Title

The regulation of Cox-2 activation in human colon cancer cells

Date of Completion

January 2004

Abstract

As a pivotal enzyme in prostaglandin synthesis, Cox-2 has been shown to be involved in colon carcinogenesis and inflammation. Cox-2 over-expression in tumors protects cancer cells from apoptosis and facilitates tumor angiogenesis. We are interested in Cox-2 regulation in colon cancer cells since this knowledge could provide insight into colon carcinogenesis and suggest ways to suppress Cox-2 expression in colon tumors. Studying the HT-29 colon cancer cell line as a model, we found that Cox-2 mRNA and protein levels were activated over 10-fold by the inflammatory cytokine, TNF-α. As Histone Deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation of human colon cancer cells, we determined how HDAC inhibition might affect Cox-2 activation. The HDAC inhibitors butyrate and TSA blocked the TNF-α-induced Cox-2 protein and mRNA synthesis, and dramatically suppressed Cox-2 activity in HT-29 cells. Moreover, we found that the HDAC inhibitors butyrate and trichostatin A blocked Cox-2 activation in a gene specific manner. The suppression of Cox-2 synthesis did not involve promoter inactivation and could be achieved even when applied after the TNF-α stimulus. The effect of the HDAC inhibitors was observed prior to the activation of p21 expression and did not require new protein synthesis. In addition, butyrate did not prevent p38 phosphorylation which regulates Cox-2 mRNA stability. ^ A number of steps in the Cox-2 gene activation pathway were examined to determine how TNF-α and butyrate modulated Cox-2 expression. Neither butyrate nor TSA significantly affected Cox-2 promoter activity, mRNA stability or ARE activity. Instead, TNF-α was found to stimulate RNA polymerase II elongation on the Cox-2 gene, whereas butyrate and TSA induced premature polymerase termination. A butyrate-induced elongational block was also observed on the c-myc gene, as reported for other cell lines. ^ We propose that overcoming a transcriptional block on the Cox-2 and c-myc genes is critical for expression of these genes in cancer cells. The ability of HDAC inhibitors to suppress polymerase II elongation on Cox-2 and c-myc genes indicates that specific protein acetylation reactions are involved in regulating gene expression. It is postulated that the inhibitory effect of HDAC inhibitors on Cox-2 and c-myc expression contributes to their anti-neoplastic activities. ^