RESUMEN
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.
Asunto(s)
Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Invasividad Neoplásica , Ácido Palmítico/farmacología , Neoplasias Pancreáticas/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Línea Celular Tumoral , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Lipopolysaccharides (LPS) are known to stimulate the production of mucin 5AC (MUC5AC) via epidermal growth factor receptor (EGFR) in human airway cells. Noteworthy, we have previously demonstrated that EGFR/Rac1/reactive oxygen species (ROS)/matrix metalloproteinase 9 (MMP-9) is a key signaling cascade regulating MUC5AC production in airway cells challenged with LPS. Various reports have shown an inverse association between the intake of polyunsaturated fatty acids (PUFA) of the n-3 (omega-3) family or fish consumption and COPD. In the present study, we investigated the influence of docosahexaenoic acid (DHA), one of the most important omega-3 PUFA contained in fish oil, on the production of MUC5AC in LPS-challenged human airway cells NCI--H292. Our results indicate that DHA is capable of counteracting MUC5AC overproduction in LPS-stimulated cells by abrogating both EGFR phosphorylation and its downstream signaling pathway. This signaling pathway not only includes Rac1, ROS and MMP-9, but also NF-κB, since we have found that ROS require NF-κB activity to induce MMP-9 secretion and activation.
Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Receptores ErbB/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Mucina 5AC/biosíntesis , Transducción de Señal/efectos de los fármacos , Línea Celular , Humanos , Lipopolisacáridos/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Quimotripsina/genética , Estrés del Retículo Endoplásmico , Precursores Enzimáticos/metabolismo , Páncreas/metabolismo , Sirtuina 2/metabolismo , Acetilación , Células Acinares/metabolismo , Amilasas/metabolismo , Animales , Línea Celular , Células Cultivadas , Quimotripsina/metabolismo , Humanos , Ratones , Microtúbulos/metabolismo , Páncreas/citología , Fosforilación , Mutación Puntual , Ratas , Sirtuina 2/genética , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Páncreas Exocrino/inmunología , Pancreatitis/psicología , Estrés Psicológico/complicaciones , Factor de Necrosis Tumoral alfa/metabolismo , Actinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Amilasas/metabolismo , Animales , Anticuerpos/farmacología , Señalización del Calcio , Caspasas/metabolismo , Ceruletida , Colecistoquinina/metabolismo , Enfermedad Crónica , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Lesión Pulmonar/etiología , Lesión Pulmonar/inmunología , Lesión Pulmonar/psicología , Masculino , FN-kappa B/metabolismo , Necrosis , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/metabolismo , Páncreas Exocrino/patología , Pancreatitis/inducido químicamente , Pancreatitis/inmunología , Pancreatitis/metabolismo , Pancreatitis/patología , Pancreatitis/prevención & control , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Transporte de Proteínas , Ratas , Ratas Wistar , Restricción Física , Índice de Severidad de la Enfermedad , Técnicas de Cultivo de Tejidos , Tripsina/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients' serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.
Asunto(s)
Lipopolisacáridos/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Mucina 5AC/metabolismo , Mucosa Respiratoria/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Línea Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Moco/metabolismo , NADP/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Mucosa Respiratoria/inmunología , Transducción de SeñalRESUMEN
Cancer metastasis involves tumor cells invading the surrounding tissue. Remodeling of tissue barriers depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects. Epidermal growth factor (EGF) has been shown to regulate tumor cell invasion through activation of matrix metalloproteinase-2 (MMP-2) in various tumor cell types. In the present study, we investigated the role of MMP-2 and the signaling pathway involved in EGF-promoted invasion by human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that EGF stimulation of these tumor cells induced secretion and activation of the collagenase MMP-2, which was required for EGF-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events downstream of EGF receptor involved PI3K- and Src-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.