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1.
BMC Cancer ; 9: 190, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19534809

RESUMO

BACKGROUND: The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells. METHODS: Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay. RESULTS: DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure. CONCLUSION: DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.


Assuntos
Ciclo-Oxigenase 2/biossíntese , Ácido Desoxicólico/farmacologia , Neoplasias Esofágicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Transcrição AP-1/biossíntese , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Esôfago de Barrett/enzimologia , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colágeno Tipo XI/metabolismo , DNA de Neoplasias/metabolismo , Indução Enzimática/efeitos dos fármacos , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/patologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
J Immunol ; 175(12): 7811-8, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16339516

RESUMO

T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C beta and delta isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the beta(1) integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Quimiotaxia de Leucócito , Proteínas do Citoesqueleto/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Humanos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Microtúbulos/metabolismo , Complexos Multiproteicos/fisiologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/fisiologia
3.
Eur J Cancer ; 41(14): 2160-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16122920

RESUMO

Deoxycholic acid (DCA) has been implicated in colonic carcinogenesis through effects mediated by protein kinase C (PKC) activation. By contrast, ursodeoxycholic acid (UDCA) is reported to reduce colon cancer incidence in ulcerative colitis. The aim of this study was to investigate whether UDCA modulated DCA-induced PKC isoenzyme translocation to its site of activity. HCT116 cells were treated with DCA, UDCA alone or pre-treated with UDCA followed by DCA. Analysis of translocation of endogenous and enhanced green fluorescent protein (EGFP) constructs of PKC isoenzymes was performed. Both DCA and phorbol myristate acetate (PMA) but not UDCA caused translocation of endogenous PKC alpha, epsilon and delta and transfected PKC beta1-, epsilon- and delta-EGFP from cytosol to plasma membrane, reflecting isoenzyme activation. Furthermore, UDCA inhibited DCA-induced translocation of PKC isoenzymes. Inhibition of DCA-induced PKC translocation may be a mechanism for UDCA-mediated chemoprevention of colon carcinogenesis.


Assuntos
Anticarcinógenos/farmacologia , Neoplasias do Colo/enzimologia , Ácido Desoxicólico/farmacologia , Proteína Quinase C/metabolismo , Ácido Ursodesoxicólico/farmacologia , Western Blotting , Linhagem Celular Tumoral , Forma Celular , Neoplasias do Colo/prevenção & controle , Proteínas de Fluorescência Verde/metabolismo , Humanos , Acetato de Tetradecanoilforbol/farmacologia , Transfecção/métodos
4.
Int J Cancer ; 114(6): 887-95, 2005 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-15645414

RESUMO

Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta. DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 microM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered. Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.


Assuntos
Butiratos/farmacologia , Colo/citologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/prevenção & controle , Ácido Desoxicólico/farmacologia , Detergentes/farmacologia , Proteína Quinase C/farmacologia , Proteína Quinase C/farmacocinética , Técnicas de Cultura de Células , Colo/patologia , Citoesqueleto , Ativação Enzimática , Epitélio , Humanos , Isobutiratos , Isoenzimas
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