Silencing the breast cancer resistance protein expression and function in caco-2 cells using lentiviral vector-based short hairpin RNA.
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.
Zhang
Wei
W
Absorption Systems, LP, 436 Creamery Way, Suite 600, Exton, PA 19341, USA.
Li
Jibin
J
Allen
Samantha M
SM
Weiskircher
Erica A
EA
Huang
Yuehua
Y
George
Rebecca A
RA
Fong
Ramon G
RG
Owen
Albert
A
Hidalgo
Ismael J
IJ
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D018938:Caco-2 Cells; D017931:DNA Primers; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D000089762:Multidrug Resistance-Associated Protein 2; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D000089762:Multidrug Resistance-Associated Protein 2; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012313:RNA