Background

Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites may induce therapeutic effects in the colon by activating peroxisome proliferator activated receptors (PPARs), specifically the PPAR-? subtype. The activation of PPAR-? changes the expression and activity of target genes and transcription factors such as COX-2, NF-KB and AP-1, resulting in anti-inflammatory and anti-tumorigenic effects. The metabolites of CYP2C40/55 such as 16-,HETE, 8,9-EET and 14,15-EET have anti-inflammatory effects suggesting a potential therapeutic role in colon tumorigenesis. This study aims to determine whether PPAR activation up-regulates the expression of CYP2C40/55.

Method

CYP2C40/55 promoter regions isolated from murine DNA via PCR were inserted into luciferase plasmid (PGL4.10). Plasmid DNA was cloned following transfection into highly competent cells. Purified plasmids were transfected into COS-7 and HCA-7 cells and treated with PPAR-?/?/? ligands Wy14643, GW0742 and Rosiglitazone (COS-7 cells had PPAR ?/?/? over-expressed). Cells were harvested after 24 hours 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-?/?/? ligands led to a significant increase in CYP2C40 RLU from baseline (mean (± SD)) 235(20) to 726(45), 458(61), 466(42) for PPAR-?/?/? respectively. CYP2C55 showed a significant increase from 154(6) to 263(10), 354(21) for PPAR-?/? respectively (p=0.001). HCA-7 cells were shown to express only endogenous PPAR-? and following incubation with PPAR-?/?/? ligands RLU significantly increased in CYP2C40 from 55(13) to 126(17) and in CYP2C55 from 62(11) to 111(4) for PPAR ? (p=0.001).

Conclusion

The results suggest that a functional peroxisome proliferator response element (PPRE) exists within the promoter regions of CYP2C40/55 and that activation of PPAR-a within the HCA-7 cell line leads to a significant increase in CYP2C40/55 expression. Given the beneficial properties of PPAR-a and CYP derived AA metabolites it seems that CYP2C40 and CYP2C55 may become future therapeutic targets in colon cancer.