COLORECTAL CANCER

Downregulation of prostaglandin E receptor subtype EP₃ during colon cancer development

Y Shoji¹, M Takahashi¹, T Kitamura¹, K Watanabe¹, T Kawamori¹, T Maruyama², Y Sugimoto³, M Negishi⁴, S Narumiya⁵, T Sugimura¹, K Wakabayashi¹

¹ Cancer Prevention Basic Research Project, National Cancer Center Research Institute, Tokyo, Japan
² Minase Research Institute, Ono Pharmaceutical Co. Ltd, Osaka, Japan
³ Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
⁴ Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
⁵ Department of Pharmacology, School of Medicine, Kyoto University, Kyoto, Japan

Correspondence to:
Correspondence to:
Dr K Wakabayashi
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; kwakabay{at}gan2.res.ncc.go.jp

Background and aims: Involvement of prostaglandin E₂ (PGE₂) receptors EP₁, EP₂, and EP₄ in the formation of aberrant crypt foci (ACF) and/or intestinal polyps has been suggested. In contrast, EP₃ appears to have no influence on the early stages of colon carcinogenesis. In the present study, we examined expression of PGE₂ receptor subtypes EP₁, EP₂, EP₃, and EP₄ in normal colon mucosa and colon cancers, and assessed the contribution of EP₃ to colon cancer development.

Methods: mRNA expression of PGE₂ receptor subtypes EP₁, EP₂, EP₃, and EP₄ in normal colon mucosa and colon cancers in azoxymethane (AOM) treated mice and rats, and in humans, were examined by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and immunohistochemical analyses. Evaluation of the role of EP₃ was performed by intraperitoneal injection of AOM, using EP₃ receptor knockout mice. Effects of EP₃ receptor activation on cell growth of human colon cancer cell lines were examined using ONO-AE-248, an EP₃ selective agonist. Moreover, EP₃ expression in colon cancer cell lines was analysed with or without 5-aza-2'-deoxycytidine (5-aza-dC) treatment.

Results: Expression levels of EP₁ and EP₂ mRNA were increased in cancer tissues. EP₄ mRNA was constantly expressed in normal mucosa and cancers. In contrast, expression of EP₃ mRNA was markedly decreased in colon cancer tissues, being 5% in mice, 9% in rats, and 28% in humans compared with normal colon mucosa, analysed by quantitative real time RT-PCR. Immunohistochemical staining demonstrated the rat EP₃ receptor protein to be expressed in epithelial cells of normal mucosa and some parts of small carcinomas but hardly detectable in large carcinomas of the colon. Colon cancer development induced by AOM in EP₃ receptor knockout mice was enhanced compared with wild-type mice, with a higher incidence of colon tumours (78% v 57%) and mean number of tumours per mouse (2.17 (0.51) v 0.75 (0.15); p<0.05). Expression of EP₃ mRNA was detected in only one of 11 human colon cancer cell lines tested. Treatment with 5 µM of an EP₃ selective agonist, ONO-AE-248, resulted in a 30% decrease in viable cell numbers in the HCA-7 human colon cancer cell line in which EP₃ was expressed. Treatment with 5-aza-dC restored EP₃ expression in CACO-2, CW-2, and DLD-1 cells but not in WiDr cells, suggesting involvement of hypermethylation in the downregulation of EP₃ to some extent.

Conclusion: The PGE₂ receptor subtype EP₃ plays an important role in suppression of cell growth and its downregulation enhances colon carcinogenesis at a later stage. Hypermethylation of the EP₃ receptor gene could occur and may contribute towards downregulating EP₃ expression to some extent in colon cancers.

Abbreviations: PGE₂, prostaglandin E₂; ACF, aberrant crypt foci; AOM, azoxymethane; COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs; RT-PCR, reverse transcription-polymerase chain reaction; 5-aza-dC, 5-aza-2'-deoxycytidine; FBS, fetal bovine serum

Keywords: EP3 receptor; prostaglandin E2; colon cancer; colon carcinogenesis; gene expression

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