RESUMO
Pancreatic cancer (PC) is an aggressive malady with proclivity for early metastasis. Overexpression of toll-like receptor 4 (TLR4) in pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, correlates to tumor size, lymph node involvement, venous invasion and pathological stage. Lipopolysaccharides (LPS) are natural TLR4 ligands that have been shown to increase the invasive ability of PC cells. However, rapid inactivation of circulating LPS and low systemic absorption of inhaled LPS from the bronchoalveolar compartment make other agonists such as saturated fatty acids more suitable to be considered for TLR4-related cell invasiveness. Interestingly, PC risk was strongly associated to intake of saturated fat from animal food sources, in particular to consumption of saturated palmitic acid (PA). In the present study, we investigated the influence of PA on the invasive capacity of human PC cells AsPC-1. Using specific inhibitors, we found that PA stimulation of these tumor cells induced a TLR4-mediated cell invasion. Our results also indicate that the signaling events downstream of TLR4 involved generation of reactive oxygen species, activation of nuclear factor-kappa beta, and secretion and activation of matrix metalloproteinase 9 (MMP-9). Furthermore, PA stimulation decreased the levels of the micro RNA 29c (miR-29c). Of note, while inhibition of miR-29c increased MMP-9 mRNA levels, MMP-9 secretion and activation, and invasiveness, miR-29c mimic abrogated all these PA-stimulated effects. These results strongly suggest that miR-29c could be an attractive potential pharmacological agent for antitumoral therapy in PC.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica , Ácido Palmítico/farmacologia , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismoRESUMO
Pancreatitis has been suspected for a long time to have an autodigestive genesis. The main events occurring in the pancreatic acinar cell that initiate acute pancreatitis include inhibition of zymogen secretion and intracellular activation of proteases. Chymotrypsin C (CTRC) is a protective protease that limits trypsin and trypsinogen proteolytic activity. Hereditary pancreatitis-associated CTRC mutants such as p.A73T and p.G61R precipitate within the endoplasmic reticulum (ER) causing ER stress. We found that expression of these mutants reduces amylase secretion from carbachol-stimulated rat pancreatic acinar cells AR42J and isolated mice pancreatic acini. Furthermore, this expression also reduces the levels of acetylated tubulin by increasing both the levels and phosphorylation of the deacetylase SIRT2. Remarkably, inhibition of SIRT2 not only greatly recovers tubulin acetylation, but also amylase secretion in pancreatic acinar cells and isolated acini. However, SIRT2 inhibition does not rescue secretion of the CTRC mutants. These results strongly suggest that CTRC variants associated to ER stress inhibit secretagogue-stimulated pancreatic zymogen secretion by altering microtubule stability. Of note, the extent of this inhibition correlates with the degree of ER stress exhibited by the particular CTRC variant.
Assuntos
Quimotripsina/genética , Estresse do Retículo Endoplasmático , Precursores Enzimáticos/metabolismo , Pâncreas/metabolismo , Sirtuína 2/metabolismo , Acetilação , Células Acinares/metabolismo , Amilases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Quimotripsina/metabolismo , Humanos , Camundongos , Microtúbulos/metabolismo , Pâncreas/citologia , Fosforilação , Mutação Puntual , Ratos , Sirtuína 2/genética , Tubulina (Proteína)/metabolismo , Regulação para CimaRESUMO
AIM: To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer. METHODS: Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 µg/kg per hour) cerulein stimulation on chronically stressed rats. RESULTS: In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases' activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases'activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues' ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats. CONCLUSION: In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.
Assuntos
Pâncreas Exócrino/imunologia , Pancreatite/psicologia , Estresse Psicológico/complicações , Fator de Necrose Tumoral alfa/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Amilases/metabolismo , Animais , Anticorpos/farmacologia , Sinalização do Cálcio , Caspases/metabolismo , Ceruletídeo , Colecistocinina/metabolismo , Doença Crônica , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Lesão Pulmonar/etiologia , Lesão Pulmonar/imunologia , Lesão Pulmonar/psicologia , Masculino , NF-kappa B/metabolismo , Necrose , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Pancreatite/induzido quimicamente , Pancreatite/imunologia , Pancreatite/metabolismo , Pancreatite/patologia , Pancreatite/prevenção & controle , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Restrição Física , Índice de Gravidade de Doença , Técnicas de Cultura de Tecidos , Tripsina/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Pancreatitis is a disease with high morbidity and mortality. In vitro experiments on pancreatic acini showed that supramaximal but not submaximal cholecystokinin (CCK) stimulation induces effects in the acinar cell that can be correlated with acinar morphological changes observed in the in vivo experimental model of cerulein-induced pancreatitis. The GTPase Rac1 was previously reported to be involved in CCK-evoked amylase release from pancreatic acinar cells. Here, we demonstrate that pretreatment with the Rac1 inhibitor NSC23766 (100 microM, 2 h) effectively blocked Rac1 translocation and activation in CCK-stimulated pancreatic acini, without affecting activation of its closely related GTPase, RhoA. This specific Rac1 inhibition decreased supramaximal (10 nM) CCK-stimulated acinar amylase release (27.% reduction), which seems to be connected to the reduction observed in serum amylase (46.6% reduction) and lipase levels (46.1% reduction) from cerulein-treated mice receiving NSC23766 (100 nmol h(-1)). The lack of Rac1 activation also reduced formation of reactive oxygen species (ROS; 20.8% reduction) and lactate dehydrogenase release (LDH; 24.3% reduction), but did not alter calcium signaling or trypsinogen activation in 10 nM CCK-stimulated acini. In the in vivo model, the cerulein-treated mice receiving NSC23766 also presented a decrease in both pancreatic and lung histopathological scores (reduction in oedema, 32.4 and 66.4%; haemorrhage, 48.3 and 60.2%; and leukocyte infiltrate, 53.5 and 43.6%, respectively; reduction in pancreatic necrosis, 65.6%) and inflammatory parameters [reduction in myeloperoxidase, 52.2 and 38.9%; nuclear factor kappaB (p65), 61.3 and 48.6%; and nuclear factor kappaB (p50), 46.9 and 44.9%, respectively], together with lower serum levels for inflammatory (TNF-alpha, 40.4% reduction) and cellular damage metabolites (LDH, 52.7% reduction). Collectively, these results suggest that pharmacological Rac1 inhibition ameliorates the severity of pancreatitis and pancreatitis-associated lung injury through the reduction of pancreatic acinar damage induced by pathological digestive enzyme secretion and overproduction of ROS.