Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers

Jamie A. Wright, Iain S. Haslam, Tanya Coleman, Nicholas L. Simmons

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km = 69.41 ± 22.3 μM and Vmax = 14.03 ± 2.27 nmol/(cm 2.h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP.

Original languageEnglish
Pages (from-to)70-76
Number of pages7
JournalEuropean Journal of Pharmacology
Volume672
Issue number1-3
Early online date10 Oct 2011
DOIs
Publication statusPublished - 15 Dec 2011
Externally publishedYes

Fingerprint

Nitrofurantoin
Breast Neoplasms
rosiglitazone
Proteins
Caco-2 Cells
Epithelium
Protein Transport
ATP Binding Cassette Transporter, Sub-Family G, Member 2
ATP-Binding Cassette Transporters
Prazosin
Verapamil
Drug Interactions
Pharmaceutical Preparations
Real-Time Polymerase Chain Reaction
Up-Regulation
Western Blotting
Diet
Cell Line
Messenger RNA

Cite this

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title = "Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers",
abstract = "In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km = 69.41 ± 22.3 μM and Vmax = 14.03 ± 2.27 nmol/(cm 2.h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92{\%} by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP.",
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Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers. / Wright, Jamie A.; Haslam, Iain S.; Coleman, Tanya; Simmons, Nicholas L.

In: European Journal of Pharmacology, Vol. 672, No. 1-3, 15.12.2011, p. 70-76.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers

AU - Wright, Jamie A.

AU - Haslam, Iain S.

AU - Coleman, Tanya

AU - Simmons, Nicholas L.

PY - 2011/12/15

Y1 - 2011/12/15

N2 - In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km = 69.41 ± 22.3 μM and Vmax = 14.03 ± 2.27 nmol/(cm 2.h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP.

AB - In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km = 69.41 ± 22.3 μM and Vmax = 14.03 ± 2.27 nmol/(cm 2.h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP.

KW - β-napthoflavone

KW - BCRP

KW - Caco-2 cells

KW - Induction

KW - Intestinal secretion

KW - Nitrofurantoin

KW - Rosiglitazone

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U2 - 10.1016/j.ejphar.2011.10.004

DO - 10.1016/j.ejphar.2011.10.004

M3 - Article

C2 - 22004608

AN - SCOPUS:81255199239

VL - 672

SP - 70

EP - 76

JO - European Journal of Pharmacology

JF - European Journal of Pharmacology

SN - 0014-2999

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