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1.
Nat Commun ; 6: 6175, 2015 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-25629724

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with <5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK1/2 and higher MMP activities. For biological function, ADAM8 requires multimerization and associates with ß1 integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerization. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a Kras(G12D)-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.


Assuntos
Proteínas ADAM/metabolismo , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas ADAM/antagonistas & inibidores , Animais , Western Blotting , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Integrina beta1/metabolismo , Estimativa de Kaplan-Meier , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Transdução de Sinais/efeitos dos fármacos
2.
Mol Nutr Food Res ; 58(8): 1658-66, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25044634

RESUMO

SCOPE: The potential benefit of vitamin K as a therapeutic in osteoporosis is controversial and the vitamin K regimen being used clinically (45 mg/day) employs doses that are many times higher than required to ensure maximal gamma-carboxylation of the vitamin K-dependent bone proteins. We therefore tested the hypothesis that vitamin K catabolites, 5-carbon (CAN5C) and 7-carbon carboxylic acid (CAN7C) aliphatic side-chain derivatives of the naphthoquinone moiety exert an osteotrophic role consistent with the treatment of osteoporosis. METHODS AND RESULTS: Osteoblast-like MG63 cell cultures were challenged with lipopolysaccharide and the levels of interleukin-6, an osteoclastogenic cytokine, measured with and without catabolites; low concentrations of CAN7C significantly inhibited interleukin-6 release, but CAN5C did not. In models of bone loss induced by ovariectomy or sciatic neurectomy in C57BL/6 mice, we found that the rarer CAN7C catabolite markedly restricted ovariectomy-induced bone loss and possibly limited sciatic neurectomy-induced bone loss. CAN7C activity depends on a free carboxylic acid and its particular side-chain structure. CONCLUSION: These in vivo data indicate for the first time that the clinical utility of vitamin K for osteoporosis may reside in an unusual catabolite.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Ácidos Carboxílicos/uso terapêutico , Modelos Animais de Doenças , Naftoquinonas/uso terapêutico , Osteoporose Pós-Menopausa/tratamento farmacológico , Vitamina K/análogos & derivados , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Ácidos Carboxílicos/administração & dosagem , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Denervação/efeitos adversos , Feminino , Humanos , Injeções Intraperitoneais , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Estrutura Molecular , Naftoquinonas/administração & dosagem , Naftoquinonas/química , Naftoquinonas/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ovariectomia/efeitos adversos , Distribuição Aleatória , Nervo Isquiático/cirurgia , Relação Estrutura-Atividade , Vitamina K/administração & dosagem , Vitamina K/farmacologia , Vitamina K/uso terapêutico
3.
J Hepatol ; 53(1): 91-7, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20451280

RESUMO

BACKGROUND & AIMS: Liver failure is associated with progressive cytotoxic brain oedema (astrocyte swelling), which underlies hepatic encephalopathy (HE). Ammonia and superimposed inflammation are key synergistic factors in HE, but the mechanism(s) involved remain unknown. We aimed to determine whether aquaporin-4 (AQP4), an astrocyte endfeet bi-directional water channel, is associated with the brain oedema of HE. METHOD: Rats (n=60) received sham-operation (sham), 5 days hyperammonaemia-inducing diet (HD), galactosamine (GALN) induced acute liver failure (ALF), 4 weeks bile duct-ligation (BDL) induced cirrhosis, or caecal ligation and puncture (CLP), a 24h model of bacterial peritonitis. Rats from every group (except CLP) were randomised to receive intraperitoneal injections of lipopolysaccharide (LPS; 1mg/kg) or saline, prior to termination 3h later. Brain water, AQP4 protein expression (western blot) and AQP4 localisation by immunogold electron microscopy were investigated. RESULTS: Significant hyperammonaemia was observed in saline-injected BDL (p<0.05), GALN (p<0.01), and HD (p<0.01), compared to sham rats. LPS injection did not affect arterial ammonia or plasma biochemistry in any of the treatment groups. Increased brain water was observed in saline-injected GALN (p<0.05), HD (p<0.01), and CLP (p<0.001) compared to sham rats. Brain water was numerically increased in BDL rats, but this failed to reach significance (p=0.09). LPS treatment further increased oedema significantly in all treatment groups (p<0.05, respectively). AQP4 expression was significantly increased in saline-injected BDL (p<0.05), but not other treatment groups, compared to sham rats. Membrane polarisation was maintained in BDL rats. CONCLUSION: The results suggest that AQP4 is not directly associated with the development of brain oedema in liver failure, hyperammonaemia, or sepsis. In cirrhosis, there is increased AQP4 protein expression, but membrane polarisation, is maintained, possibly in a compensatory attempt to limit severe brain oedema.


Assuntos
Aquaporina 4/metabolismo , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Falência Hepática/complicações , Falência Hepática/metabolismo , Animais , Western Blotting , Edema Encefálico/patologia , Modelos Animais de Doenças , Lobo Frontal/irrigação sanguínea , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Hiperamonemia/complicações , Hiperamonemia/metabolismo , Masculino , Microscopia Imunoeletrônica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sepse/complicações , Sepse/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
J Clin Invest ; 119(10): 3011-23, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19741298

RESUMO

Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-alpha is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-alpha was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-alpha-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25- cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-alpha signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-kappaB system, TGF-beta1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-alpha in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.


Assuntos
Interleucina-17/imunologia , Neoplasias Ovarianas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Quimera/genética , Quimera/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Infliximab , Interleucina-17/genética , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/genética
5.
J Pathol ; 219(2): 143-52, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19662665

RESUMO

A large number of variables have been identified which appear to influence macrophage phenotype within the tumour microenvironment. These include reciprocal chemical and physical interactions with tumour cells and with non-malignant cells of the tumour microenvironment, tissue oxygen tension, and the origin and prior experience of the particular macrophage population. In this review we outline the key evidence for these influences and consider how macrophage phenotype is acquired and the relevance of the TLR-NF-kappaB pathway.


Assuntos
Macrófagos/imunologia , NF-kappa B/imunologia , Neoplasias/imunologia , Receptores Toll-Like/imunologia , Comunicação Celular/imunologia , Hipóxia Celular/imunologia , Trato Gastrointestinal/imunologia , Humanos , Fígado/imunologia , Ativação de Macrófagos/imunologia
6.
J Lipid Res ; 46(5): 1053-60, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15722567

RESUMO

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eletroquímica/métodos , Vitamina K/urina , Calibragem , Humanos , Hidrólise , Metilação , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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