RESUMO
As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 µM) or therapeutic (5, 10 or 20 µM). Retinol at 10 and 20 µM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 µM), SB203580 (10 µM) or siRNA to either p38α (MAPK14) or p38ß (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 µg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.
Assuntos
Regulação para Baixo/efeitos dos fármacos , NF-kappa B/metabolismo , Receptores Imunológicos/metabolismo , Vitamina A/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteína Quinase 11 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , Oxirredução , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , alfa-Tocoferol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of beta-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6%) or normal (17%; control) protein diets and maintained for 48 h in culture medium containing 5.6 mmol/l glucose, with or without 0.6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2.8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways.
Assuntos
Dieta com Restrição de Proteínas , Proteínas de Homeodomínio/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Ácido Palmítico/farmacologia , Transativadores/metabolismo , Animais , Western Blotting/métodos , Células Cultivadas , Ácidos Graxos não Esterificados/metabolismo , Feminino , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Homeodomínio/genética , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Proteína Quinase 11 Ativada por Mitógeno/genética , Modelos Animais , RNA Mensageiro/análise , Ratos , Ratos Wistar , Transativadores/genéticaRESUMO
Angiogenesis, the growth of new capillaries from pre-existing ones, occurs through dynamic functions of the endothelial cells (EC), including migration, which is essential to achieve an organized formation of the vessel sprout. We demonstrated previously that an aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), inhibits vascular endothelial growth factor (VEGF)-induced EC migration. In the present study, we investigated the effects of ATL-1 in the actin cytoskeleton reorganization of EC stimulated with VEGF. Pretreatment of EC with ATL-1 caused a reduction in VEGF-induced stress fibers and therefore reduced the intracellular content of filamentous actin. A concomitant impairment in stress-activated protein kinase (SAPK2/p38) phosphorylation suggests that ATL inhibition of VEGF-stimulated actin polymerization involves the SAPK2/p38 pathway. Moreover, ATL-1 treatment inhibited focal adhesion clustering due to inhibition of focal adhesion kinase (FAK) phosphorylation and the subsequent association of FAK with the actin cytoskeleton. This final event, which ultimately allows cell migration, was reverted by an LX receptor antagonist, but not by a cys-LT1R antagonist, indicating an effect via the G-protein-linked LXA4 receptor. Together our results provide evidence that ATL-1 inhibits EC migration via the concerted inhibition of actin polymerization and proper assembly of focal adhesions, supporting a role for these novel lipid mediators as angiogenesis modulators.