Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodiumbutyrate and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Purpose. To examine the changes in expression levels of CYP3A4 and efflux. transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the pr esence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol- 13-acetate (TPA). To characterize the transport of [H-3]-digoxin and the me tabolism of midazolam in the cells under different inducing conditions. Methods. CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were brown for 13-14 days. Transport and metabolism studies were performed on c ells induced with NaB and/or TPA for 24 h. The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy. Results. In the presence of both inducers, CYP3A4 protein levels were incre ased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, an d P-gp and MRP1 expression were unchanged, Midazolam 1-OH formation exhibit ed a rank order correlation with increased CYP3A4 protein, whereas [H-3]-di goxin transport (a measure of P-gp activity) was unchanged with induction. P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found per inuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferent ial polarization towards the apical side of the cell. Conclusions. The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA. These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism.