Abstract
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 (Papp) ratio of the three compounds at 2 weeks was the highest in the periods tested. The Papp 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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.027490.
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ABBREVIATIONS: hMDR1, human multidrug resistance 1; hMRP2, human multidrug resistance-associated protein 2; hBCRP, human breast cancer resistance protein; TEER, transepithelium electrical resistance; ES, estrone-3-sulfate; LY, Lucifer yellow; ZO-1, zonula occludens; MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; FTC, fumitremorgin C; TB, transport buffer; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Papp, permeability coefficient.
- Accepted June 4, 2009.
- Received March 9, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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