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. 2001 Aug;18(8):1102-9. doi: 10.1023/a:1010914624111.


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 perinuclear 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • Blotting, Western
  • Butyrates / pharmacology*
  • Caco-2 Cells
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • DNA-Binding Proteins / biosynthesis*
  • Densitometry
  • Enzyme Induction / drug effects
  • GABA Modulators / metabolism
  • Humans
  • Intestinal Absorption / genetics
  • Microscopy, Confocal
  • Midazolam / metabolism
  • Mitochondrial Proteins*
  • Mixed Function Oxygenases / biosynthesis*
  • Multidrug Resistance-Associated Proteins*
  • MutS Homolog 3 Protein
  • Ribosomal Proteins / biosynthesis*
  • Saccharomyces cerevisiae Proteins*
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Transfection


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Butyrates
  • DNA-Binding Proteins
  • GABA Modulators
  • MRP2 protein, S cerevisiae
  • MSH3 protein, human
  • Mitochondrial Proteins
  • Multidrug Resistance-Associated Proteins
  • MutS Homolog 3 Protein
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Tetradecanoylphorbol Acetate
  • Midazolam
  • multidrug resistance-associated protein 1