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Drug Metabolism and Disposition Fast Forward
First published on January 8, 2009; DOI: 10.1124/dmd.108.023309

0090-9556/09/3704-737-744$20.00
DMD 37:737-744, 2009

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Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA

Wei Zhang, Jibin Li, Samantha M. Allen, Erica A. Weiskircher, Yuehua Huang, Rebecca A. George, Ramon G. Fong, Albert Owen, and Ismael J. Hidalgo

Absorption Systems, LP, Exton, Pennsylvania

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.

Address correspondence to: Ismael J. Hidalgo, Absorption Systems, LP, 436 Creamery Way, Suite 600, Exton, PA 19341. E-mail: ihidalgo{at}absorption.com






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