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
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