ROLE OF LIPIDS IN COLON CANCER

花生四烯酸和亚油酸代谢的重要性得到动物和人类流行病学研究的支持，这些研究表明阿司匹林和其他非甾体抗炎药可以降低结肠癌的发病率和死亡率。最近的研究报道Cox-2在结肠癌和其他癌症中显著表达。一些数据表明前列腺素的作用是通过调节细胞凋亡和血管生成。我们正在研究花生四烯酸和亚油酸的代谢，以及Cox-1和-2，脂氧合酶，人类细胞系的表达。我们以CaCo-2细胞作为人结肠上皮细胞模型，研究了Cox-2、-1和15-LO在细胞凋亡和分化过程中的表达。凋亡和分化伴随着Cox-2表达的减少和15-LO表达的增加。测定了人类结直肠肿瘤组织及UNC邻近正常组织中Cox-1、Cox-2和15-脂氧合酶的表达。Cox-1在正常组织和肿瘤组织中均有高表达，而Cox-2在大多数肿瘤组织中的表达均高于正常组织。15-脂氧合酶在大多数结直肠组织中表达，表征为15- lo -1，在肿瘤中表达量高出2-3倍。免疫组织化学研究发现15-脂氧合酶-1在上皮细胞中高表达。15-脂氧合酶和Cox-2表达的调节也在研究中。本研究以羧基截断APC基因突变的人结直肠癌细胞系HT-29细胞为研究对象，在诱导启动子的控制下导入完整的APC基因。这些HT-29-APC细胞提供了一个合适的模型系统来研究在APC功能丧失后Cox-2表达是如何失调的。全长APC诱导HT-29-APC细胞发生凋亡。全长APC蛋白已被证明与细胞内蛋白b-连环蛋白结合，结果导致f/Tcf转录因子下调。APC免疫沉淀分析显示，与全长APC相互作用的b-连环蛋白呈时间依赖性增加。因此，在这些细胞中，此时left /Tcf信号通路是完整的。此外，全长APC表达后，Cox-2蛋白表达下调，而Cox-2 mRNA水平保持不变。这一数据表明APC直接或间接地参与了Cox-2的翻译调控。凋亡诱导剂丁酸钠处理HT-29-APC细胞，不改变Cox-2蛋白的表达。因此，Cox-2下调似乎是APC特异性的，而不仅仅是由于凋亡诱导。APC似乎是唯一调控Cox-2表达的基因。在HT-29-APC细胞中Cox-2蛋白表达下调的机制正在研究中。我们研究了NaBT和其他组蛋白去乙酰化酶（HDAC）抑制剂在人结直肠癌细胞中对15-LO的转录调节。NaBT、trichostatin A和HC毒素处理可诱导Caco-2和SW-480细胞明确表达15-LO—1。酸性尿素聚丙烯酰胺凝胶电泳结果显示，HDAC抑制剂处理后Caco-2和SW-480细胞的组蛋白发生乙酰化。此外，在表达15-脂氧合酶的人结肠癌手术样本中检测到组蛋白乙酰化。这些观察结果表明，HDAC抑制剂如丁酸酯的组蛋白乙酰化可能直接或间接地调节人类结直肠癌细胞和结直肠组织中15-脂氧合酶的转录调节。我们目前正在研究已知参与分化的GATA转录因子家族是否也直接导致15-脂氧合酶的表达增加。-细胞凋亡，结肠癌，C0X-2, 15-LO，细胞分化

The importance of arachidonic acid and linoleic acid metabolism is supported by animal and human epidemiology studies that indicate that aspirin and other NSAIDs reduce the incidence and mortality of colon cancer. Recent studies reported that Cox-2 is significantly expressed in colon cancer and other cancers. Some data suggest effects of prostaglandins are via modulation of apoptosis and angiogenesis. We are examining arachidonic and linoleic acid metabolism, and the expression of Cox-1 and -2, lipoxygenases, human cell lines. Using CaCo-2 cells as a model for human colorectal epithelium, we have studied the expression of Cox-2, -1 and 15-LO during apoptosis and differentiation. Apoptosis and differentiation is accompanied by a decrease in the expression of Cox-2 and an increase in expression of 15-LO. The expression of Cox-1, Cox-2 and 15-lipoxygenase was measured in human colorectal tumors and the adjacent normal tissue obtained from UNC. Cox-1 is highly expressed in both the normal and tumor tissue while higher expression of Cox-2 was detected in most of the tumors compared to normal tissue.15- lipoxygenase was expressed in most colorectal tissue and characterized as 15-LO-1, with a 2-3 fold higher expression noted in tumors. Immunohistochemical studies found the 15-lipoxygenase-1 to be highly expressed in the epithelial cells. The regulation of 15-lipoxygenase and Cox-2 expression is also under investigation. We have used HT-29 cells, a human colorectal carcinoma cell line with mutant carboxy- truncated APC gene, in which intact APC gene has been introduced under the control of an inducible promoter. These HT-29-APC cells provide a suitable model system to examine how Cox-2 expression becomes dis- regulated after loss of APC function. Induction of full-length APC causes the HT-29-APC cells to undergo apoptosis. Full-length APC protein has been shown to bind the intracellular protein b-catenin and as a result, the Lef/Tcf transcription factors are downregulated. Analysis of APC immunoprecipitates demonstrates a time dependent increase of b-catenin interacting with full-length APC. Thus, the Lef/Tcf signaling pathway is intact at this point in these cells. Furthermore, upon expression of full-length APC, Cox-2 protein expression is downregulated while Cox-2 mRNA levels remain the same. This data indicates that APC plays a role, either directly or indirectly, in the translational regulation of Cox-2. Treatment of the HT-29-APC cells with sodium butyrate, an inducer of apoptosis, does not alter Cox-2 protein expression. Thus, Cox-2 downregulation appears to be APC specific and not just due to apoptotic induction. APC appears to uniquely regulate Cox-2 expression. The mechanism by which Cox-2 protein expression is downregulated in the HT-29-APC cells is under investigation.We investigated the transcriptional regulation of 15-LO in human colorectal carcinoma cells by treatment with NaBT and other histone deacetylase (HDAC) inhibitors. The treatment with NaBT, trichostatin A and HC toxin induced the clear expression of 15-LO--1 in Caco-2 and SW-480 cells. Acid urea polyacrylamide gel electrophoresis indicated that histone proteins from Caco-2 and SW-480 cells were acetylated by treatment with HDAC inhibitors. Moreover, histone acetylation was detected in the surgical samples of human colon cancer expressing 15-lipoxygenase. These observations suggested that histone acetylation by HDAC inhibitors such as butyrate may, directly or indirectly, modulate the transcriptional regulation of 15-lipoxygenase in human colorectal carcinoma cells and colorectal tissue. We are currently investigating if the family of GATA transcriptions factor know to be involved in differentiation are also directly responsible for the increased expression of 15-lipoxygenase. - apoptosis, colon cancer, C0X-2, 15-LO, cell differentiation