Assuntos
Doenças Ósseas/metabolismo , Regulação da Expressão Gênica , Inflamação/metabolismo , Animais , Proteínas da Matriz Extracelular/metabolismo , Genes Supressores de Tumor , Humanos , Sistema de Sinalização das MAP Quinases , Esclerose Múltipla/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Many factors implicated in the pathogenesis of cerebral ischaemia such as glutamate, tumour necrosis factor and interleukin-1 have also been shown to activate nuclear factor-kappaB (NF-kappaB). In the present study we have investigated NF-kappaB activity at various times following permanent focal cerebral ischaemia in rats using immunohistochemistry, western blotting and electrophoretic mobility shift assay (EMSA). Three hours following middle cerebral artery occlusion nuclear translocation of NF-kappaB was detected using immunohistochemical and western blotting techniques. This was reflected in a trend towards increased NF-kappaB binding activity (EMSA) in the ischaemic cortex compared to histologically normal tissue. In contrast however, from 6 to 48 h post-occlusion nuclear translocation and NF-kappaB binding activity was decreased in the ischaemic cortex. Decreased NF-kappaB binding activity detected in degenerating neurones, suggests that decreased NF-kappaB activity may exacerbate ischaemia induced neuronal cell death.
Assuntos
Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , NF-kappa B/metabolismo , Animais , Western Blotting , Química Encefálica/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/metabolismo , Masculino , NF-kappa B/análise , NF-kappa B/genética , Neurônios/química , Neurônios/metabolismo , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
Assuntos
Ácidos Borônicos , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/metabolismo , Complexos Multienzimáticos/metabolismo , Animais , Células Cultivadas , Quimotripsina/metabolismo , Complexo de Endopeptidases do ProteassomaRESUMO
Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.
Assuntos
Anti-Inflamatórios/farmacologia , Benzoatos/farmacologia , Piridinas/farmacologia , Receptores do Leucotrieno B4/antagonistas & inibidores , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Animais , Ligação Competitiva , Calcimicina/farmacologia , Cálcio/sangue , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Cobaias , Humanos , Ionóforos/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Leucotrieno B4/sangue , Leucotrieno B4/farmacologia , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
The predominant response of the heart to sustained increased work load is development of ventricular hypertrophy, principally as a result of hypertrophy of cardiomyocytes. The molecular mechanisms and factors involved in cardiomyocyte hypertrophy are poorly understood. Myotrophin is a novel 12-kilodalton protein recently implicated as a factor associated with and able to induce cardiac hypertrophy. Cloning of rat myotrophin revealed that this protein is identical to the functionally undefined rat, murine and chicken V-1 proteins. Although human myotrophin has been purified to homogeneity, its gene has not been characterized. In this report we describe the cloning, expression, purification and characterization of the human homolog of myotrophin/V-1 protein. Sequence analysis indicators high homology (>90%) between all species at both the nucleotide and amino acid levels, and Southern blot analysis of genomic DNA from diverse species verifies that myotrophin/V-1 is a highly conserved gene. Northern analysis indicates wide-spread expression of a single human transcript, and examination of mRNA distribution in 50 human tissues by dot blot analysis indicates ubiquitous expression with relatively high expressioon in adult and fetal heart. We verify that recombinant human myotrophin produces cardiomyocyte hypertrophy, and we demonstrate for the first time that elevated levels of myotrophin/V-1 protein mRNA are expressed in human dilated cardiomyopathic hearts. We report the novel findings that myotrophin expression is elevated in ischemic hearts, and that myotrophin expression correlates positively with ventricular mass in a hypoxic rat model of induced right ventricular hypertrophy.
Assuntos
Cardiomegalia/genética , DNA Complementar/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Animais , Sequência de Bases , Cardiomegalia/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Primers do DNA/genética , Modelos Animais de Doenças , Expressão Gênica , Substâncias de Crescimento/farmacologia , Humanos , Hipertrofia Ventricular Direita/genética , Masculino , Camundongos , Dados de Sequência Molecular , Miocárdio/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.
Assuntos
Antineoplásicos/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/farmacologia , Células HT29/efeitos dos fármacos , Humanos , Ligantes , Teste de Cultura Mista de Linfócitos , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Ligação Proteica , Membro 14 de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Distribuição Tecidual , Fator de Transcrição AP-1/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Activation of the transcription factor NF-kappaB is known to be important in the regulated expression of a large number of pro-inflammatory genes including interleukin-8 (IL-8). Previously, we showed that the protein kinase inhibitor staurosporine potentiates IL-1-stimulated IL-8 production in human keratinocytes. Moreover, recent studies by other investigators demonstrated that staurosporine treatment alone results in a concentration-dependent increase in IL-8 mRNA and protein production. Therefore, in order to understand the mechanism underlying this observation, the effect of staurosporine on the activation of NF-kappaB was investigated. Electrophoretic mobility shift assays using an oligonucleotide containing the NF-kappaB consensus motif demonstrated that staurosporine treatment resulted in the activation of NF-kappaB by 15 min post-treatment. The ability of staurosporine to activate NF-kappaB was investigated further, using luciferase reporters under the control of the HIV-LTR or IL-8 core promoter transfected into human U937 cells. Stimulation with staurosporine resulted in a concentration-dependent induction of luciferase activity. In contrast, the very selective protein kinase C inhibitor 3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2-a]indol -10-yl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione hydrochloride (Ro32-0432) did not stimulate the activation of NF-kappaB, as measured in the luciferase reporter assay. The mechanism underlying NF-kappaB activation does not appear to involve the classical activation pathways in that staurosporine does not induce the disappearance of IkappaB family members. In conclusion, staurosporine appears to stimulate the activation of NF-kappaB in at least two cell types, and this effect appears to be independent of protein kinase C.
Assuntos
Queratinócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Estaurosporina/farmacologia , Fatores de Transcrição , Sequência de Bases , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Genes Reporter , Humanos , Indóis/farmacologia , Interleucina-8/biossíntese , Interleucina-8/genética , Queratinócitos/metabolismo , Luciferases/genética , NF-kappa B/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pirróis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição RelBRESUMO
OBJECTIVE AND DESIGN: The ability of azaspiranes to modulate the acute inflammatory response in models of skin inflammation was examined. MATERIAL: The in vivo experiments involved the use of 5-6 age-matched male Balb/c inbred mice (22-25 g) per treatment group and a control group of 8-10 animals. In vitro mechanistic studies used RBL-1 and U937 cells lines and freshly isolated human monocytes. TREATMENT: Arachidonic acid (AA) (2 mg/20 microl in acetone) or PMA (phorbol myristate acetate) (4 microg/20 microl) were applied topically. SK&F 106615 and SK&F 106610 were administered topically either dissolved in acetone or dimethylacetamide just after the application of the irritant. Isolated cells were treated with the compounds dissolved in DMSO. METHODS: The thickness and influx of neutrophils into the treated ears was measured as was the effects of the azaspiranes on 5-lipoxygenase activity, cyclooxygenase activity, prostaglandin and leukotriene synthesis, and the activation of the transcription factor NF-kappaB. RESULTS: SK&F 106615 and SK&F 106610 significantly reduced inflammation in the AA- and PMA-induced inflammation models (p < 0.05) with ED50's of 179 and 120 mg/ear for edema and myeloperoxidase, respectively. The compounds did not inhibit eicosanoid biosynthesis, have a direct effect on 5-lipoxygenase or cyclooxygenase enzymes, or inhibit NF-kappaB. CONCLUSIONS: The potent anti-inflammatory and immunomodulatory activities of the azaspiranes observed in these and other studies appear to be mediated by a novel mechanism.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Dermatite de Contato/tratamento farmacológico , Edema/tratamento farmacológico , Imunossupressores/farmacologia , Monócitos/efeitos dos fármacos , Piperidinas/farmacologia , Compostos de Espiro/farmacologia , Administração Tópica , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico , Linhagem Celular/efeitos dos fármacos , Ciclo-Oxigenase 1 , Dermatite de Contato/etiologia , Dinoprostona/biossíntese , Orelha , Edema/etiologia , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Isoenzimas/metabolismo , Leucotrieno B4/biossíntese , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Peroxidase/metabolismo , Piperidinas/administração & dosagem , Piperidinas/uso terapêutico , Prostaglandina-Endoperóxido Sintases/metabolismo , Compostos de Espiro/administração & dosagem , Compostos de Espiro/uso terapêutico , Acetato de TetradecanoilforbolRESUMO
Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM). Alternatively, both an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, were without affect. Direct action of hymenialdisine on IL-1-induced NFkappaB activation was demonstrated by a significant reduction (approximately 80%) in NFkappaB binding to the classical kappaB consensus motif (as assessed by electrophoretic mobility shift assay) and inhibition of stimulated p65 migration from the cytosol of treated cells (as assessed by Western analysis). Consistent with the role of NFkappaB in the transcriptional regulation of COX II and 85-kDa PLA2, hymenialdisine-treated RSF did not transcribe the respective mRNAs in response to IL-1. This led to reductions in their respective protein levels and subsequent reductions in the ability to produce PGE2. Specificity of action is suggested as IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFkappaB-regulated event, was also inhibited by hymenialdisine, whereas IL-1-induced production of vascular endothelial growth factor, a non-NFkappaB-regulated gene, was not affected by exposure to hymenialdisine. Taken together, hymenialdisine inhibits IL-1-stimulated-RSF PGE2 formation acting predominately through modulation of NFkappaB activation and offers an interesting novel tool to evaluate the role of NFkappaB in inflammatory disease.
Assuntos
Artrite Reumatoide/metabolismo , Azepinas/farmacologia , Dinoprostona/biossíntese , Interleucina-1/farmacologia , NF-kappa B/antagonistas & inibidores , Pirróis/farmacologia , Membrana Sinovial/metabolismo , Adulto , Células Cultivadas , Fatores de Crescimento Endotelial/biossíntese , Humanos , Interleucina-8/biossíntese , Linfocinas/biossíntese , Fosfolipases A/metabolismo , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The nuclear factor-kappaB (NF-kappaB) family of transcription factors have been implicated in the inducible expression of genes involved in inflammatory and immune responses. As such, a specific inhibitor of NF-kappaB would be a useful therapeutic agent in a variety of inflammatory disorders. The marine natural product hymenialdisine was evaluated as an inhibitor of NF-kappaB in U937 cells. U937 cells were transfected with either a luciferase reporter plasmid containing the human immunodeficiency virus long terminal repeat or the interleukin-8 (IL-8) core promoter, both of which are activated by NF-kappaB. Hymenialdisine caused a concentration-dependent decrease in luciferase production from both reporters when the cells were stimulated with tumor necrosis factor-alpha, lipopolysaccharide or phorbol myristate acetate. An electrophoretic mobility shift assay confirmed its activity by inhibiting DNA binding of NF-kappaB. Hymenialdisine was shown to be a selective inhibitor of NF-kappaB in that it had no effect on the binding of other transcription factors to their DNA concensus motifs; these included activator protein-1, CCAAT/enhancer binding protein and Sp1. Functional studies showed hymenialdisine to be an inhibitor of IL-8 production and IL-8 mRNA formation in the U937 cell. Investigation into the mechanism of action of hymenialdisine showed that it was not due to inhibition of protein kinase C because the selective protein kinase C inhibitor RO 32-0432 was inactive against tumor necrosis factor-alpha-stimulated luciferase and IL-8 production. The compound also had no effect on IkappaB alpha or IkappaB beta phosphorylation and degradation. Thus, hymenialdisine is a potent inhibitor of NF-kappaB and IL-8 production in U937 cells.
Assuntos
Azepinas/farmacologia , Interleucina-8/biossíntese , NF-kappa B/antagonistas & inibidores , Pirróis/farmacologia , Fatores de Transcrição , Humanos , Interleucina-8/genética , Luciferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/análise , Fator de Transcrição RelB , Células Tumorais CultivadasRESUMO
A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.
Assuntos
Benzofuranos/farmacologia , Inibidores de Lipoxigenase , Inibidores de Lipoxigenase/farmacologia , Ureia/análogos & derivados , Animais , Benzofuranos/química , Benzofuranos/farmacocinética , Cromatografia Líquida de Alta Pressão , Cães , Humanos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacocinética , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Estereoisomerismo , Ureia/química , Ureia/farmacocinética , Ureia/farmacologiaRESUMO
Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.
Assuntos
Artrite Reumatoide/metabolismo , Dinoprostona/biossíntese , Interleucina-1/farmacologia , NF-kappa B/metabolismo , Membrana Sinovial/efeitos dos fármacos , Northern Blotting , Ciclo-Oxigenase 2 , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Interleucina-8/biossíntese , Isoenzimas/metabolismo , Proteínas de Membrana , NF-kappa B/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Fator de Transcrição RelA , Regulação para Cima/efeitos dos fármacosRESUMO
A recombinant baculovirus was constructed to express a cDNA encoding RelA (p65), a member of the NF-kappa B/Rel family of proteins. Infection of Spodoptera frugiderda insect cells with the recombinant baculovirus resulted in the production of the biologically active protein as measured by immunoblotting using RelA-specific antisera and by electrophoretic mobility shift assays. The recombinant protein bound specifically to an oligonucleotide containing the NF-kappa B consensus motif but not to that containing the unrelated Oct-1 consensus motif. Thus insect cell-derived RelA possess properties similar to the native protein and may be used in physical, biochemical, and pharmacological studies.
Assuntos
Vetores Genéticos/genética , NF-kappa B/biossíntese , Nucleopoliedrovírus/genética , Proteínas Recombinantes de Fusão/biossíntese , Spodoptera/citologia , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , DNA/metabolismo , DNA Complementar/genética , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/metabolismo , Fator de Transcrição RelARESUMO
ET-18-O-CH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) is an antiproliferative agent, blocking the growth of cancer cells both in vitro and in vivo. However, there is controversy regarding the mechanism leading to its antiproliferative effects. CoA-independent transacylase (CoA-IT) is an enzyme that remodels arachidonate between specific phospholipid donor and acceptor molecules in a variety of mammalian cells. ET-18-O-CH3 was found to be a potent inhibitor of CoA-IT (IC50, 0.5 microM), and kinetic analysis revealed that its inhibition was competitive with the lyso-phospholipid substrate. The goal of the current study was to explore the connection between inhibition of CoA-IT and antiproliferative effects using several structurally distinct inhibitors of CoA-IT. ET-18-O-CH3 and other inhibitors of CoA-IT were found to inhibit cell proliferation and thymidine incorporation into the DNA, as well as to induce apoptosis in human HL-60 monocytic leukemia cells. The mechanism of apoptosis induced by ET-18-O-CH3 appeared to be different from that induced by tumor necrosis factor; the former failed to activate NF-kappa B, whereas tumor necrosis factor did. Closer examination of the pharmacology of apoptosis in this model revealed that compounds that were structurally related to CoA-IT inhibitors, but lacked CoA-IT inhibitory activity, also failed to induce apoptosis. In addition, compounds that inhibited other enzymes that participate in arachidonic acid metabolism, cyclooxygenase, 5-lipoxygenase and phospholipase A2, did not induce apoptosis. Taken together, these results demonstrate that inhibition of CoA-IT can be linked to blockade of proliferation and the induction of apoptosis in HL-60 cells.
Assuntos
Aciltransferases/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Coenzima A/fisiologia , Inibidores Enzimáticos/farmacologia , Éteres Fosfolipídicos/farmacologia , Ácido Araquidônico/metabolismo , Divisão Celular/efeitos dos fármacos , Células HL-60 , Humanos , Metabolismo dos Lipídeos , NF-kappa B/metabolismo , Fosfolipídeos/metabolismoRESUMO
A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Inibidores de Ciclo-Oxigenase , Citocinas/antagonistas & inibidores , Imidazóis/síntese química , Inibidores de Lipoxigenase , Morfolinas/síntese química , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Artrite/tratamento farmacológico , Densidade Óssea/efeitos dos fármacos , Imidazóis/metabolismo , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Morfolinas/metabolismo , Morfolinas/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas Quinases/metabolismo , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The cellular origin of leukotriene B4 (LTB4), a potent pro-inflammatory molecule present in psoriatic lesions, has yet to be determined. In the present study, cultured human keratinocytes were evaluated for their ability to produce LTB4. Keratinocytes stimulated under a variety of conditions did not produce detectable amounts of LTB4, as measured by enzyme immunoassay and liquid chromatographic techniques. Prostaglandin E2 and 15-hydroxyeicosatetraenoic acid were the only eicosanoids detected. The capacity of keratinocytes to synthesize 5-lipoxygenase (5-LO) products, or lack thereof, was further evaluated by preparing subcellular fractions and examining them for the presence of 5-LO activity and the proteins responsible for LTB4 production. Using Western blot analysis, we detected no bands that migrated with the 78-kDa 5-LO enzyme. Subcellular fractions were also examined for the presence of the 5-LO-activating protein (FLAP). This protein, which is essential to 5-LO activity, could not be detected in any keratinocyte preparation examined. Consistent with the absence of proteins, the mRNAs for 5-LO and FLAP were undetectable by reverse transcriptase polymerase chain reactions analysis. These results demonstrate that human keratinocytes lack the crucial proteins necessary for LTB4 production.
Assuntos
Queratinócitos/metabolismo , Leucotrieno B4/biossíntese , Proteínas Ativadoras de 5-Lipoxigenase , Araquidonato 5-Lipoxigenase/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Eicosanoides/biossíntese , Humanos , Leucotrieno B4/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.5 microM) but exhibits a 40-fold weaker inhibition of 85 kDa PLA2 (IC50 = 20 microM) using [3H]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA2 was confirmed both by observing the pH dependence of its IC50 and by demonstrating linear inhibition in a "scooting" kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating the effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA2 activity in acid extracted-intact human neutrophil homogenate (IC50 = 4.7 microM). SB 203347 inhibited A23187-induced intact human neutrophil AA mass release in a concentration-dependent manner (IC50 = 1 microM), which coincided with reductions in the biosynthesis of platelet-activating factor (IC50 = 1.5 microM) and leukotriene B4 (IC50 = 2.3 microM). Finally, SB 203347 prolonged survival in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA2 in the formation of AA-derived proinflammatory lipid mediator.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ácido Araquidônico/metabolismo , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfolipases A/antagonistas & inibidores , Choque Séptico/metabolismo , Sulfonamidas/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Sistema Livre de Células , Modelos Animais de Doenças , Humanos , Leucotrieno B4/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosfolipases A2 , Fator de Ativação de Plaquetas/biossíntese , Choque Séptico/induzido quimicamente , Choque Séptico/mortalidade , SobreviventesRESUMO
The enzyme CoA-independent transacylase (CoA-IT) has been proposed to mediate the movement of arachidonate between specific phospholipid subclasses, and we have shown that two inhibitors of CoA-IT (SK&F 98625 and SK&F 45905) block this movement. In this report, we use these inhibitors to further characterize the role of CoA-IT in the production of lipid mediators. SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-1-yl)heptane- phosphonate) and SK&F 45905 [2(-)[3-(4-chloro-3-trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5-dichlorobenzenesulfonic acid) inhibited CoA-IT activity (IC50 values of 9 microM and 6 microM, respectively). Neither compound had any effect on cyclooxygenase, 14-kDa PLA2 or acetyltransferase activities at concentrations below 20 microM. However, SK&F 45905 inhibited 85-kDa PLA2 activity (IC50 = 3 microM), and both compounds inhibited 5-lipoxygenase activity (IC50 values of 2-4 microM). In ionophore-stimulated neurotrophils, SK&F 98625 and SK&F 45905 blocked the liberation of arachidonic acid from phospholipids, which suggests that the movement of arachidonate into specific phospholipid pools is a prerequisite for release. Both compounds also inhibited the production of platelet-activating factor in ionophore-stimulated neutrophils and antigen-stimulated mast cells. This inhibition of platelet-activating factor and arachidonic acid release was not mimicked by an inhibitor of 5-lipoxygenase, zileuton, which indicates that the primary mode of action of SK&F 98625 and SK&F 45905 is via inhibition of CoA-IT. SK&F 98625 and SK&F 45905 were able to decrease prostaglandin production in several inflammatory cells and to block signs of inflammation in ears of phorbol ester-challenged mice. Taken together, these results show that blockade of CoA-IT, which leads to inhibition of arachidonate remodelling between phospholipids, results in the attenuation of platelet-activating factor production, arachidonic acid release and the formation of eicosanoid products.
Assuntos
Aciltransferases/antagonistas & inibidores , Benzenossulfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Mediadores da Inflamação/metabolismo , Compostos Organofosforados/farmacologia , Fosfolipídeos/biossíntese , Ureia/análogos & derivados , Animais , Células Cultivadas , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Ureia/farmacologiaRESUMO
The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays. In addition, the potential therapeutic value of SB 202235 was assessed in preclinical models of allergic asthma. The activity of the 5-LO enzyme isolated from rat basophilic leukemia-1 cells was inhibited by SB 202235 in a concentration-dependent manner with an IC50 value of 1.9 microM. Consistent with its ability to inhibit 5-LO, SB 202235 inhibited the production of leukotriene B4 by human monocytes and in human whole blood (IC50 values of 1.5 microM and 1.1 microM, respectively). The selectivity of SB 202235 was confirmed by its lack of effect against several other enzymes and receptors. SB 202235 potently and effectively inhibited the contraction produced by a single concentration of ovalbumin in guinea pig trachea (IC50 = 20 microM) and of anti-IgE in human bronchus (IC50 = 2 microM). SB 202235 (3-30 microM) also inhibited the contraction of guinea pig trachea in response to increasing concentration of ovalbumin. When administered orally (30 mg/kg) to conscious guinea pigs, SB 202235 attenuated antigen-induced broncho-constriction and the subsequent eosinophil influx.(ABSTRACT TRUNCATED AT 250 WORDS)