RESUMO
OBJECTIVES: Pancreatic stellate cells are source of dense fibrotic stroma, a constant pathological feature of chronic pancreatitis and pancreatic adenocarcinoma. We observed correlation between levels of cyclooxygenase 2 (COX-2) and its product prostaglandin E2 (PGE2) and the extent of pancreatic fibrosis. The aims of this study were to delineate the effects of PGE2 on immortalized human pancreatic stellate cells (HPSCs) and to identify the receptor involved. METHODS: Immunohistochemistry, reverse transcription-polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction were used to assess COX-2, extracellular matrix, and matrix metalloproteinase gene expression. Eicosanoid profile was determined by liquid chromatography-tandem mass spectrometry. Human pancreatic stellate cell proliferation was assessed by MTS assay, migration by Boyden chamber assay, and invasion using an invasion chamber. Transient silencing was obtained by small interfering RNA. RESULTS: Human pancreatic stellate cells express COX-2 and synthesize PGE2. Prostaglandin E2 stimulated HPSC proliferation, migration, and invasion and stimulated expression of both extracellular matrix and matrix metalloproteinase genes. Human pancreatic stellate cells expressed all 4 EP receptors. Only blocking the EP4 receptor resulted in abrogation of PGE2-mediated HPSC activation. Specificity of EP4 for the effects of PGE2 on stellate cells was confirmed using specific antagonists. CONCLUSIONS: Our data indicate that PGE2 regulates pancreatic stellate cell profibrotic activities via EP4 receptor, thus suggesting EP4 receptor as useful therapeutic target for pancreatic cancer to reduce desmoplasia.
Assuntos
Ciclo-Oxigenase 2/genética , Dinoprostona/farmacologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Naftalenos/farmacologia , Células Estreladas do Pâncreas/metabolismo , Fenilbutiratos/farmacologia , Interferência de RNA , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.
