The Effects of Culture Conditions on CYP3A4 and MDR1 mRNA Induction by 1α,25-Dihydroxyvitamin D3 in Human Intestinal Cell Lines, Caco-2 and LS180

doi:10.2133/dmpk.20.268

Drug Metabolism and Pharmacokinetics

Volume 20, Issue 4, 2005, Pages 268-274

https://doi.org/10.2133/dmpk.20.268 Get rights and content

Summary:

To evaluate an in vitro model suitable for investigating intestinal first-pass drug metabolism, CYP3A4 and MDR1 mRNA induction by 1α,25-dihydroxyvitamin D₃ (VD3) was examined in two human intestinal cell lines, Caco-2 and LS180, under various culture conditions. CYP3A4 mRNA expression was induced by 100 nM VD3 at levels between 234–549 times above normal in Caco-2 cells for 2 weeks and by 74–200 times above normal in LS180 cells for 2 days. The CYP3A4 induction effect of 250 nM VD3 was similar to or slightly higher than that of 100 nM VD3 in both Caco-2 and LS180 cells. Also, CYP3A4 was induced in Caco-2 and LS180 cells when they were cultured on a polystyrene plate slightly less than when they were cultured on a porous membrane. The increase in fetal bovine serum (FBS) content in the culture medium resulted in little or only slight increase of CYP3A4 induction in both Caco-2 and LS180 cells. MDR1 mRNA expression was marginally increased by VD3 in LS180 cells, but not in Caco-2 cells, and neither increased FBS content nor use of a porous membrane significantly affected MDR1 induction in LS180 cells. The transepithelial electrical resistance of LS180 cells was almost zero, whereas that of Caco-2 cells was high and was marginally decreased by VD3. These findings indicate that Caco-2 cells cultured on a porous membrane with 100 nM VD3 for 2 weeks may be used as a model to investigate the intestinal absorption and first-pass metabolism of drugs, while LS180 cells may be utilized to elucidate the mechanisms which regulate intestinal CYP3A4 mRNA expression.

References (31)

H. Komura et al.
Species difference in nisoldipine oxidation activity in the small intestine
Drug Metab. Pharmacokinet.
(2002)
M. Goto et al.
Decreased expression of P-glycoprotein during differentiation in the human intestinal cell line Caco-2
Biochem. Pharmacol.
(2003)
M. Watabe et al.
Role of c-Myc in nitric oxide-mediated suppression of cytochrome P450 3A4
Life Sci.
(2003)
K. Takara et al.
Digoxin up-regulates MDR1 in human colon carcinoma Caco-2 cells
Biochem. Biophys. Res. Commun.
(2002)
X.T. Wang et al.
The mRNA of L-type calcium channel elevated in colon cancer: protein distribution in normal and cancerous colon
Am. J. Pathol.
(2000)
H. Hara et al.
Alteration of cellular phosphorylation state affects vitamin D receptor-mediated CYP3A4 mRNA induction in Caco-2 cells
Biochem. Biophys. Res. Commun.
(2002)
S. Lu et al.
Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes
J. Pharm. Sci.
(1996)
C.M. Banwell et al.
Targeting 1α,25-dihydroxyvitamin D₃ antiproliferative insensitivity in breast cancer cells by co-treatment with histone deacetylation inhibitors
J. Steroid. Biochem. Mol. Biol.
(2004)
J.C. Kolars et al.
CYP3A gene expression in human gut epithelium
Pharmacogenetics
(1994)
M.F. Paine et al.
Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism
J. Pharmacol. Exp. Ther.
(1997)

V. Andersen et al.

Intestinal first pass metabolism of midazolam in liver cirrhosis-effect of grapefruit juice

Br. J. Clin. Pharmacol

(2002)

T. Aiba et al.

Poor correlation between intestinal and hepatic metabolic rates of CYP3A4 substrates in rats

Pharm. Res

(2003)

Y. Hashimoto et al.

Effects of intestinal and hepatic metabolism on the bioavailability of tacrolimus in rats

Pharm. Res

(1998)

J.H. Lin

Applications and limitations of interspecies scaling and in vitro extrapolation in pharmacokinetics

Drug Metab. Dispos

(1998)

T. Matsubara et al.

Isolation and characterization of a new major intestinal CYP3A form, CYP3A62, in the rat

J. Pharmacol. Exp. Ther.

(2004)

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The expression of P-gp in different cell lines is also different, leading to different effects of VD on P-gp in different cell lines. For example, the effect of VD on MDR1 mRNA in LS180 cells is more significant than that in Caco-2 cells [158], and is stronger in LS174T cells than in HepG2 cells [159]. VD could regulate MRP 2, 3, and 4.
Vitamin D supplementation and vitamin D deficiency are common in clinical experience and in daily life. Vitamin D not only promotes calcium absorption and immune regulation, but also changes drug effects (pharmacodynamics and adverse reactions) and drug disposal in vivo when combined with various commonly used clinical drugs. The extensive physiological effects of vitamin D may cause synergism effects or alleviation of adverse reactions, and vitamin D's affect on drugs in vivo disposal through drug transporters or metabolic enzymes may also lead to changes in drug effects. Herein, the effects of vitamin D combined with commonly used drugs were reviewed from the perspective of drug efficacy and adverse reactions. The effects of vitamin D on drug transport and metabolism were summarized and analyzed. Hopefully, more attention will be paid to vitamin D supplementation and deficiency in clinical treatment and drug research and development.
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Induction patterns for MDR1 (Fig. 10C), OATP1A2 (Fig. 10D), and TRPV6 (Fig. 10E) were also similar, although the change in MDR1 mRNA expression was of a much lower magnitude. Similar magnitudes of the induction for CYP3A4 (extremely high mRNA fold-changes (200–400) and MDR1 (50% mRNA fold-change) were also observed by Aiba et al. [39]. TRPV6 fold changes in Caco-2 cells were lowest for TPD-090, then TPD-014, TPD-002, and TPD-012, with the remaining compounds showing similar response to 1,25(OH)2D3.
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^†: This work was supported in part by a grant from the Nakatomi Health Science Foundation.

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Regular ArticleThe Effects of Culture Conditions on CYP3A4 and MDR1 mRNA Induction by 1α,25-Dihydroxyvitamin D3 in Human Intestinal Cell Lines, Caco-2 and LS180†

Summary:

Drug Metab. Pharmacokinet.

Biochem. Pharmacol.

Life Sci.

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Biochem. Biophys. Res. Commun.

J. Pharm. Sci.

J. Steroid. Biochem. Mol. Biol.

CYP3A gene expression in human gut epithelium

Pharmacogenetics

Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism

J. Pharmacol. Exp. Ther.

Intestinal first pass metabolism of midazolam in liver cirrhosis-effect of grapefruit juice

Br. J. Clin. Pharmacol

Poor correlation between intestinal and hepatic metabolic rates of CYP3A4 substrates in rats

Pharm. Res

Effects of intestinal and hepatic metabolism on the bioavailability of tacrolimus in rats

Pharm. Res

Applications and limitations of interspecies scaling and in vitro extrapolation in pharmacokinetics

Drug Metab. Dispos

Isolation and characterization of a new major intestinal CYP3A form, CYP3A62, in the rat

J. Pharmacol. Exp. Ther.

Regular Article
The Effects of Culture Conditions on CYP3A4 and MDR1 mRNA Induction by 1α,25-Dihydroxyvitamin D₃ in Human Intestinal Cell Lines, Caco-2 and LS180^†