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1.
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
2.
Comput Biol Chem ; 81: 16-20, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31422018

RESUMO

Many biochemical events involve multistep reactions. Among them, an important biological process that involves multistep reaction is the transcriptional process. A widely used approach for simplifying multistep reactions is the delayed reaction method. In this work, we devise a model reduction strategy that represents several OFF states by a single state, accompanied by specifying a time delay for burst frequency. Using this model reduction, we develop Clumped-MCEM which enables simulation and parameter inference. We apply this method to time-series data of endogenous mouse glutaminase promoter, to validate the model assumptions and infer the kinetic parameters. Further, we compare efficiency of Clumped-MCEM with state-of-the-art methods - Bursty MCEM2 and delay Bursty MCEM. Simulation results show that Clumped-MCEM inference is more efficient for time-series data and is able to produce similar numerical accuracy as state-of-the-art methods - Bursty MCEM2 and delay Bursty MCEM in less time. Clumped-MCEM reduces computational cost by 57.58% when compared with Bursty MCEM2 and 32.19% when compared with delay Bursty MCEM.


Assuntos
Glutaminase/química , Modelos Químicos , Transcrição Genética , Algoritmos , Animais , Simulação por Computador , Glutaminase/genética , Cinética , Camundongos , Método de Monte Carlo , Regiões Promotoras Genéticas , Fatores de Tempo
3.
Crit Rev Biotechnol ; 39(7): 944-963, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31327254

RESUMO

This article focuses on significant advances in the production and applications of microbial glutaminases and provides insight into the structures of different glutaminases. Glutaminases catalyze the deamidation of glutamine to glutamic acid, and this unique ability forms the basis of their applications in various industries such as pharmaceutical and food organizations. Microbial glutaminases from bacteria, actinomycetes, yeast, and fungi are of greater significance than animal glutaminases because of their stability, affordability, and ease of production. Owing to these notable benefits, they are considered to possess considerable potential in anticancer and antiviral therapy, flavor enhancers in oriental foods, biosensors and in the production of a nutraceutical theanine. This review also aims to fully explore the potential of microbial glutaminases and to set the pace for future prospects.


Assuntos
Glutaminase/biossíntese , Microbiologia Industrial/métodos , Animais , Clonagem Molecular , Glutaminase/química , Glutaminase/genética , Glutaminase/farmacologia , Humanos , Conformação Proteica , Tolerância ao Sal
4.
Mol Med Rep ; 20(2): 1915-1924, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257527

RESUMO

Kidney­type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non­cancerous tissues. Second, a wound­healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial­mesenchymal transition (EMT)­related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT­related markers, E­cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E­cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.


Assuntos
Biomarcadores Tumorais/genética , Colangiocarcinoma/genética , Glutaminase/genética , Recidiva Local de Neoplasia/genética , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Vimentina/genética
5.
Prensa méd. argent ; 105(3): 130-137, may 2019. fig, tab
Artigo em Inglês | LILACS, BINACIS | ID: biblio-1025428

RESUMO

Trichomonas vaginalis (T. vaginalis), the etiologic agent of human trichomoniasis, is a flagellated protozoan parasite, has been associated sith advese pregnancy outcomes, HIV transmission, and infertilityh. A total of one hundred and fifty-seven (157) women at childbearing age (14-49 years), were included in the presnt study, eighty six (86) symptomatic fertile while the other seventy-one (71) were infertile with or without sumptoms attending the Gynecology outpatient Department in Al-Emamayn Al-Kadhimayn Medical City, the High Institute of Infertility Diagnosis and Assisted Reproductive Technoligies at Al-Nahrain University in Baghdad, the maternity Teaching hospital, and Dr. Khawer center for infertility and IVF in Erbil province in Iraq. Two vaginal swab specimens were obtained from each of them:; one swab was immediately examined by wet mount microscopy, the other swab for molecular study (DNA extraction and p3 nested PCR). One hundred (100) samples positive in one or more test were identified: 20 (12.7%) infecions were detected by wet mount microscopy, while nested PCR was positive in 100 (63.7%) samples. These positive samples were seguenced and phylogenetic tree were done and, there was no association between the variations in glut (p3) gene of T. vaginalis isolated from infected women (fertile and infertile)


Assuntos
Humanos , Feminino , Gravidez , Adolescente , Adulto , Complicações na Gravidez/etiologia , Manejo de Espécimes/classificação , Tricomoníase/etiologia , Trichomonas vaginalis/genética , Alelos , Fertilidade , Glutaminase/genética , Infertilidade Feminina
6.
N Engl J Med ; 380(15): 1433-1441, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30970188

RESUMO

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Ataxia/genética , Deficiências do Desenvolvimento/genética , Glutaminase/deficiência , Glutaminase/genética , Glutamina/metabolismo , Repetições de Microssatélites , Mutação , Atrofia/genética , Cerebelo/patologia , Pré-Escolar , Feminino , Genótipo , Glutamina/análise , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Sequenciamento Completo do Genoma
7.
Cancer Res ; 79(7): 1302-1304, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936077

RESUMO

The cancer target glutaminase (GLS) has proven to be a fascinating protein. Since it was first described to be regulated by the oncogene Myc 10 years ago, several other transcriptional, posttranscriptional, and posttranslational regulatory mechanisms have emerged, and the list is growing. A recent study by Deng and colleagues revealed that an antisense (AS) long noncoding RNA named GLS-AS, which is negatively regulated by Myc, downregulates GLS in pancreatic cancer. The Myc/GLS-AS/GLS regulatory axis is activated by nutrient stress, which is important for the often hypovascular pancreatic cancer, displaying the significance of GLS for the progression of this highly lethal type of cancer.See related article by Deng et al., p. 1398.


Assuntos
Glutaminase/genética , Mitocôndrias , Linhagem Celular Tumoral
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.
Neuroscience ; 396: 175-186, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472430

RESUMO

Significant alterations in glutamatergic neurotransmission have been reported in major depressive disorder (MDD) that could underlie psychiatric traits. Studies were mainly interested in synaptic dysfunction in the prefrontal cortex, a key structure involved in depressive-like behavior, however hippocampus has been shown to be important in MDD. As cognitive deficits such as hippocampus-memory process were observed in MDD, we investigated in a mild hypoglutamatergic model behaviors related to depression and memory, synaptic transmission parameters and glutamatergic state specifically in the hippocampus. We thus characterized these phenotypes in adult male mice partially depleted in glutaminase type 1 or GLS1 (GLS1 HET), the enzyme responsible for glutamate synthesis in neurons, that we previously characterized as displaying moderate lower levels of glutamate in brain. We showed that GLS1 mutant mice display AMPA-R-mediated response deficits after prolonged repetitive stimulation with electrophysiological recording and inability to sustain glutamate release by microdialysis experiments with no consequences on behavioral spatial learning performances. However, their ability to escape from unpleasant but repeated escapable condition was attenuated whereas they were more immobile in the unescapable situation in the FST during re-test. These results show that GLS1 mutant mice display moderate impairments of hippocampal glutamatergic neurotransmission and moderate changes in adaptive behaviors that have been shown to participate to the development of depressive-like state.


Assuntos
Aprendizagem da Esquiva/fisiologia , Ácido Glutâmico/fisiologia , Glutaminase/fisiologia , Hipocampo/fisiologia , Resposta de Imobilidade Tônica/fisiologia , Aprendizagem Espacial/fisiologia , Transmissão Sináptica/fisiologia , Animais , Corticosterona/sangue , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ácido Glutâmico/metabolismo , Glutaminase/genética , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Microdiálise , Mutação , Restrição Física/fisiologia
10.
Hum Mol Genet ; 28(1): 96-104, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30239721

RESUMO

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.


Assuntos
Glutaminase/genética , Glutaminase/fisiologia , Adolescente , Animais , Encéfalo/metabolismo , Catarata/genética , Pré-Escolar , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Feminino , Fibroblastos , Mutação com Ganho de Função/genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/fisiologia , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Masculino , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
11.
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
12.
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
13.
J Bioinform Comput Biol ; 16(5): 1850023, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30419780

RESUMO

Many biochemical events involve multistep reactions. One of the most important biological processes that involve multistep reaction is the transcriptional process. Models for multistep reaction necessarily need multiple states and it is a challenge to compute model parameters that best agree with experimental data. Therefore, the aim of this work is to design a multistep promoter model which accurately characterizes transcriptional bursting and is consistent with observed data. To address this issue, we develop a model for promoters with several OFF states and a single ON state using Erlang distribution. To explore the combined effects of model and data, we combine Monte Carlo extension of Expectation Maximization (MCEM) and delay Stochastic Simulation Algorithm (DSSA) and call the resultant algorithm as delay Bursty MCEM. We apply this algorithm to time-series data of endogenous mouse glutaminase promoter to validate the model assumptions and infer the kinetic parameters. Our results show that with multiple OFF states, we are able to infer and produce a model which is more consistent with experimental data. Our results also show that delay Bursty MCEM inference is more efficient.


Assuntos
Algoritmos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Animais , Biologia Computacional/métodos , Glutaminase/genética , Análise de Séries Temporais Interrompida , Cinética , Funções Verossimilhança , Camundongos , Método de Monte Carlo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processos Estocásticos , Transcrição Genética
14.
Cell Physiol Biochem ; 51(2): 513-527, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30458442

RESUMO

BACKGROUND/AIMS: Increasing evidence showed that miR-1-3p plays a major role in malignant tumor progression. However, the specific biological function of miR-1-3p in bladder cancer is yet unknown. METHODS: The expression levels of miR-1-3p in bladder cancer tissues and cell lines were examined by qRT-PCR. Bisulfite sequencing PCR was used for DNA methylation analysis. The target of miR-1-3p was validated by a dual luciferase reporter assay, and the effects of miR-1-3p on phenotypic changes in bladder cancer cells were investigated in vitro and in vivo. RESULTS: The expression of miR-1-3p in bladder cancer cells was downregulated as compared to normal SV-HUC-1 cells. Also, the expression of miR-1-3p was significantly lower in bladder cancer tissues than the corresponding non-cancerous tissues. The methylation status of CpG islands was involved in the regulation of miR-1-3p expression. miR-1-3p inhibited the bladder cancer cell proliferation, migration, and invasion by directly targeting the 3'-UTR of glutaminase. It also exerted an anti-tumor effect by negatively regulating the glutaminase in a xenograft mouse model. Furthermore, GLS depletion resulted in the prolonged expression of γH2AX. CONCLUSION: Taken together, these results demonstrated that miR-1-3p acts as a tumor suppressor via regulation of glutaminase expression in bladder cancer progression, and miR-1-3p might represent a novel therapeutic target for the treatment of bladder cancer.


Assuntos
Proliferação de Células , Glutaminase/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Antagomirs/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Ilhas de CpG , Dano ao DNA , Metilação de DNA , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
15.
Cell ; 175(7): 1780-1795.e19, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30392958

RESUMO

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Glutaminase/imunologia , Ativação Linfocitária , Células Th1/imunologia , Células Th17/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Glutaminase/genética , Masculino , Camundongos , Camundongos Transgênicos , Células Th1/citologia , Células Th17/citologia
16.
PLoS Genet ; 14(10): e1007667, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30289878

RESUMO

The role of host genetic variation in the development of complicated Staphylococcus aureus bacteremia (SAB) is poorly understood. We used whole exome sequencing (WES) to examine the cumulative effect of coding variants in each gene on risk of complicated SAB in a discovery sample of 168 SAB cases (84 complicated and 84 uncomplicated, frequency matched by age, sex, and bacterial clonal complex [CC]), and then evaluated the most significantly associated genes in a replication sample of 240 SAB cases (122 complicated and 118 uncomplicated, frequency matched for age, sex, and CC) using targeted sequence capture. In the discovery sample, gene-based analysis using the SKAT-O program identified 334 genes associated with complicated SAB at p<3.5 x 10-3. These, along with eight biologically relevant candidate genes were examined in the replication sample. Gene-based analysis of the 342 genes in the replication sample using SKAT-O identified one gene, GLS2, significantly associated with complicated SAB (p = 1.2 x 10-4) after Bonferroni correction. In Firth-bias corrected logistic regression analysis of individual variants, the strongest association across all 10,931 variants in the replication sample was with rs2657878 in GLS2 (p = 5 x 10-4). This variant is strongly correlated with a missense variant (rs2657879, p = 4.4 x 10-3) in which the minor allele (associated here with complicated SAB) has been previously associated with lower plasma concentration of glutamine. In a microarray-based gene-expression analysis, individuals with SAB exhibited significantly lower expression levels of GLS2 than healthy controls. Similarly, Gls2 expression is lower in response to S. aureus exposure in mouse RAW 264.7 macrophage cells. Compared to wild-type cells, RAW 264.7 cells with Gls2 silenced by CRISPR-Cas9 genome editing have decreased IL1-ß transcription and increased nitric oxide production after S. aureus exposure. GLS2 is an interesting candidate gene for complicated SAB due to its role in regulating glutamine metabolism, a key factor in leukocyte activation.


Assuntos
Glutaminase/genética , Infecções Estafilocócicas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Animais , Bacteriemia , Feminino , Frequência do Gene/genética , Variação Genética/genética , Glutaminase/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células RAW 264.7 , Fatores de Risco , Staphylococcus aureus/patogenicidade , Transcriptoma/genética , Sequenciamento Completo do Exoma/métodos
17.
Biochimie ; 154: 69-76, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30092248

RESUMO

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.


Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Glutaminase/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Substituição de Aminoácidos , Linhagem Celular Tumoral , Glutaminase/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação
18.
Biochem Pharmacol ; 156: 204-214, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30144404

RESUMO

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G0/G1 phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Endoteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/farmacologia , Aorta/citologia , Benzenoacetamidas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ciclina A/genética , Ciclina A/metabolismo , Diazo-Oxo-Norleucina/farmacologia , Células Endoteliais/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Interferência de RNA , Tiadiazóis/farmacologia , Veias/citologia
20.
J Mol Med (Berl) ; 96(8): 777-790, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29942976

RESUMO

Papillary thyroid cancer is a prevalent endocrine malignancy. Although alterations in glutamine metabolism have been reported in several types of hematological and solid tumors, little is known about the functions of glutamine and glutaminolysis-associated proteins in papillary thyroid cancer. Here, we demonstrated the glutamine dependence of papillary thyroid cancer cells, and with the use of RT2-PCR arrays, we screened for the aberrant overexpression of glutaminase in human papillary thyroid cancer tissues and cells. These results were later confirmed via real-time PCR, Western blots, and immunohistochemical staining. We found that the levels of glutaminase were significantly correlated with extrathyroidal extension. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. The proliferative, viable, migratory, and invasive abilities of papillary thyroid cancer cells were impaired by both the pharmacological inhibition and the genetic knockdown of glutaminase. Additionally, the inhibition of glutaminase deactivated the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, promoting autophagy and apoptosis. Collectively, these findings show that glutaminase-mediated glutamine dependence may be a potential therapeutic target for papillary thyroid cancer. KEY MESSAGES: PTC cells are glutamine-dependent, and GLS is aberrantly overexpressed in PTC. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. Inhibition of GLS impairs the viability of PTC cells. GLS blockade causes deactivation of mTORC1 and induction of autophagy and apoptosis. GLS may be a potential therapeutic target for PTC.


Assuntos
Glutaminase/metabolismo , Glutamina/metabolismo , Câncer Papilífero da Tireoide/metabolismo , Adulto , Idoso , Apoptose , Autofagia , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular , Respiração Celular , Feminino , Glutaminase/genética , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estadiamento de Neoplasias , Transdução de Sinais , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Carga Tumoral
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