Abstract
A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based short hairpin RNA (shRNA). Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1), and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp in particular play a significant role in pharmacokinetics because of their expression at several key interfaces. Overexpression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. The shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence microscopy, and bidirectional transport of probe substrates, estrone-3-sulfate (E3S), and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates.
Footnotes
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This work was supported in part by the U.S. Department of Health and Human Services, Food and Drug Administration [Grant 1R43 FD-003482-01].
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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.108.023309.
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ABBREVIATIONS: DDI, drug-drug interaction; P-gp, P-glycoprotein; BCRP, breast cancer resistance protein; ABC, ATP-binding cassette; MRP, multidrug resistance protein; MDR, multidrug resistance; RNAi, RNA interference; siRNA, small interfering RNA; shRNA, short hairpin RNA; E3S, estrone-3-sulfate; TBS, Tris-buffered saline; FTC, fumitremorgin C; PhA, pheophorbide A; RT-PCR, reverse transcription-polymerase chain reaction; qPCR, quantitative polymerase chain reaction; PBS, phosphate-buffered saline; OATP, organic anion transporting polypeptide; BSA, bovine serum albumin; TEER, transepithelial electrical resistance; HBSSg, Hanks' balanced salt solution with glucose; Papp, apparent permeability coefficient.
- Received July 10, 2008.
- Accepted January 5, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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