Colon Cancer Chemoprevention and COX-2 Suppression by Ursodeoxycholic Acid

描述（由申请人提供）： 考克斯-2选择性抑制剂的最新进展显示出预防结肠癌的前景，但不利的心血管副作用抑制了对这些药物的热情。由于选择性考克斯-2抑制剂出现问题，越来越多的努力正在作出，以确定其他有效和更安全的化学预防剂。一项熊去氧胆酸（UDCA）预防结直肠腺瘤复发的大型、III期、双盲、安慰剂对照试验显示，伴有高度异型增生的腺瘤复发率在统计学上显著降低。我的长期研究目标是阐明UDCA对结肠癌发挥抗癌作用的机制。我们以前已经证明，补充膳食胆酸促进氧化偶氮甲烷诱导的大鼠结肠肿瘤的发展。相反，饮食UDCA抑制肿瘤发生和抑制胆酸诱导的肿瘤促进。为了检查UDCA引起抗癌作用的机制，该提案中的研究表明，UDCA抑制野生型Ras激活的肿瘤，并产生RasGT 3激活蛋白（RasGAP）-N和-C末端片段。RasGAP C-末端片段使结肠癌细胞对UDCA诱导的细胞周期阻滞敏感，并抑制这些细胞中的考克斯-2表达。本研究的主要目标之一是提供RasGAP C片段蛋白将有助于提高UDCA预防结肠癌的疗效的原理证明。这将通过研究用RasGAP C转染子建立的裸鼠中肿瘤异种移植物的生长以及UDCA对细胞培养物中G 0/G1细胞周期阻滞调节剂的影响来实现。此外，我们还将研究肿瘤内递送可渗透的RasGAP C片段蛋白对肿瘤异种移植物的影响。本研究的另一个目的是确定和表征RasGAP C-末端片段改变结肠癌细胞中组成型考克斯-2基因表达和UDCA抑制异常隐窝病灶（ACF）（结肠癌的推定恶变前体）中考克斯-2的转录和转录后机制。这将通过在结肠癌发生的氧化偶氮甲烷模型中研究激光捕获显微切割的ACF的基质和上皮细胞来实现。我们将通过从患者中分离ACF并研究RasGAP片段和考克斯-2调节剂来将我们的研究扩展到人类样本，以确保在体外和体内鉴定的调节组分与人类疾病相关。因此，这项研究的建议是重要的，以获得洞察基本的分子调控考克斯-2的RasGAP C-片段，一种新的和未探索的机制。这将有助于开发更好的治疗干预措施，因为潜在的考克斯-2抑制剂被认为对人类使用是有毒的。

DESCRIPTION (provided by applicant): Recent progress in Cox-2 selective inhibitors have shown promise to prevent colon cancer, but untoward cardiovascular side effects have dampened the enthusiasm for these agents. Since selective Cox-2 inhibitors appear problematic, increasing efforts are being made to identify other effective and safer chemopreventive agents. A large, phase III, double blind, placebo-controlled trial of Ursodeoxycholic acid (UDCA) to prevent the recurrence of colorectal adenoma was associated with a statistically significant reduction in recurrence of adenomas with high-grade dysplasia. My long-term research objectives are to elucidate the mechanisms by which UDCA exerts anticarcinogenic effects with respect to colon cancer. We have previously demonstrated that supplemental dietary cholic acid promoted the development of azoxymethane-induced rat colonic tumors. In contrast, dietary UDCA inhibited tumorigenesis and suppressed cholic acid-induced tumor promotion. To examine the mechanisms by which UDCA causes anticarcinogenic effects, studies in the proposal demonstrated that UDCA inhibited wild-type Ras activated tumors and generated RasGTPase activating protein (RasGAP) -N and -C terminal fragments. RasGAP C-terminal fragment sensitizes colon cancer cells towards UDCA-induced cell cycle arrest and suppresses Cox-2 expression in these cells. One of the major goals of this investigation is to provide proof of principle that RasGAP C-fragment protein will be useful in improving the efficacy of UDCA in colon cancer prevention. This will be achieved by investigating the growth of tumor xenografts in nude mice established with RasGAP C-transfectants and the effect of UDCA on regulators of G0/G1 cell cycle arrest in cell culture. Additionally, we will also investigate the effect of Intratumoral delivery of a permeable RasGAP C-fragment protein on tumor xenografts. Another goal of this investigation is to identify and characterize the transcriptional and post-transcriptional mechanisms by which RasGAP C-terminal fragment alters constitutive Cox-2 gene expression in colon cancer cells and UDCA suppresses Cox-2 in Aberrant Crypt Foci (ACF), the putative premalignant precursors of colon cancer. This will be achieved by investigating the stromal and epithelial cells of laser capture microdissected ACF in azoxymethane model of colonic carcinogenesis. We will extend our investigation to human samples by isolating ACFs from patients and study RasGAP fragments and Cox-2 regulators to ensure that regulatory components identified in vitro and in vivo are relevant to human disease. Studies in this proposal are, therefore, important for gaining insights into basic molecular regulation of Cox-2 by RasGAP C-fragments, a novel and unexplored mechanism. It will be useful in the development of better therapeutic interventions, as potential Cox-2 inhibitors are considered toxic for human use.