Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development
Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development
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DOI：
10.1136/gut.2003.028787
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发表时间：
2004-08-01
期刊：
影响因子：
24.5
通讯作者：
Wakabayashi, K
中科院分区：
文献类型：
--
作者：
Shoji, Y;Takahashi, M;Wakabayashi, K
背景和目的：有人提出前列腺素 E-2 (PGE(2)) 受体 EP1、EP2 和 EP4 参与异常隐窝病灶 (ACF) 和/或肠息肉的形成。相反，EP3似乎对结肠癌发生的早期阶段没有影响。在本研究中，我们检测了正常结肠粘膜和结肠癌中 PGE(2) 受体亚型 EP1、EP2、EP3 和 EP4 的表达，并评估了 EP3 对结肠癌发展的贡献。方法：检测了氧化偶氮甲烷 (AOM) 处理的小鼠和大鼠以及人类中正常结肠粘膜和结肠癌中 PGE(2) 受体亚型 EP1、EP2、EP3 和 EP4 的 mRNA 表达通过逆转录聚合酶链反应 (RT-PCR)、实时定量 RT-PCR 和免疫组织化学分析。使用 EP3 受体敲除小鼠腹腔注射 AOM 来评估 EP3 的作用。使用 EP3 选择性激动剂 ONO-AE-248 检查了 EP3 受体激活对人结肠癌细胞系细胞生长的影响。此外，分析了有或没有5-aza-2'-脱氧胞苷(5-aza-dC)处理的结肠癌细胞系中EP3的表达。结果：癌组织中EP1和EP2 mRNA的表达水平增加。 EP4 mRNA 在正常粘膜和癌症中持续表达。相反，通过实时定量RT-PCR分析，与正常结肠粘膜相比，结肠癌组织中EP3 mRNA的表达显着降低，小鼠中为5％，大鼠中为9％，人类中为28％。免疫组织化学染色显示大鼠EP3受体蛋白在正常粘膜上皮细胞和部分小癌中表达，但在大结肠癌中几乎检测不到。与野生型小鼠相比，AOM 在 EP3 受体敲除小鼠中诱导的结肠癌发展增强，结肠肿瘤发生率更高 (78% vs 57%)，每只小鼠的平均肿瘤数量 (2.17 (0.51) vs 0.75 (0.15)；p < 0.05）。仅在所测试的 11 种人类结肠癌细胞系中的一种中检测到 EP3 mRNA 的表达。用5μM EP3选择性激动剂ONO-AE-248处理，导致表达EP3的HCA-7人结肠癌细胞系中活细胞数量减少30%。 5-aza-dC处理可以恢复CACO-2、CW-2和DLD-1细胞中的EP3表达，但不能恢复WiDr细胞中的EP3表达，这表明高甲基化在一定程度上参与了EP3的下调。结论：PGE(2)受体亚型EP3在抑制细胞生长中发挥重要作用，其下调增强了后期结肠癌的发生。 EP3 受体基因的高甲基化可能发生，并可能在一定程度上导致结肠癌中 EP3 表达的下调。
Background and aims: Involvement of prostaglandin E-2 (PGE(2)) 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 PGE(2) 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 PGE(2) 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 mu M 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 PGE(2) 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.