Efeito da suplementação com óleo de peixe sobre a expressão Gênica de Cox-2 em ratos portadores de tumor de Walker 256

Abstract

Resumo: Evidencias epidemiologicas e experimentais tem demonstrado o potencial farmacologico e o valor nutricional de uma dieta enriquecida com oleo de peixe. Estudos previos do Laboratorio de Metabolismo Celular, UFPR, demonstraram que o oleo de peixe, rico em AGPI n-3, e capaz de reduzir o crescimento tumoral e o grau de caquexia em ratos portadores de tumor de Walker 256. Apesar de inumeras pesquisas sobre a eficacia dos AGPI n-3 no tratamento de doencas inflamatorias, muitos mecanismos moleculares responsaveis pelos efeitos biologicos, ainda nao sao completamente entendidos. Alteracoes na expressao genica pode ser um importante mecanismo na qual AGPI n-3 exercem seus efeitos beneficos em condicoes patologicas, como o cancer. Devido a importancia da expressao de COX- 2 na genese do cancer ha enorme interesse em modular sua atividade, pois existem fortes evidencias de sua participacao na iniciacao, transformacao, progressao, e metastase. O objetivo deste trabalho foi investigar o efeito da uplementacao com oleo de peixe sobre a expressao genica de COX-2 em ratos portadores de tumor de Walker 256. Ratos Wistar machos de 70 dias foram divididos em tres grupos: alimentados com racao (W), suplementados com oleo de peixe (WP) ou gordura de coco (WS) ate completarem 100 dias de vida na dose de 1g/Kg p.c. Apos este periodo, foi inoculado uma suspensao de celulas de Tumor de Walker 256 contendo 3X107 celulas/mL. Apos 14 dias da inoculacao, os animais foram ortotanasiados e a massa tumoral retirada para as analises experimentais. O RNA total foi isolado do tecido tumoral e a expressao genica foi avaliada por transcricao reversa-PCR em tempo real (RT-qPCR) utilizando o metodo 2-ĢĢCT. Foi realizado validacao da eficiencia de amplificacao dos genes alvo e controle interno, assim como do gene de referencia. Os dados revelaram que a suplementacao com oleo de peixe foi capaz de reduzir significativamente o peso do tumor, a sintese proteica e a expressao genica da enzima COX-2 (p<0.05) no tecido tumoral. Apesar dos mecanismos moleculares permanecerem desconhecidos, este resultado sugere que os AGPI n-3 possuem a capacidade de alterar a expressao de genes envolvidos na inflamacao, participantes dos processos de carcinogenese. Alem disso, estes resultados demonstram pela primeira vez que a suplementacao com oleo de peixe e capaz de reduzir a expressao genica de COX-2 no tecido tumoral em ratos portadores de Tumor de Walker 256.

Abstract: Epidemiological and experimental evidence has shown the pharmacological potential and the nutritional value of diets enriched in fish oil. Previous research in our laboratory has shown that a diet rich in fish oil decreases the growth of Walker 256 tumors and cancer cachexia in rats. Although several studies have reported the efficacy of n-3 PUFA in inflammatory diseases, many of the molecular mechanisms responsible for the biological effects remain unknown. Alterations in the gene expression could be an important mechanism where n-3 PUFA (fish oil) applies its beneficial effects in pathological conditions, such as cancer. Due to the important role of COX-2 in cancer genesis, there is an enormous interest in its modulation because of strong evidence shown in the role of COX-2 expression with initiation, transformation invasiveness and metastasis. The objective of this study was to investigate the effects of diets supplemented with fish oil in COX-2 gene expression in Walker 256 tumor-bearing rats. Male wistar rats at 70 days old were separated into 3 groups: fed with regular chow (W) supplemented with fish oil (WP) and coconut fat (WS) (1 g/kg b.w.) until they reached 100 days of age. Then, they were inoculated with a suspension of Walker 256 ascitic tumor cells (3 ~107 ml) and after 14 days they were euthanized and the tumor mass was removed for experimental analysis. Total RNA was isolated from tumor tissues and a quantitative real-time reverse transcription (RT-qPCR) conducted to compare the relative expression using the 2-ĢĢCT method. The efficiency of target and internal control genes as well as the design of a reference gene was alidated. The data revealed that diets supplemented with fish oil resulted in a significant decrease in the tumor weight, protein and gene expression of COX-2 (p<0.05) in tumor tissue. Despite the molecular echanisms involved are not fully understood, the data suggests that fish oil may exert their effects via their capacity to regulate the expression of inflammatory genes, associated in carcinogenisis. These results demonstrate for the first time the decrease in gene expression of COX-2 in tumor tissue in Walker 256 tumor-bearing rats by fish oil.