Cyclopentenone Prostaglandins and Colon Cancer

描述：（申请人的描述）结直肠癌（CRC）是西方世界第二常见的致命恶性肿瘤。 虽然在理解与这种恶性肿瘤相关的遗传改变方面取得了重大进展，但参与CRC发展的生化途径尚不清楚。 然而，最近的证据表明，诱导型环氧合酶（考克斯），考克斯-2，及其产物，前列腺素（PGs），可能在CRC的发病机制中发挥关键作用。 我们已经报道了在极化的HCA-7 CRC细胞中，考克斯-2和PG的产生受表皮生长因子受体的调节（PNAS 94：657，1997）。 我们现在已经发现，来自亲本HCA-7细胞的提取脂质的条件培养基刺激经工程改造以表达诱导型考克斯-2反义cDNA的HCA-7细胞以及PG缺陷型CRC细胞系HCT-15的增殖。此外，我们已经观察到，环戊烯酮PG，这是PGD 2和PGE 2的脱水产物，在这些细胞系中以低纳摩尔或亚纳摩尔范围的浓度诱导增殖。 现在提出的研究，以进一步探索环戊烯酮PG在结肠上皮细胞增殖的作用。 我们将研究各种环戊烯酮PG在不产生PG的其他结肠癌细胞系（包括ALA、FET和HCT-15细胞）以及其他胃肠道上皮细胞系中诱导增殖的能力。 为了补充这些研究，我们将在ALA、FET和HCT-15细胞中过表达考克斯，并确定它们是否形成环戊烯酮PG以及增殖是否改变。 我们已经发现亲本HCA-7细胞产生环戊烯酮PG，PGJ 2。 我们将利用色谱和质谱方法来明确确定其他环戊烯酮PG。 我们已经获得了间接证据表明，环戊烯酮PG通过与假定的受体相互作用诱导增殖。 为了获得对这一假设的直接支持，我们合成了放射性标记的环戊烯酮PG，[氚标记]-15-脱氧-δ 12，14-前列腺素J2，具有高比活性，我们将在HCA-7细胞的亚细胞提取物中进行放射性配体结合研究。 最后，我们将研究环戊烯酮PG的形成，在CRC的动物模型和人类结肠肿瘤样本，通过定量这些化合物，利用质谱分析，我们将开发。 这些研究将有助于深入了解环戊烯酮PG在结直肠癌发生发展中的作用，并进一步阐明考克斯在结直肠癌发病机制中的作用。

DESCRIPTION: (Applicant's Description) Colorectal cancer (CRC) is the second most common fatal malignancy in the Western world. While significant advances have been made in understanding genetic alterations associated with this malignancy, biochemical pathways involved in the development of CRC are less well understood. Recent evidence suggests, however, that the inducible cyclooxygenase (COX), COX-2, and its products, prostaglandins (PGs), may play a key role in the pathogenesis of CRC. We have reported that production of COX-2 and PGs is regulated by the epidermal growth factor receptor in polarized HCA-7 CRC cells (PNAS 94:657, 1997). We now have found that lipid-extracted conditioned medium from parental HCA-7 cells stimulates proliferation of HCA-7 cells engineered to express an inducible COX-2 antisense cDNA, as well as in the PG-deficient CRC cell line, HCT-15. Moreover, we have observed that cyclopentenone PGs, which are dehydration products of PGD2 and PGE2, induce proliferation at concentrations in the low nanomolar or subnanomolar range in these cell lines. Studies are now proposed to further explore the role of cyclopentenone PGs in colonic epithelial proliferation. We will examine the ability of various cyclopentenone PGs to induce proliferation in other colon cancer cell lines that do not make PGs, including ALA, FET, and HCT-15 cells, and also in other gastrointestinal epithelial cell lines. To complement these studies, we will overexpress COX in ALA, FET, and HCT-15 cells and determine whether they form cyclopentenone PGs and whether proliferation is altered. We have found that parent HCA-7 cells produce the cyclopentenone PG, PGJ2. We will utilize chromatographic and mass spectrometric methods to definitively identify other cyclopentenone PGs. We have obtained indirect evidence that cyclopentenone PGs induce proliferation by interaction with a putative receptor. To obtain direct support for this hypothesis, we have synthesized a radiolabeled cyclopentenone PG, [tritium-labeled]-15-deoxy-delta 12,14-prostaglandin J2, with a high specific activity and we will perform radioligand binding studies in sub-cellular extracts from HCA-7 cells. Finally, we will study the formation of cyclopentenone PGs in animal models of CRC and in human colon tumor samples by quantifying these compounds utilizing mass spectrometric assays that we will develop. These studies will provide insight into the role of cyclopentenone PGs in the development of CRC and further clarify the contributions of COX to the pathogenesis of CRC.