Abstract
The aim of this work is to develop a highly sensitive assay system for P-gp-mediated transport by using two methods, induction of P-gp and short-term culture of Caco-2 cells. To induce P-gp in Caco-2 cells, cells were cultured in vinblastine-containing medium. The mRNA level of P-gp was approximately 7-fold higher in Caco-2 cells cultured with vinblastine (P-gp-induced Caco-2 cells) than in control cells. Western blot analysis showed a significant increase in P-gp expression. After cell differentiation, the mRNA level of P-gp was downregulated, however, P-gp-induced Caco-2 cells still possessed a 5.6-fold higher mRNA level of P-gp compared to control cells. Polarized transport of substrate drugs was greater in the monolayer of P-gp-induced cells than in that of control cells. Moreover, we found that P-gp expression in Caco-2 cells could be further enhanced by applying the higher concentration of vinblastine. Transport activity of P-gp in Caco-2 cells cultured with higher concentration of vinblastine was markedly higher than that in P-gp-induced Caco-2 cells and was comparable with that in MDR1-MDCKII cells. In conclusion, this study provided a stable and highly sensitive in vitro assay system that can identify compounds that are subject to P-gp-mediated efflux.
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
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ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
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ATP Binding Cassette Transporter, Subfamily B, Member 1 / physiology
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Animals
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Antineoplastic Agents, Phytogenic / pharmacokinetics
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Antineoplastic Agents, Phytogenic / pharmacology
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Biological Transport / drug effects
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Biological Transport / physiology
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Blotting, Western
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Caco-2 Cells
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Cell Differentiation / drug effects
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Cell Differentiation / genetics
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Cell Differentiation / physiology
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Cell Growth Processes / drug effects
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Cell Growth Processes / genetics
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Cell Growth Processes / physiology
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Cell Line
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Cytochrome P-450 CYP3A
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism
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Digoxin / pharmacokinetics
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Digoxin / pharmacology
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Gene Expression / drug effects
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Humans
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Membrane Transport Proteins / genetics
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Membrane Transport Proteins / metabolism
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Membrane Transport Proteins / physiology
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins / genetics
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Multidrug Resistance-Associated Proteins / metabolism
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Multidrug Resistance-Associated Proteins / physiology
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Peptide Transporter 1
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Pharmaceutical Preparations / metabolism*
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Quinidine / pharmacokinetics
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Quinidine / pharmacology
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Symporters / genetics
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Symporters / metabolism
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Symporters / physiology
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Verapamil / pharmacokinetics
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Verapamil / pharmacology
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Vinblastine / pharmacokinetics
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Vinblastine / pharmacology
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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Antineoplastic Agents, Phytogenic
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Membrane Transport Proteins
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Microfilament Proteins
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins
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Peptide Transporter 1
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Pharmaceutical Preparations
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RNA, Messenger
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Symporters
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villin
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Vinblastine
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Digoxin
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Cytochrome P-450 Enzyme System
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Verapamil
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Cytochrome P-450 CYP3A
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CYP3A4 protein, human
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Quinidine
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multidrug resistance-associated protein 1