce a pronounced reduction in proliferation and an increase in cell death of cancer cells. Our data uncover GPT2 as an important component of the adaptive metabolic response for glutamine deprivation and indicate that targeting this pathway in combination with GLS inhibition may be an effective therapeutic approach for cancer treatment.


Assuntos
Adaptação Fisiológica/genética , Glutamina/metabolismo , Mitocôndrias/metabolismo , Transaminases/fisiologia , Células A549 , Células Cultivadas , Glutaminase/metabolismo , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Mitocôndrias/genética , Espécies Reativas de Oxigênio/metabolismo , Transaminases/metabolismo
11.
EBioMedicine ; 41: 200-213, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30796006

RESUMO

BACKGROUND: LncRNAs have been found to be involved in various aspects of biological processes. In this study, we aimed to uncover the molecular mechanisms of lncRNA EPB41L4A-AS1 in regulating glycolysis and glutaminolysis in cancer cells. METHODS: The expression of EPB41L4A-AS1 in cancer patients was analyzed in TCGA and GEO datasets. The level of cellular metabolism was determined by extracellular flux analyzer. The relationship between p53 and EPB41L4A-AS1 was explored by qRT-PCR, luciferase assay and ChIP assay. The interactions between EPB41L4A-AS1 and HDAC2 or NPM1 were determined by RNA immunoprecipitation, RNA pull-down assay and RNA-FISH- immunofluorescence. FINDINGS: EPB41L4A-AS1 was a p53-regulated gene. Low expression and deletion of lncRNA EPB41L4A-AS1 were found in a variety of human cancers and associated with poor prognosis of cancer patients. Knock down EPB41L4A-AS1 expression triggered Warburg effect, demonstrated as increased aerobic glycolysis and glutaminolysis. EPB41L4A-AS1 interacted and colocalized with HDAC2 and NPM1 in nucleolus. Silencing EPB41L4A-AS1 reduced the interaction between HDAC2 and NPM1, released HDAC2 from nucleolus and increased its distribution in nucleoplasm, enhanced HDAC2 occupation on VHL and VDAC1 promoter regions, and finally accelerated glycolysis and glutaminolysis. Depletion of EPB41L4A-AS1 increased the sensitivity of tumor to glutaminase inhibitor in tumor therapy. INTERPRETATION: EPB41L4A-AS1 functions as a repressor of the Warburg effect and plays important roles in metabolic reprogramming of cancer.


Assuntos
Núcleo Celular/metabolismo , Glicólise , Histona Desacetilase 2/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Transporte Ativo do Núcleo Celular , Animais , Glutaminase/metabolismo , Células HeLa , Células Hep G2 , Humanos , Camundongos , Camundongos Nus , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Longo não Codificante/metabolismo
12.
J Med Chem ; 62(2): 589-603, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30543285

RESUMO

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.


Assuntos
Antineoplásicos/química , Azóis/química , Inibidores Enzimáticos/química , Glutaminase/antagonistas & inibidores , Regulação Alostérica , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Azóis/farmacologia , Azóis/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glutaminase/metabolismo , Humanos , Rim/enzimologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Selênio/química , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/farmacologia , Tiadiazóis/uso terapêutico , Transplante Heterólogo
13.
J Pharmacol Exp Ther ; 368(3): 382-390, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30552293

RESUMO

Glutamate is the principal excitatory neurotransmitter in the brain and is at the base of a wide variety of neuropathologies, including epilepsy, autism, Fragile X, and obsessive compulsive disorder. Glutamate has also become the target for novel drugs in treatment and in fundamental research settings. However, much remains unknown on the working mechanisms of these drugs and the effects of chronic administration on the glutamatergic system. This study investigated the chronic effects of two glutamate-modulating drugs with imaging techniques to further clarify their working mechanisms for future research opportunities. Animals were exposed to saline (1 ml/kg), (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (0.3 mg/kg), or ebselen (10 mg/kg) for 7 consecutive days. At the sixth injection, animals underwent a positron emission tomography (PET)/computed tomography (CT) with (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime) (ABP-688) to visualize the metabotropic G protein-coupled glutamate receptor 5 (mGluR5). After the seventh injection, animals underwent a magnetic resonance spectroscopy (MRS) scan to visualize glutamate and glutamine content. Afterward, results were verified by mGluR5 immunohistochemistry (IHC). PET/CT analysis revealed that animals receiving chronic MK-801 or ebselen had a significant (P < 0.05) higher binding potential (2.90 ± 0.47 and 2.87 ± 0.46, respectively) when compared with saline (1.97 ± 0.39) in the caudate putamen. This was confirmed by mGluR5 IHC, with 60.83% ± 6.30% of the area being highlighted for ebselen and 57.14% ± 9.23% for MK-801 versus 50.21% ± 5.71% for the saline group. MRS displayed significant changes on the glutamine level when comparing chronic ebselen (2.20 ± 0.40 µmol/g) to control (2.72 ± 0.34 µmol/g). Therefore, although no direct effects on glutamate were visualized, the changes in glutamine suggest changes in the total glutamate-glutamine pool. This highlights the potential of both drugs to modulate glutamatergic pathologies.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Glutaminase/metabolismo , Imagem Molecular/métodos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Maleato de Dizocilpina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Glutaminase/antagonistas & inibidores , Glutamina/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
14.
Cell Mol Neurobiol ; 39(2): 255-263, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30552621

RESUMO

Many PET tracers enable determination of fluctuations in neurotransmitter release, yet glutamate specifically can not be visualized in a noninvasive manner. Several studies point to the possibility of visualizing fluctuations in glutamate release by changes in affinity of the mGluR5 radioligand [11C]ABP688. These studies use pharmacological challenges to alter glutamate levels, and so probe release, but have not measured chronic alterations in receptor occupancy due to altered neurotransmission relevant to chronic neuropsychiatric disorders or their treatment. In this regard, the GLS1 heterozygous mouse has known reductions in activity of the glutamate-synthetic enzyme glutaminase, brain glutamate levels and release. We imaged this model to elucidate glutamatergic systems. Dynamic [11C]ABP688 microPET scans were performed for mGluR5. Western blot was used as an ex vivo validation. No significant differences were found in BPND between WT and GLS1 Hets. SPM showed voxel-wise increased in BPND in GLS1 Hets compared to WT consistent with lower synaptic glutamate. This was not due to alterations in mGluR5 levels, as western blot results showed lower mGluR5 levels in GLS1 Hets. We conclude that because of the chronic glutaminase deficiency and subsequent decrease in glutamate, the mGluR5 protein levels are lowered. Due to these decreased endogenous glutamate levels, however, there is increased [11C]ABP688 binding to the allosteric site in selected regions. We speculate that lower endogenous glutamate leads to less conformational change to the receptors, and thus higher availability of the binding site. The lower mGluR5 levels, however, lessen [11C]ABP688 binding in GLS1 Hets, in part masking the increase in binding due to diminished endogenous glutamate levels as confirmed with voxel-wise analysis.


Assuntos
Radioisótopos de Carbono/química , Glutaminase/metabolismo , Imagem Molecular , Oximas/química , Piridinas/química , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Glutamina/metabolismo , Heterozigoto , Camundongos
15.
EBioMedicine ; 39: 239-254, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30555042

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignant disease with poor prognosis. Recent advances suggest the existence of cancer stem cells (CSCs) within liver cancer, which are considered to be responsible for tumor relapse, metastasis, and chemoresistance. However, novel therapeutic approaches for eradicating CSCs are yet to be established. Here, we aimed to identify the role of glutaminase 1 (GLS1) in stemness, and the feasibility that GLS1 serves as a therapeutic target for elimination CSCs as well as the possible mechanism. METHODS: Publicly-available data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was mined to unearth the association between GLS1 and stemness phenotype. Using big data, human tissues and multiple cell lines, we gained a general picture of GLS1 expression in HCC progression. We generated stable cell lines by lentiviral-mediated overexpression or CRISPR/Cas9-based knockout. Sphere formation assays and colony formation assays were employed to analyze the relationship between GLS1 and stemness. A series of bioinformatics analyses and molecular experiments including qRT-PCR, immunoblotting, flow cytometry, and immunofluorescence were employed to investigate the role of GLS1 in regulating stemness in vitro and in vivo. FINDINGS: We observed GLS1 (both KGA and GAC isoform) is highly expressed in HCC, and that high expression of GAC predicts a poor prognosis. GLS1 is exclusively expressed in the mitochondrial matrix. Upregulation of GLS1 is positively associated with advanced clinicopathological features and stemness phenotype. Targeting GLS1 reduced the expression of stemness-related genes and suppressed CSC properties in vitro. We further found GLS1 regulates stemness properties via ROS/Wnt/ß-catenin signaling and that GLS1 knockout inhibits tumorigenicity in vivo. INTERPRETATION: Targeting GLS1 attenuates stemness properties in HCC by increasing ROS accumulation and suppressing Wnt/ß-catenin pathway, which implied that GLS1 could serve as a therapeutic target for elimination of CSCs.


Assuntos
Carcinoma Hepatocelular/patologia , Glutaminase/genética , Glutaminase/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adulto , Idoso , Animais , Big Data , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Glutaminase/antagonistas & inibidores , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Prognóstico , Regulação para Cima , Via de Sinalização Wnt
16.
FASEB J ; 33(1): 557-571, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30001166

RESUMO

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Ácido Glutâmico/metabolismo , Isocitrato Desidrogenase/genética , Ácido Láctico/metabolismo , Mutação , Estresse Fisiológico , 4-Aminobutirato Transaminase/genética , 4-Aminobutirato Transaminase/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Med Chem ; 62(1): 46-59, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29969024

RESUMO

Kidney-type glutaminase (GLS), the first enzyme in the glutaminolysis pathway, catalyzes the hydrolysis of glutamine to glutamate. GLS was found to be upregulated in many glutamine-dependent cancer cells. Therefore, selective inhibition of GLS has gained substantial interest as a therapeutic approach targeting cancer metabolism. Bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide (BPTES), despite its poor physicochemical properties, has served as a key molecular template in subsequent efforts to identify more potent and drug-like allosteric GLS inhibitors. This review article provides an overview of the progress made to date in the development of GLS inhibitors and highlights the remarkable transformation of the unfavorable lead into "druglike" compounds guided by systematic SAR studies.


Assuntos
Inibidores Enzimáticos/química , Glutaminase/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Glutaminase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/metabolismo , Tiadiazóis/química , Tiadiazóis/metabolismo
18.
Am J Respir Cell Mol Biol ; 60(1): 49-57, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30130138

RESUMO

Fibrotic responses involve multiple cellular processes, including epigenetic changes. Epigenetic changes are sensitive to alterations in the tissue microenvironment such as the flux of tricarboxylic acid (TCA) cycle metabolites. TCA metabolites directly regulate epigenetic states, in part by regulating histone modification-related enzymes. Glutaminolysis is a critical metabolic process by which glutamine is converted to glutamate by glutaminase and then to α-ketoglutarate (α-KG), a TCA cycle metabolite. Idiopathic pulmonary fibrosis (IPF) is a disease characterized by aberrant metabolism, including enhanced glutaminolysis. IPF fibroblasts are apoptosis resistant. In this study, we explored the relationship between glutaminolysis and the resistance to apoptosis of IPF fibroblasts. Inhibition of glutaminolysis decreased expression of XIAP and survivin, members of the inhibitor of apoptosis protein (IAP) family. α-KG is a cofactor for JMJD3 histone demethylase, which targets H3K27me3. In the absence of glutamine, JMJD3 activity in fibroblasts is significantly decreased, whereas H3K27me3 levels are increased. Chromatin immunoprecipitation assays confirmed that JMJD3 directly interacts with XIAP and survivin promoter regions in a glutamine-dependent manner. Exogenous α-KG partially restores JMJD3 function and its interaction with the XIAP and survivin promoter regions under glutamine-deficient conditions. Interestingly, α-KG upregulates XIAP, but not survivin, suggesting differential α-KG-dependent and -independent mechanisms by which glutamine regulates these IAPs. Our data demonstrate a novel mechanism of metabolic regulation in which glutaminolysis promotes apoptosis resistance of IPF fibroblasts through epigenetic regulation of XIAP and survivin.


Assuntos
Epigênese Genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glutamina/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Survivina/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose , Células Cultivadas , Fibroblastos/patologia , Glutaminase/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Survivina/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
19.
Folia Microbiol (Praha) ; 64(3): 313-320, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30361879

RESUMO

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 µmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.


Assuntos
Asparaginase/metabolismo , Glutaminase/metabolismo , Pyrobaculum/enzimologia , Asparaginase/isolamento & purificação , Clonagem Molecular , Estabilidade Enzimática , Glutamina/metabolismo , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Pyrobaculum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura Ambiente
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