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1.
J Agric Food Chem ; 67(38): 10685-10693, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31479251

RESUMO

Theanine is the most abundant non-protein amino acid in Camellia sinensis, but it is not known how a tea plant accumulates such high levels of theanine. The endophyte isolated from in vitro grown plantlets of C. sinensis cultivars was identified as Luteibacter spp., showing strong biocatalytic activity for converting both glutamine and ethylamine to theanine. Theanine was secreted outside of the bacteria. The endophyte isolated from in vitro plantlets of Camellia oleifera cultivar was identified as Bacillus safensis and did not convert glutamine and ethylamine to theanine. Enzymatic assays in vitro indicated that γ-glutamyltranspeptidases rCsEGGTs from the endophyte Luteibacter strains converted glutamine and ethylamine to theanine at higher rates than rCsGGTs from C. sinensis. This is the first report on theanine biosynthesis by an endophyte from C. sinensis, which provides a new pathway to explore the mechanism of theanine biosynthesis in C. sinensis and the interactions between an endophyte and tea plants.


Assuntos
Bactérias/metabolismo , Camellia sinensis/microbiologia , Endófitos/metabolismo , Glutamatos/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Camellia sinensis/química , Camellia sinensis/classificação , Endófitos/classificação , Endófitos/genética , Endófitos/isolamento & purificação , Etilaminas/metabolismo , Glutamina/metabolismo , Folhas de Planta/química , Folhas de Planta/microbiologia
2.
Cancer Sci ; 110(11): 3453-3463, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31444923

RESUMO

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Glutationa/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Terapia de Alvo Molecular/métodos , Células-Tronco Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Glutamato Desidrogenase/metabolismo , Glutamina/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma , Camundongos , Camundongos Nus , Antígenos de Histocompatibilidade Menor/genética , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Sulfassalazina/farmacologia
3.
Cell Physiol Biochem ; 53(1): 200-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31287628

RESUMO

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.


Assuntos
Proteínas de Transporte/metabolismo , Glutamina/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Animais , Proteínas de Transporte/análise , Linhagem Celular , Cromatografia Líquida , Glutamina/análise , Insulina/análise , Camundongos , Fibras Musculares Esqueléticas/química , Fosfoproteínas/análise , Fosforilação , Proteínas Proto-Oncogênicas c-akt/análise , Proteína S6 Ribossômica/análise , Espectrometria de Massas em Tandem
4.
Methods Mol Biol ; 1975: 321-330, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062317

RESUMO

Stem cell research has been greatly facilitated by comprehensive and integrative multi-omics studies. As a unique approach of functional analysis, metabolomics measures many metabolites and activities of metabolic pathways which can directly indicate cellular energetic status, cell proliferation and fitness, and stem cell fate choices such as self-renewal versus differentiation. Here we describe the methods of applying metabolomics, 13C-labeled glucose and glutamine tracing with mouse embryonic stem cells (ES cells), metabolite analysis using mass spectrometry tools, and the following statistical and computational modeling analysis. Integration of these methods into the more common gene expression and epigenetics analysis toolbox will help to generate a more complete picture and in-depth understanding of one's stem cells of interest.


Assuntos
Diferenciação Celular , Linhagem da Célula , Biologia Computacional/métodos , Metabolômica/métodos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Pesquisa Biomédica , Glucose/metabolismo , Glutamina/metabolismo , Camundongos
5.
Methods Mol Biol ; 1978: 219-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119666

RESUMO

Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply 13C isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.


Assuntos
Técnicas de Cultura de Células/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Marcação por Isótopo/métodos , Lipogênese/genética , Animais , Isótopos de Carbono/química , Glutamina/metabolismo , Humanos , Redes e Vias Metabólicas/genética
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.
Oncol Rep ; 41(6): 3555-3564, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002368

RESUMO

Neoplastic transformation is characterized by metabolic rewiring to sustain the elevated biosynthetic demands of highly proliferative cancer cells. To obtain the precursors for macromolecule biosynthesis, cancer cells avidly uptake and metabolize glucose and glutamine. Thus, targeting the availability or metabolism of these nutrients is an attractive anticancer therapeutic strategy. To improve our knowledge concerning how cancer cells respond to nutrient withdrawal, the response to glutamine and/or glucose starvation was studied in human in vitro transformed fibroblasts, deeply characterized at the cellular and molecular level. Concomitant starvation of both nutrients led to rapid loss of cellular adhesion (~16 h after starvation), followed by cell death. Deprivation of glucose alone had the same effect, although at a later time (~48 h after starvation), suggesting that glucose plays a key role in enabling cell attachment to the extracellular matrix. Glutamine deprivation did not induce rapid cell death, but caused a prolonged arrest of cellular proliferation; the cells started dying only 96 h after starvation. Before massive cell death occurred, the effects of all the starvation conditions were reversible. Autophagy activation was observed in cells incubated in the absence of glucose for more than 48 h, while autophagy was not detected under the other starvation conditions. Markers of apoptotic cell death, such as caspase 3, caspase 9 and poly(ADP­ribose) polymerase 1 (PARP­1) proteolytic fragments, were not observed under any growth condition. Glucose and/or glutamine deprivation caused very rapid PARP­1 activation, with marked PARP­1 (poly­ADP) ribosylation and protein (poly­ADP) ribosylation. This activation was not due to starvation­induced DNA double­strand breaks, which appeared at the late stages of deprivation, when most cells died. Collectively, these results highlight a broad range of consequences of glucose and glutamine starvation, which may be taken into account when nutrient availability is used as a target for anticancer therapies.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Glucose/metabolismo , Glutamina/metabolismo , Apoptose/genética , Autofagia/genética , Caspases/genética , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/genética , Glutamina/genética , Humanos , Terapia de Alvo Molecular , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inanição/genética , Inanição/metabolismo
8.
Proc Natl Acad Sci U S A ; 116(13): 6313-6318, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30862735

RESUMO

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.


Assuntos
Amônia/metabolismo , Deficiências Nutricionais , Inativação Metabólica , Fígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Deficiências Nutricionais/patologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Técnicas de Silenciamento de Genes , Glutamato-Amônia Ligase/metabolismo , Glutamina/metabolismo , Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Hiperamonemia/metabolismo , Cinética , Fígado/patologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Estresse Oxidativo , Perfusão , Ureia/metabolismo
9.
Proc Natl Acad Sci U S A ; 116(14): 6964-6968, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877243

RESUMO

The growth-stimulating capacity of calf serum (CS) in cell culture reaches a maximum of 10% with Balb 3T3 cells, remains at a plateau to 40% CS, and declines steeply to 100% CS. Growth capacity can be largely restored to the latter by a combination of cystine and glutamine. Glutamine is a conditionally essential amino acid that continues to function at very low concentrations to support the growth of nontransformed cells, but transformed cells require much larger concentrations to survive. These different requirements hold true over a 10-fold variation in background concentrations of CS and amino acids. The high requirement of glutamine for transformed cells applies to the development of neoplastically transformed foci. These observations have given rise to a novel protocol for cancer therapy based on the large difference in the need for glutamine between nontransformed and transformed cells. This protocol would stop the cumulative growth and survival of the transformed cells without reducing the growth rate of the nontransformed cells. The results call for studies of glutamine deprivation as a treatment for experimental cancer in rodents and clinical trials in humans.


Assuntos
Transformação Celular Neoplásica/metabolismo , Glutamina/deficiência , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Células 3T3 BALB , Técnicas de Cultura de Células , Transformação Celular Neoplásica/patologia , Meios de Cultura , Glutamina/metabolismo , Camundongos , Neoplasias/patologia
10.
Nature ; 567(7749): 535-539, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867594

RESUMO

Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.


Assuntos
Regulação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Serotonina/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Feminino , Proteínas de Ligação ao GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Neurônios Serotoninérgicos/citologia , Transglutaminases/metabolismo
12.
Poult Sci ; 98(9): 4066-4072, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30843058

RESUMO

An experiment was conducted to investigate the effect of supplemental L-glutamine (L-Gln) and a higher concentration of zinc (Zn) on excreta moisture under nutritionally induced wet droppings via decreased intestinal water reabsorption. A 2 × 2 × 2 factorial arrangement of treatments was used to investigate 3 dietary factors of L-Gln supplementation (0 or 10 g/kg), and added Zn concentration (80 and 160 mg/kg) with or without magnesium chloride (MgCl) (2 g/kg-only in grower diets). A total of 576 male day-old Ross 308 broiler chickens were assigned to the experimental diets. Each diet was replicated 6 times with 12 birds per replicate. Wheat-based diets were formulated to be isocaloric and isonitrogenous. Starter diets were given from day 0 to 9 followed by grower (day 10 to 23) and finisher diets (day 24 to 35). Excreta moisture was measured for all the growth phases. The moisture content of different segments of intestine was assessed for starter and grower phases of feeding. There was no significant effect of any of the 3 main treatments on body weight gain or feed intake of birds during the experiment. Birds fed higher Zn (160 mg/kg) tended (P = 0.09) to have higher weight gain only in the first 9 days of age. Feeding 10 g/kg L-Gln increased the feed conversion ratio of the birds only from hatch until day 9 after which there was no significant effect. No effect of experimental treatments was found on digesta or excreta moisture, except a reduction in ileal moisture at the starter phase resulting from higher Zn concentration in the diets. MgCl at 2 g/kg was not effective in inducing wet droppings in birds fed grower diets. Under the conditions of this study, no positive response was observed in terms of performance or reduction in excreta moisture when birds were fed diets containing 10 g/kg L-Gln or higher concentration of Zn.


Assuntos
Galinhas/fisiologia , Fezes/química , Glutamina/metabolismo , Magnésio/administração & dosagem , Zinco/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Digestão/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutamina/administração & dosagem , Masculino , Zinco/administração & dosagem
13.
Biologicals ; 59: 1-5, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30910309

RESUMO

Therapeutic recombinant monoclonal antibodies are subject to various modifications during cell culture, and to a lesser degree, during purification. These modifications are expected to remain relatively constant during storage with the protection of appropriate formulations. However, after administration to patients, the levels of modifications may vary over time in circulation, where the recombinant monoclonal antibodies are exposed to the physiological conditions. Scientific understanding of those in vivo modifications can help drug candidate selection to choose the most stable molecules and set appropriate specifications for product release, which ultimately ensures safety and efficacy.


Assuntos
Anticorpos Monoclonais/química , Preparações Farmacêuticas/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Asparagina/química , Asparagina/metabolismo , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutamina/química , Glutamina/metabolismo , Glicosilação , Humanos , Lisina/química , Lisina/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/metabolismo , Succinimidas/química , Succinimidas/metabolismo , Fatores de Tempo
14.
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
15.
Nat Commun ; 10(1): 1351, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30903027

RESUMO

The inability to inspect metabolic activities within subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Here, we describe a spatial-fluxomics approach for inferring metabolic fluxes in mitochondria and cytosol under physiological conditions, combining isotope tracing, rapid subcellular fractionation, LC-MS-based metabolomics, computational deconvolution, and metabolic network modeling. Applied to study reductive glutamine metabolism in cancer cells, shown to mediate fatty acid biosynthesis under hypoxia and defective mitochondria, we find a previously unappreciated role of reductive IDH1 as the sole net contributor of carbons to fatty acid biosynthesis under standard normoxic conditions in HeLa cells. In murine cells with defective SDH, we find that reductive biosynthesis of citrate in mitochondria is followed by a reversed CS activity, suggesting a new route for supporting pyrimidine biosynthesis. We expect this spatial-fluxomics approach to be a highly useful tool for elucidating the role of metabolic dysfunction in human disease.


Assuntos
Compartimento Celular , Glutamina/metabolismo , Análise do Fluxo Metabólico , Neoplasias/metabolismo , Animais , Isótopos de Carbono , Hipóxia Celular , Citrato (si)-Sintase/metabolismo , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Citosol/metabolismo , Células HeLa , Humanos , Isocitrato Desidrogenase/metabolismo , Metaboloma , Camundongos , Mitocôndrias/metabolismo , Frações Subcelulares/metabolismo , Succinato Desidrogenase/metabolismo
16.
Biochim Biophys Acta Bioenerg ; 1860(5): 391-401, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885735

RESUMO

Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a "signaling protein" under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1 h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies.


Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Proteínas de Neoplasias/biossíntese , Neuroblastoma/metabolismo , Proteína Desacopladora 2/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Metabolismo Energético/genética , Glucose/genética , Glucose/metabolismo , Glutamina/genética , Camundongos , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteína Desacopladora 2/genética
17.
Biol Res ; 52(1): 15, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30917872

RESUMO

BACKGROUND: Tumourigenic cells modify metabolic pathways in order to facilitate increased proliferation and cell survival resulting in glucose- and glutamine addiction. Previous research indicated that glutamine deprivation resulted in potential differential activity targeting tumourigenic cells more prominently. This is ascribed to tumourigenic cells utilising increased glutamine quantities for enhanced glycolysis- and glutaminolysis. In this study, the effects exerted by glutamine deprivation on reactive oxygen species (ROS) production, mitochondrial membrane potential, cell proliferation and cell death in breast tumourigenic cell lines (MCF-7, MDA-MB-231, BT-20) and a non-tumourigenic breast cell line (MCF-10A) were investigated. RESULTS: Spectrophotometry demonstrated that glutamine deprivation resulted in decreased cell growth in a time-dependent manner. MCF-7 cell growth was decreased to 61% after 96 h of glutamine deprivation; MDA-MB-231 cell growth was decreased to 78% cell growth after 96 h of glutamine deprivation, MCF-10A cell growth was decreased 89% after 96 h of glutamine deprivation and BT-20 cell growth decreased to 86% after 24 h of glutamine deprivation and remained unchanged until 96 h of glutamine deprivation. Glutamine deprivation resulted in oxidative stress where superoxide levels were significantly elevated after 96 h in the MCF-7- and MDA-MB-231 cell lines. Time-dependent production of hydrogen peroxide was accompanied by aberrant mitochondrial membrane potential. The effects of ROS and mitochondrial membrane potential were more prominently observed in the MCF-7 cell line when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines. Cell cycle progression revealed that glutamine deprivation resulted in a significant increase in the S-phase after 72 h of glutamine deprivation in the MCF-7 cell line. Apoptosis induction resulted in a decrease in viable cells in all cell lines following glutamine deprivation. In the MCF-7 cells, 87.61% of viable cells were present after 24 h of glutamine deprivation. CONCLUSION: This study demonstrates that glutamine deprivation resulted in decreased cell proliferation, time-dependent- and cell line-dependent ROS generation, aberrant mitochondrial membrane potential and disrupted cell cycle progression. In addition, the estrogen receptor positive MCF-7 cell line was more prominently affected. This study contributes to knowledge regarding the sensitivity of breast cancer cells and non-tumorigenic cells to glutamine deprivation.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , Glutamina/deficiência , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Glutamina/metabolismo , Humanos , Espectrofotometria
18.
Cell Prolif ; 52(3): e12594, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847992

RESUMO

OBJECTIVE: Ex vivo expansion is an effective way to produce cytokine-induced killer (CIK) cells needed for clinical trials. Here, ex vivo expansion and metabolism characters of CIK cells in static and dynamic cultures and the relationship between cell expansion and metabolism were investigated. MATERIALS AND METHODS: Oxygen transfer efficiency was assessed by computational fluid dynamics technique. Cell phenotype, apoptosis and of transporter expression were determined by flow cytometry and Western blotting. Metabolites and enzyme activities were assessed by biochemical methods. RESULTS: Dynamic cultures favoured better CIK cell expansion without impairing their phenotype and cytotoxicity, enhanced oxygen transfer efficiency. The glucose metabolism flux of cells in dynamic cultures was enhanced by upregulating surface glucose transporter 1 expression and phosphofructokinase activity. Moreover, pentose phosphate pathway (PPP) metabolic flux was enhanced through upregulating glucose-6-phosphate dehydrogenase activity. Glutaminolysis was also accelerated via boosting glutamine transporters expression, glutaminase (GLS) and glutamate dehydrogenase activities. Together with higher oxygen consumption rate and extracellular acidification rate, it was suggested that cells in dynamic cultures were in a more vigorous metabolic state for ATP production. CONCLUSION: Dynamic cultures accelerated glucose and glutamine metabolic flux to promote ATP production, elevated glucose metabolic flux through PPP to promote biosynthesis for better cell expansion. These findings may provide the basis for ex vivo CIK cell expansion process optimization.


Assuntos
Trifosfato de Adenosina/biossíntese , Células Matadoras Induzidas por Citocinas/metabolismo , Via de Pentose Fosfato , Técnicas de Cultura de Células , Proliferação de Células , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/imunologia , Sangue Fetal/citologia , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glutamina/metabolismo , Glicólise , Humanos , Imunoterapia Adotiva , Modelos Biológicos , Neoplasias/imunologia , Neoplasias/terapia , Consumo de Oxigênio
19.
Cell Mol Neurobiol ; 39(3): 415-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30771196

RESUMO

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.


Assuntos
Glioblastoma/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glucosamina/biossíntese , Glucose/metabolismo , Glucose-6-Fosfato/biossíntese , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo
20.
Eur J Pharmacol ; 850: 23-34, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30716311

RESUMO

Despite the massive efforts to develop the treatment of pancreatic cancers, no effective application exhibits satisfactory clinical outcome. Macropinocytosis plays a critical role for continuous proliferation of pancreatic ductal adenocarcinoma (PDAC). In this study, we generated a screening method and identified phellodendrine chloride (PC) as a potential macropinocytosis inhibitor. PC significantly inhibited the viability of KRAS mutant pancreatic cancer cells (PANC-1 and MiaPaCa-2) in a dose-dependent manner; however, it did not affect the wild type KRAS pancreatic cancer cells (BxPC-3). Further experiments indicated that PC reduced the growth of PANC-1 cells through inhibition of macropinocytosis and diminishing the intracellular glutamine level. Disruption of glutamine metabolism led to enhance the reactive oxygen species level and induce mitochondrial membrane potential depolarization in PANC-1 cells. PC treatment caused increased Bax and decreased Bcl-2 expression, along with the activation of cleaved caspase-3, 7, 9 and cleaved-PARP, thus induced mitochondrial apoptosis. Moreover, PC inhibited macropinocytosis in vivo and effectively reduced the growth of PANC-1 xenograft tumors. All together, we demonstrated that inhibition of macropinocytosis might be an effective strategy to treat pancreatic cancers. Thus, PC could be a potential compound with improved therapeutic efficacy in patients with pancreatic cancers.


Assuntos
Mutação , Nutrientes/metabolismo , Neoplasias Pancreáticas/patologia , Pinocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinolizinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glutamina/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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