OP41 - Methylation at positon 7’ but not 5’ and the isoprenoid chain are required for vitamin E to induce P-glycoprotein protein expression and activity in the human colon carcinoma cell line LS180

Abstract

The eight congeners of vitamin E differ in their methylation pattern and either possess an alkyl (tocopherols, T) or an isoprenoid (tocotrienols, T3) side chain. The different chemical structures influence the biological activity of the vitamin, like the binding to the nuclear pregnane X receptor (PXR). P-glycoprotein (P-gp), a PXR target gene, is an efflux transporter expressed at various barriers in the body including small intestine, bile ducts and the blood-brain-barrier. We therefore investigated the impact of methylation of the chromanol ring (position 5’ and 7’ = α; position 7’ = γ) and the structure of the side chain on the expression and activity of P-gp. LS180 cells, which have a low basal P-gp expression, are a good model to study the induction of P-gp protein expression and activity. LS180 cells were either treated with the positive control rifampicin (25 µmol/L), a known P-gp inducer, the appropriate solvent control, or the respective vitamin E congener for 48 hours. αT was investigated in the range of 10–100 µmol/L, γT 10-50 µmol/L, αT3 5-50 µmol/L and γT 5-25 µmol/L due to differences in cell toxicity. Protein expression was determined by Western blot analysis and P-gp activity via the efflux of the fluorescent substrate rhodamine 123 in the presence or absence of the P-gp specific inhibitor elacridar. αT, γT and αT3 neither influenced the protein expression nor the activity of P-gp. γT3 induced P-glycoprotein activity and protein expression which is in accordance with the literature. To induce P-glycoprotein, both methylation at position 7’ but not 5’ of the chromanol ring as well as an isoprenoid side chain are required as none of the two structural features individually showed an effect.