Investigation of the effect of sodium butyrate on the regulation of cyclooxygenase-2 in colon cancer cell lines Caco-2 and HT-29

Ülgen, Doğukan Hazar
Sodium butyrate (NaBt) is a four-carbon short-chain fatty acid histone deacetylase inhibitor (HDACi) that is available in the colon through the commensal microbiota-mediated fermentation of dietary fibers. It is the main source of energy for colonocytes, and is regarded to have tumor suppressive effects, most prominently in colorectal cancer (CRC). Cyclooxygenase-2 (COX-2) is a gene important in the inflammatory response due to its ability to convert arachidonic acid to prostaglandins. Overexpression and overactivity of COX-2 were observed in chronic inflammatory diseases and CRC. To understand whether the expression of COX-2 was regulated through NaBt in CRC, we treated Caco-2 and HT-29 cells with 3mM NaBt. We observed that Caco-2 cells treated with 3 mM NaBt showed increased mRNA and protein expression of COX-2 while a decrease in the same was observed in HT-29 cells. To understand the mechanism behind this dual regulation, we analyzed the transcriptional regulation of COX-2 through NF-κB. Contrary to our v expectations, we found that even though the nuclear localization of p65 subunit of NF-κB showed an increase in Caco-2 cells and not in HT-29 cells, the recruitment of NF-κB to the COX-2 promoter region decreased in Caco-2 cells, and increased in HT-29 cells We therefore hypothesized that COX-2 was regulated post-trascriptionally. We observed that mitogen-activated protein kinase (MAPK) p38, a pathway that activates a number of proteins involved in mRNA stabilization, was activated only in Caco-2 cells and not HT-29 cells. We investigated the downstream signaling and discovered that the use of distal polyadenylation signal, and the stability of COX-2 were increased in NaBt-treated Caco-2 cells and decreased in HT-29 cells. We then showed that the said stability was lost when the p38 activity was inhibited in Caco-2 cells. The stabilizing AU-rich element binding protein (AREBPs) HuR was found to decline in the two cell lines, whereas destabilizing AREBP TTP exhibited a decrease only in Caco-2 cells. Collectively, this study indicates that NaBt can modify the transcriptome not only through its HDACi ability but also via the activation of p38-dependent post-transcriptional regulation networks.