Delineating the Contribution of Secretory Transporters in the Efflux of Etoposide Using Madin-Darby Canine Kidney (MDCK) Cells Overexpressing P-Glycoprotein (Pgp), Multidrug Resistance-Associated Protein (MRP1), and Canalicular Multispecific Organic Anion Transporter (cMOAT)

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Abstract
Multidrug resistance conferred to cancer cells is often mediated by the expression of efflux transporter “pumps”. It is also believed that many of the same transporters are involved in drug efflux from numerous normal endothelial and epithelial cell types in the intestine, brain, kidney, and liver. Etoposide transport kinetics were characterized in Caco-2 cells and in well established Madin-Darby canine kidney (MDCKII) cell lines that were stably-transfected with a human cDNA encoding P-glycoprotein (Pgp), human multidrug resistance protein (MRP1), or the canalicular multispecific organic anion (cMOAT) transporters to determine the roles of these transporters in etoposide efflux. Etoposide transport kinetics were concentration-dependent in the MDCKII-MDR1 and MDCKII-cMOAT cells. The apparent secretory Michaelis constant (Km) and carrier-mediated permeability (Pc) values for Pgp and cMOAT were 254.96 ± 94.39 μM and 5.96 ± 0.41 × 10−6 cm/s and 616.54 ± 163.15 μM and 1.87 ± 0.10 × 10−5 cm/s, respectively. The secretory permeability of etoposide decreased significantly in the basal to apical (B to A) (i.e., efflux) direction, whereas the permeability increased 2.3-fold in the apical to basal (A to B) direction in MDCKII-MDR1 cells in the presence of elacridar (GF120918). Moderate inhibition of etoposide efflux by leukotriene C4 (LTC4) was observed in MDCKII-cMOAT cells. Furthermore, etoposide inhibited LTC4efflux, confirming the involvement of cMOAT. The flux of etoposide in MDCKII-MRP1 cells was similar to that in MDCKII/wt control cells. The current results demonstrate that the secretory transport mechanism of etoposide involves multiple transporters, including Pgp and cMOAT but not MRP1. These results demonstrate that Pgp and cMOAT are involved in the intestinal secretory transport of etoposide. Since the intestinal secretion of etoposide was previously reported in the literature, it also suggests that they may be involved in the in vivo intestinal secretion of etoposide; however, mechanistic in vivo studies are required to confirm this.