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Characterizing the Expression of CYP3A4 and Efflux Transporters (P-gp, MRP1, and MRP2) in CYP3A4-Transfected Caco-2 Cells After Induction with Sodium Butyrate and the Phorbol Ester 12-O-Tetradecanoylphorbol-13-Acetate

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Abstract

Purpose. To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). To characterize the transport of [3H]-digoxin and the metabolism of midazolam in the cells under different inducing conditions.

Methods. CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days. Transport and metabolism studies were performed on cells 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 increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged. Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas [3H]-digoxin 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 peri-nuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential 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.

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  1. Department of Biopharmaceutical Sciences, University of California, San Francisco, California, 94143

    Carolyn L. Cummins, Lara M. Mangravite & Leslie Z. Benet

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Cummins, C.L., Mangravite, L.M. & Benet, L.Z. Characterizing the Expression of CYP3A4 and Efflux Transporters (P-gp, MRP1, and MRP2) in CYP3A4-Transfected Caco-2 Cells After Induction with Sodium Butyrate and the Phorbol Ester 12-O-Tetradecanoylphorbol-13-Acetate. Pharm Res 18, 1102–1109 (2001). https://doi.org/10.1023/A:1010914624111

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