Cyclopentenone Prostaglandins and Colon Cancer

描述：(申请人描述)结直肠癌(CRC)是西方世界第二常见的致命恶性肿瘤。虽然在了解与这种恶性肿瘤相关的基因改变方面取得了重大进展，但对参与结直肠癌发展的生化途径了解较少。然而，最近的证据表明，诱导型环氧合酶(COX)、COX-2及其产物前列腺素(PGs)可能在结直肠癌的发病机制中起关键作用。我们已经报道在极化的HCa-7CRC细胞中，COX-2和PGs的产生受表皮生长因子受体的调节(PNAS 94：657,1997)。我们现在发现，从亲代Hca-7细胞中提取脂类的条件培养液可以刺激Hca-7细胞的增殖，该细胞被设计为表达可诱导的COX-2反义cDNA，以及在PG缺陷的CRC细胞系HCT-15中。此外，我们还观察到，作为PGD2和PGE2的脱水产物的环戊烯酮PGs在低纳摩尔或亚纳摩尔范围内诱导这些细胞系的增殖。现在有研究建议进一步探讨环戊酮PGs在结肠上皮细胞增殖中的作用。我们将检测不同的环戊酮PGs在其他不产生PGs的结肠癌细胞系中诱导增殖的能力，包括ALA，FET和HCT-15细胞，以及其他胃肠道上皮细胞系。为了补充这些研究，我们将在ALA、FET和HCT-15细胞中过表达COX，并确定它们是否形成环戊酮PG以及增殖是否发生改变。我们发现亲本Hca-7细胞产生环戊酮PG，PGJ2。我们将利用色谱和质谱学方法来确定其他环戊酮PG。我们已经获得了间接证据，证明环戊酮PGs通过与假定的受体相互作用而诱导增殖。为了获得这一假说的直接支持，我们合成了具有高比活性的放射性标记环戊酮PG，[氚标记]-15-脱氧-三角洲12，14-前列腺素J2，并将在HCa-7细胞的亚细胞提取物中进行放射性配基结合研究。最后，我们将研究环戊酮PGs在结直肠癌动物模型和人类结肠肿瘤样本中的形成，方法是利用我们将开发的质谱分析方法对这些化合物进行定量。这些研究将深入了解环戊酮PGs在结直肠癌发生发展中的作用，并进一步阐明COX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.