RESUMO
B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.
Assuntos
Linfócitos B/fisiologia , Centro Germinativo/imunologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Apoptose/genética , Ligante de CD40/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/genética , Glicólise , Interleucina-4/metabolismo , Camundongos , Camundongos Knockout , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.
Assuntos
Carcinogênese/metabolismo , Carcinoma de Células Renais/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição/metabolismo , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.
Assuntos
ATP Citrato (pro-S)-Liase/fisiologia , Linfócitos B/imunologia , Glucose/metabolismo , Lipogênese/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária , ATP Citrato (pro-S)-Liase/antagonistas & inibidores , Animais , Linfócitos B/citologia , Diferenciação Celular , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Neoplasias do Colo/metabolismo , Proteína Quinase C/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Vias Biossintéticas , Transformação Celular Neoplásica , Glucose/metabolismo , Humanos , Camundongos , Serina/biossíntese , Organismos Livres de Patógenos Específicos , Estresse FisiológicoRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP(+). Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC(50) values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC(50) values. The most potent hG6PD inhibitors presented IC(50) values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC(50) ~25 µM) more strongly than that of normal MCF10-A cells (IC(50) >50 µM).
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/química , 6-Aminonicotinamida/química , 6-Aminonicotinamida/farmacologia , Linhagem Celular Tumoral , Desidroepiandrosterona/química , Desidroepiandrosterona/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Concentração Inibidora 50 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
Assuntos
Melanoma/metabolismo , Prolina/biossíntese , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Espaço Extracelular/metabolismo , Humanos , Prolina/química , Transporte Proteico , Pirrolina Carboxilato Redutases/metabolismo , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic ß cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
Assuntos
Benzimidazóis/farmacologia , Descoberta de Drogas , Fator 4 Nuclear de Hepatócito/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Insulina/genética , Regiões Promotoras Genéticas/genética , Sulfonamidas/farmacologia , Benzimidazóis/química , Relação Dose-Resposta a Droga , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , PPAR gama/agonistas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.
Assuntos
Glutamina/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Fermentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucose/química , Glucose/metabolismo , Glicólise , Humanos , Hipóxia , Ácidos Cetoglutáricos/química , Ácido Láctico/metabolismo , Melanócitos/citologia , Modelos BiológicosAssuntos
Coenzimas/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Neoplasias/metabolismo , Tiamina/metabolismo , Hipóxia Celular/fisiologia , Progressão da Doença , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana Transportadoras/genética , Neoplasias/genética , Neoplasias/patologiaRESUMO
p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Fosforilação Oxidativa , Animais , Carbono/metabolismo , Proteínas de Transporte/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Espectrometria de Massas , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/química , Metástase Neoplásica , Neoplasias/enzimologia , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Rotenona/farmacologiaRESUMO
There is a growing belief that the metabolic program of breast tumor cells could be a therapeutic target. Yet, without detailed information on central carbon metabolism in breast tumors it is impossible to know which metabolic pathways to target, and how their inhibition might influence different stages of breast tumor progression. Here we perform the first comprehensive profiling of central metabolism in the MCF10 model of mammary carcinoma, where the steps of breast tumor progression (transformation, tumorigenicity and metastasis) can all be examined in the context of the same genetic background. The metabolism of [U-(13)C]-glucose by a series of progressively more aggressive MCF10 cell lines was tracked by 2D NMR and mass spectrometry. From this analysis the flux of carbon through distinct metabolic reactions was quantified by isotopomer modeling. The results indicate widespread changes to central metabolism upon cellular transformation including increased carbon flux through the pentose phosphate pathway (PPP), the TCA cycle, as well as increased synthesis of glutamate, glutathione and fatty acids (including elongation and desaturation). The de novo synthesis of glycine increased upon transformation as well as at each subsequent step of breast tumor cell progression. Interestingly, the major metabolic shift in metastatic cells is a large increase in the de novo synthesis of proline. This work provides the first comprehensive view of changes to central metabolism as a result of breast tumor progression.
Assuntos
Neoplasias da Mama/fisiopatologia , Carbono/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucose/metabolismo , Humanos , Lactatos/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Metástase Neoplásica , Prolina/química , Fatores de TempoRESUMO
Comparative metabolic profiling of cancerous and normal cells improves our understanding of the fundamental mechanisms of tumorigenesis and opens new opportunities in target and drug discovery. Here we report a novel methodology of comparative metabolome analysis integrating the information about both metabolite pools and fluxes associated with a large number of key metabolic pathways in model cancer and normal cell lines. The data were acquired using [U-13C]glucose labeling followed by two-dimensional NMR and GC-MS techniques and analyzed using isotopomer modeling approach. Significant differences revealed between breast cancer and normal human mammary epithelial cell lines are consistent with previously reported phenomena such as upregulation of fatty acid synthesis. Additional changes established for the first time in this study expand a remarkable picture of global metabolic rewiring associated with tumorigenesis and point to new potential diagnostic and therapeutic targets.
Assuntos
Neoplasias da Mama/metabolismo , Metabolismo , Mama/metabolismo , Isótopos de Carbono , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional , Células Epiteliais/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Via de Pentose FosfatoRESUMO
The patellazoles are a family of compounds consisting of a 24-member macrolide ring with a thiazole-epoxide tail. The opening of this epoxide does not greatly affect the bioactivity of these compounds, although the cellular toxicity is generally decreased. The patellazoles are extremely cytotoxic towards HCT 116 human colon tumor cells. Treatment with nanomolar amounts of these compounds results in immediate inhibition of protein synthesis and cell cycle arrest at the G1 and S phase. HCT 116 wild-type cells underwent apoptosis after extended patellazole treatment. Although treatment with the patellazoles resulted in an increased amount of p53, the p53 null cells were still strongly affected by treatment. The inhibition of translation by patellazole treatment is linked to the inhibition of the mTOR/p70 pathway. Like the mTOR inhibitor rapamycin, the patellazoles inhibit translation through the 4EBP1 and S6 kinase pathways. However, the cytotoxicity of rapamycin and the patellazoles differs greatly in HCT 116 cells. The cellular target of the patellazoles is still unknown; the patellazole-induced inhibition of this pathway occurs either downstream or parallel to AKT.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Macrolídeos/farmacologia , Nanotecnologia , Proteínas de Neoplasias/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Tiazóis/farmacologia , Urocordados/química , Animais , Antineoplásicos/isolamento & purificação , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Relação Estrutura-AtividadeRESUMO
Lissoclinolide is a small non-nitrogenous lactone isolated from the marine ascidian Lissoclinum patella. Previous studies of lissoclinolide (isolated from a fungus and an actinomycete) have identified varying activity against both Gram-negative and Gram-positive bacteria. In this study, lissoclinolide was able to inhibit cell growth in various mammalian tumor lines at an average IC(50) of 395 nM (determined by MTT conversion after 48-h treatment). Treatment of HCT 116 human colon tumor cells with 2.4 microM lissoclinolide resulted in a strong arrest in the G(2)/M phase of the cell cycle after 24-h exposure. A daughter cell line lacking p53 showed an identical response while there was a slight increase in cytotoxicity towards a p21 null cell line. Although treatment with 2.4 microM lissoclinolide did not result in apoptosis after 48 h, this arrest was not reversible when drug wash out was attempted. The mechanism of action does not appear to involve tubulin, ubiquitin-specific isopeptidases, p53 or p21. COMPARE analysis in the NCI 60 cell line tumor panel revealed a moderate selectivity towards colon tumor cell lines.
Assuntos
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Antineoplásicos/farmacologia , 4-Butirolactona/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Concentração Inibidora 50 , Urocordados/químicaRESUMO
[structure: see text] The crude methanol extract of a marine sponge Cymbastela sp. collected in Papua New Guinea was selected for chemical investigation due to its significant cytotoxicity. Fractionation of the extract led to the isolation of jaspamide (1), hemiasterlin (2), milnamide A (3), and a new metabolite, milnamide D (4). The structure was solved by interpretation of NMR and mass spectra data. The cytotoxic and antitubulin activities of milnamide D (4) were evaluated.