Introduction Certain Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are thought to induce therapeutic effects in the colon via activation of peroxisome proliferator activated receptors (PPARs), specifically the PPAR α subtype. The activation of PPAR α leads to changes in the expression and activity of target genes and other transcription factors such as COX-2, NF-KB and AP-1, resulting in anti-inflammatory and anti-tumorigenic effects. CYP2C40 and CYP2C55 are two recently discovered CYPs, isolated from the murine intestinal tract. Their metabolites include 16-HETE, 8,9-EET and 14,15-EET which have been shown to have anti-inflammatory effects both in vitro and in vivo. Evidence suggests that CYP2C40 and CYP2C55 may have a potential therapeutic role to play in colon tumorigenesis. This study aims to determine whether PPAR activation leads to an up-regulation in the expression of CYP2C40 and CYP2C55.
Methods CYP2C40 and CYP2C55 promoter regions were isolated from murine DNA via PCR and inserted into a luciferase plasmid (pGL4.10). Plasmid DNA was cloned following transfection into highly competent cells and purified via Midi-Prep recovery. Purified plasmids were transfected into COS-7 and HCA-7 cells and the cells were treated with PPAR α, β and γ ligands Wy14643, GW0742 and Rosiglitazone (COS-7 cells had PPAR α/β/γ over-expressed). Cells were harvested after 24 h incubation and luciferase activity (equivalent to gene expression) was measured in relative light units (RLU) using a reporter assay system.
Results In COS-7 cells PPAR α, β and γ ligands led to a significant increase in CYP2C40 RLU from a baseline measurement of (mean (±SD)) 235 (20) to 726 (45), 458 (61) and 466 (42) for PPAR α, β and γ respectively. CYP2C55 showed a significant increase from 154 (6) to 263 (10) and 354 (21) for PPAR α and β respectively (p=0.001). HCA-7 cells were shown to only express endogenous PPAR α and following incubation with PPAR α, β and γ ligands a significant RLU increase was observed in CYP2C40 from 55 (13) to 126 (17) and in CYP2C55 from 62 (11) to 111 (4) for PPAR α (p=0.001).
Conclusion The results suggest that a functional peroxisome proliferator response element (PPRE) exists within the promoter regions of CYP2C40 and CYP2C55 and that activation of PPAR α within the HCA-7 cell line leads to a significant increase in CYP2C40 and CYP2C55 expression. Given the beneficial properties of PPAR α and CYP derived AA metabolites it seems that CYP2C40 and CYP2C55 may become important future therapeutic targets in colon cancer.
Competing interests None declared.