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Isolation and Characterization of Caco-2 Subclones Expressing High Levels of Multidrug Resistance Protein Efflux Transporter

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Abstract

Purpose. The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors.

Methods. The subclones were selected by a dilution cloning technique. The polarized efflux of [3H]-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (Papp) of [3H]-vinblastine in the basolateral (BL)-to-apical (AP) direction and in the AP-to-BL direction (Papp BL-to-AP/Papp AP-to-BL) across the cell monolayers. The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis. The kinetics parameters (K m, V max) of [3H]-vinblastine and the inhibitory constants (K I) of several known MDR1 substrates/inhibitors on the transport of [3H]-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared.

Results. Three subclones (#1, #20, #21) were selected based on their polarized efflux of [3H]-vinblastine. The Papp BL-to-AP/Papp AP-to-BL ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells. In the presence of GF-120918 (2 μM), a known MDR1-specific inhibitor, the Papp BL-to-AP/Papp AP-to-BL ratios were significantly decreased, suggesting that these cells were overexpressing MDR1. The K m values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells. In contrast, the V max values observed in the subclones were approximate 26-69% higher. The K I values observed for various known MDR1 substrates/inhibitors on [3H]-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones. The high functional efflux activities of these subclones were stable up to 6 months.

Conclusions. Subclones #1, #20, #21 express high levels of MDR1. These Caco-2 subclones may be useful models for profiling drugs for their MDR1 substrate activity and for establishing structure-transport relationships for this efflux transporter.

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  1. Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas, 66047

    Kazutoshi Horie, Fuxing Tang & Ronald T. Borchardt

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  1. Kazutoshi Horie
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Correspondence to Ronald T. Borchardt.

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Horie, K., Tang, F. & Borchardt, R.T. Isolation and Characterization of Caco-2 Subclones Expressing High Levels of Multidrug Resistance Protein Efflux Transporter. Pharm Res 20, 161–168 (2003). https://doi.org/10.1023/A:1022359300826

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