PROSTAGLANDINS AND GASTROINTESTINAL DNA DAMAGE

前列腺素和胃肠道 DNA 损伤

• 批准号: 7010018
• 负责人: Courtney Wayne Houchen
• 金额: $ 9.72万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2006-06-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7010018
• 关键词: DNA damage; SDS polyacrylamide gel electrophoresis; apoptosis; azo compounds; biological signal transduction; carcinogenesis inhibitor; carcinogens; cell surface receptors; colorectal neoplasms; gastrointestinal epithelium; immunocytochemistry; laboratory mouse; ligands; nonsteroidal antiinflammatory agent; polymerase chain reaction; prostaglandin E; prostaglandin endoperoxide synthase; radiobiology; radioprotective agents; western blottings

DESCRIPTION (provided by applicant): Colorectal cancer, the second leading cause of cancer death in the western world, arises through the acquisition of multiple, independent genetic mutations and subsequent clonal expansion of mutated crypt epithelial stem cells. Programmed cell death, or apoptosis, is an effective method of removal of these genetically damaged epithelial cells. Azoxymethane (AOM) is a potent DNA damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. NSAIDs inhibit AOM-mediated carcinogenesis. This occurs by inhibition of prostaglandin (PG) production through cyclooygenases (COX) COX-1 or COX-2. COX-2 and not COX-1 however is frequently reported to have a key role in tumor progression. PGE2 is critically important in the maintenance of gastrointestinal integrity and is thought to be a major protective component against environmentally-induced damage. PGs, specifically PGE2 has been shown to regulate intestinal stem cell survival and crypt epithelial apoptosis in response to radiation injury. Furthermore PGE2 mediated radioprotection is COX-1 dependent. In order to determine the effects of AOM on intestinal and colonic injury, we administered AOM (10mg/kg) IP to adult C57/BL6 mice. AOM treatment resulted in marked apoptosis in crypt epithelial cells of the jejunum and distal colon within 8 h. Furthermore, we observed a 3-fold induction in COX-1 mRNA, and a 4-fold increase in intestinal PGE2 levels in WT mice. AOM did not increase PGE2 levels in Cox-1 -/- mice. These data taken together suggest that Cox-1 mediated PGE2 synthesis plays a role in the intestinal response to azoxymethane induced DNA damage. The biologic effects of PGE2 are mediated by binding to membrane bound G-protein coupled receptors, termed EP receptors. The EP receptors are encoded by four separate genes (EP1, EP2, EP3 and EP4). These receptors differ in their ligand binding specificity and in down stream signaling pathways. We have recently demonstrated that the EP2 receptor is expressed in the mouse gastrointestinal tract and is upregulated in the mouse intestine following radiation injury. Moreover, we have demonstrated that the EP2 receptor mediates PGE2's radioprotective effects on apoptosis and crypt stem cell survival. These data taken together support our central hypothesis that the EP2 receptor an important component of an adaptive response in crypt epithelial cells in response to genotoxic injury, in which PGE2 signaling through the EP2 receptor regulates stem cell survival and crypt epithelial cell apoptosis. In this proposal we have chosen to examine the role of PGE2 and EP2 receptors in the regulation of gastrointestinal epithelial apoptosis in response to DNA damage induced by AOM through the following specific aims. 1.To correlate the regional differences in AOM -induced crypt epithelial cell apoptosis along the duodenal to colonic axis with regional differences in EP2 receptor distribution 2.To define the mechanisms of AOM-induced radioprotection in the mouse gastrointestinal tract. 3. To determine the role of COX-1 and PGE2 in AOM-mediated tumorigenesis. We believe that a more complete understanding of the role of PGE2 and EP receptor signaling in the regulation of stem cell fate, will be of great importance in improving our knowledge of the pathogenesis of gastrointestinal malignancy.

描述（由申请人提供）： 结直肠癌是西方世界第二大癌症死亡原因，是通过获得多个独立的基因突变以及随后突变的隐窝上皮干细胞的克隆扩增而产生的。程序性细胞死亡或细胞凋亡是去除这些基因受损的上皮细胞的有效方法。氧化偶氮甲烷 (AOM) 是一种有效的 DNA 损伤剂和致癌物质，可诱发啮齿动物肠道和结肠肿瘤。 NSAIDs 抑制 AOM 介导的致癌作用。这是通过环化酶 (COX) COX-1 或 COX-2 抑制前列腺素 (PG) 产生而发生的。然而，经常有报道称 COX-2 而不是 COX-1 在肿瘤进展中发挥关键作用。 PGE2 对于维持胃肠道完整性至关重要，被认为是抵御环境引起的损害的主要保护成分。 PG，特别是 PGE2 已被证明可以调节肠道干细胞的存活和隐窝上皮细胞凋亡以应对辐射损伤。此外，PGE2 介导的辐射防护是 COX-1 依赖性的。为了确定 AOM 对肠道和结肠损伤的影响，我们给成年 C57/BL6 小鼠腹腔注射 AOM (10mg/kg)。 AOM处理在8小时内导致空肠和远端结肠的隐窝上皮细胞显着凋亡。此外，我们观察到 WT 小鼠中 COX-1 mRNA 诱导增加了 3 倍，肠道 PGE2 水平增加了 4 倍。 AOM 不会增加 Cox-1 -/- 小鼠中的 PGE2 水平。这些数据综合表明 Cox-1 介导的 PGE2 合成在肠道对氧化偶氮甲烷诱导的 DNA 损伤的反应中发挥作用。 PGE2 的生物效应是通过与膜结合的 G 蛋白偶联受体（称为 EP 受体）结合来介导的。 EP 受体由四个独立的基因（EP1、EP2、EP3 和 EP4）编码。这些受体的配体结合特异性和下游信号传导途径有所不同。我们最近证明EP2受体在小鼠胃肠道中表达，并且在辐射损伤后小鼠肠道中表达上调。此外，我们还证明EP2受体介导PGE2对细胞凋亡和隐窝干细胞存活的辐射防护作用。这些数据共同支持了我们的中心假设，即 EP2 受体是隐窝上皮细胞响应基因毒性损伤的适应性反应的重要组成部分，其中通过 EP2 受体的 PGE2 信号传导调节干细胞存活和隐窝上皮细胞凋亡。在本提案中，我们选择通过以下具体目标来研究 PGE2 和 EP2 受体在响应 AOM 诱导的 DNA 损伤而调节胃肠道上皮细胞凋亡中的作用。 1. 将 AOM 诱导的沿十二指肠至结肠轴的隐窝上皮细胞凋亡的区域差异与 EP2 受体分布的区域差异关联起来 2. 定义 AOM 诱导的小鼠胃肠道辐射防护机制。 3. 确定COX-1和PGE2在AOM介导的肿瘤发生中的作用。我们相信，更全面地了解PGE2和EP受体信号在干细胞命运调节中的作用，对于提高我们对胃肠道恶性肿瘤发病机制的认识具有重要意义。