Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Abstract

Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 mM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-29,79-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

Applicability of free drug hypothesis to drugs with good membrane permeability that are not efflux transporter substrates: A microdialysis study in rats

“…It is of interest to note that the ER values of fexofenadine and digoxin measured by Caco‐2 cell assay were much greater than those by MDR1‐MDCK cell assay. It is well known that Caco‐2 cells express many major efflux transporters, including P‐gp, BCRP, and MRPs 31‐33 . These results suggest that fexofenadine and digoxin are not only substrates of P‐gp but also substrates of other efflux transporters, possibly BCRP and/or MRPs.…”

In Vitro Interactions of Epacadostat and its Major Metabolites with Human Efflux and Uptake Transporters: Implications for Pharmacokinetics and Drug Interactions

“…To further evaluate the relative contribution of P-gp and BCRP in the efflux of EPAC in Caco-2 cells, we conducted an additional study in MDR1/MRP2 double-KO and BCRP/MRP2 double-KO Caco-2 cell lines. It has been demonstrated that these KO cell lines, derived from Caco-2 cells (C2BBe1 clone), are similar to the wild type Caco-2 cells with respect to growth rate, morphology, differentiation, tight junction formation, passive permeability of model compounds, and stability of phenotype (Sampson et al, 2015). Two control substrates, digoxin (P-gp substrate) and estrone sulfate (BCRP substrate), were used in this study to ensure the validity of these KO models.…”

Concentration-dependent roles of DMT1 and ZIP14 in cadmium absorption in Caco-2 cells

“…The Caco-2-kh cell line may not be an unusual cell line since another subclone of Caco-2 cells, which showed the characteristics of matured intestinal epithelial cells from the beginning of culture, has also been reported (Sampson et al, 2015). This feature of the Caco-2-kh cell line allowed us to examine the uptake of Cd in the trans-well culture system within a few days after the introduction of siRNAs of metal transporters.…”