Culture Period-Dependent Change of Function and Expression of ATP-Binding Cassette Transporters in Caco-2 Cells

• Published in: Drug metabolism and disposition Vol. 37; no. 9; pp. 1956 - 1962
• Main Authors: KAMIYAMA, Emi, SUGIYAMA, Daisuke, NAKAI, Daisuke, MIURA, Shin-Ichi, OKAZAKI, Osamu
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01-09-2009
• Subjects: Angiotensin II Type 1 Receptor Blockers - metabolism; ATP Binding Cassette Transporter, Sub-Family G, Member 2; ATP-Binding Cassette Transporters - biosynthesis; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Biological and medical sciences; Blotting, Western; Caco-2 Cells; Calcium Channel Blockers - pharmacology; Digoxin - metabolism; Electric Conductivity; Estrone - analogs & derivatives; Estrone - metabolism; Humans; Imidazoles - metabolism; Leukotriene Antagonists - pharmacology; Medical sciences; Multidrug Resistance-Associated Proteins - metabolism; Neoplasm Proteins - metabolism; Ochratoxins - metabolism; Pharmacology. Drug treatments; Propionates - pharmacology; Quinolines - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; Tetrazoles - metabolism; Tight Junctions - metabolism; Time Factors; Verapamil - pharmacology; Digestive system; ABC transporter; Gut
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.109.027490

The objective of this study was to determine an appropriate culture period to assess whether a compound of interest is transported
by efflux transporters such as human multidrug resistance 1 (hMDR1), human multidrug resistance-associated protein 2 (hMRP2),
and human breast cancer resistance protein (hBCRP) in Caco-2 cells. Caco-2 cells were cultured on a Transwell for 1 to 6 weeks.
The expression of these transporters in the mRNA and protein levels was examined using a real-time polymerase chain reaction
and Western blotting, respectively. Transcellular transport activities using digoxin, ochratoxin A, olmesartan, and estrone-3-sulfate
were also examined. Except for digoxin, the permeability coefficient ( P app ) ratio of the three compounds at 2 weeks was the highest in the periods tested. The P app ratio of digoxin at 2 weeks was higher than that at 3 weeks. The temporal expression profile of each transporter in the mRNA
level was similar to that in the protein level, and the functions of hMRP2 and hBCRP were roughly correlated with the expression
in the mRNA and protein levels, but that of hMDR1 was not. These data suggest that among all the culture periods evaluated
a 2-week culture is the best culture period for transport studies to identify whether a compound is a substrate for hMDR1,
hMRP2, and hBCRP.