RT Journal Article
SR Electronic
T1 Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 737
OP 744
DO 10.1124/dmd.108.023309
VO 37
IS 4
A1 Wei Zhang
A1 Jibin Li
A1 Samantha M. Allen
A1 Erica A. Weiskircher
A1 Yuehua Huang
A1 Rebecca A. George
A1 Ramon G. Fong
A1 Albert Owen
A1 Ismael J. Hidalgo
YR 2009
UL http://dmd.aspetjournals.org/content/37/4/737.abstract
AB 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. The American Society for Pharmacology and Experimental Therapeutics