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1.
J Biol Chem ; 283(14): 8961-8, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18230607

RESUMO

Overexpression of an anti-apoptotic protein cIAP1 caused by its genetic amplification was reported in certain cancers, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, cervical cancer, and lung cancer, which confers resistance to chemotherapy and radiotherapy. Here we report cIAP1 to be selectively down-regulated by a class of small molecules ((-)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-l-leucine methyl ester (ME-BS)), resulting in a sensitization of cancer cells to apoptosis. ME-BS directly interacts with the BIR3 domain of cIAP1, promotes auto-ubiquitylation dependent on its RING domain, and facilitates proteasomal degradation of cIAP1. Other IAPs such as XIAP and cIAP2 were not affected by ME-BS. These results suggest targeted destabilization of cIAP1 by small molecules as a novel method to treat cancers expressing cIAP1, which interferes with treatment. Manipulation of the intrinsic ubiquitin-ligase activity could be a novel strategy to develop small molecules for therapeutic purposes.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Neoplasias/tratamento farmacológico , Ubiquitinação/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Ubiquitina/metabolismo
2.
Genes Cells ; 12(6): 735-44, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17573774

RESUMO

Cellular FLIP (cFLIP) is a homologue of caspase-8 without protease activity that inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) inhibits ubiquitylation of beta-catenin and enhances Wnt signaling. Here we show that cFLIP-L impairs the function of the ubiquitin-proteasome system (UPS), and increases the accumulation of various short-lived proteins, such as GFP conjugated with destabilization sequence, beta-catenin and HIF1 alpha, that are subjected to rapid ubiquitylation and degradation by proteasomes. Accordingly, beta-catenin- and HIF1 alpha-mediated gene expressions are induced in the cFLIP-L-expressing cells. Exogenously expressed cFLIP-L accumulates in aggregates at the peri-nuclear region in the cells, and the cFLIP-L aggregates are refractory to solubilization. Like exogenously expressed cFLIP-L, the endogenous cFLIP in A549 lung cancer cells displays particulate distribution in the cells and more than 60% of cFLIP-L is refractory to solubilization. Down-regulation of cFLIP in A549 cells by RNA-mediated interference reduced beta-catenin- and HIF1 alpha-mediated gene expression. These results suggest that cFLIP-L is prone to aggregate and impairs UPS function, which could be involved in the pathological function of cFLIP-L expressed in certain cancer cells.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ubiquitina/química , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interferência de RNA , Transdução de Sinais , Ubiquitina/metabolismo , beta Catenina/metabolismo
3.
Biol Pharm Bull ; 29(3): 543-6, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16508163

RESUMO

The mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that regulates the thioredoxin system, one of the major systems that maintain the intracellular redox balance. We previously reported that cytosolic TrxR (TrxR1), one of three mammalian TrxR isozymes, was induced by treatment with cadmium. In the present study, to study the role of cadmium-induced TrxR1 in cellular defense, we silenced the expression of TrxR1 in HeLa cells by using small interfering RNA and examined the effect of TrxR1 silencing on the sensitivity of the cells toward cadmium. We found that the gene silencing of TrxR1 had a dual effect on cadmium-induced cell death, depending on the concentration of cadmium. The TrxR1 silencing increased the sensitivity toward a low dose (less than 10 microM) of cadmium but decreased the sensitivity toward a high dose of cadmium. These results suggested that TrxR1 might play an important role in the cellular defense system against cadmium in two ways. TrxR1 might rescue the cells from a low dose of cadmium-induced moderate injury, while it might promote the death of cells severely injured by a high dose of cadmium.


Assuntos
Intoxicação por Cádmio/enzimologia , Intoxicação por Cádmio/genética , RNA Interferente Pequeno/farmacologia , Tiorredoxina Dissulfeto Redutase/genética , Intoxicação por Arsênico/enzimologia , Intoxicação por Arsênico/genética , Intoxicação por Arsênico/patologia , Northern Blotting , Intoxicação por Cádmio/patologia , Morte Celular/efeitos dos fármacos , Inativação Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Intoxicação por Mercúrio/enzimologia , Intoxicação por Mercúrio/genética , Intoxicação por Mercúrio/patologia , Oxidantes/toxicidade , Sais de Tetrazólio , Tiazóis , Tiorredoxina Redutase 1 , Tiorredoxina Dissulfeto Redutase/biossíntese , Transfecção
4.
J Cell Physiol ; 203(3): 529-37, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15521073

RESUMO

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor alpha (TNFalpha) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium-induced TrxR1 expression. We here showed that cadmium, differently from TNFalpha, enhanced the promoter activity of the 5'-flanking region of human TrxR1 gene (nucleotides -1692 to +49). Deletion and site-directed mutation of antioxidant responsive element (ARE) (nucleotides -62 to -48) in this region abolished the response to cadmium. Overexpression of NF-E2-related factor-2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium-induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti-Nrf2 antibody precipitated ARE from the chromatin of the cadmium-treated cells. These results indicated that cadmium-induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.


Assuntos
Cádmio/farmacologia , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Genes Reguladores/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Transativadores/metabolismo , Região 5'-Flanqueadora/efeitos dos fármacos , Região 5'-Flanqueadora/genética , Animais , Arsenitos/farmacologia , Bovinos , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Reguladores/efeitos dos fármacos , Humanos , Malatos/farmacologia , Mutagênese Sítio-Dirigida/genética , Mutação/genética , Fator 2 Relacionado a NF-E2 , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Tiorredoxina Redutase 1 , Tiorredoxina Dissulfeto Redutase/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/genética , Transativadores/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
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