Differences in substrate specificity among glutathione conjugates (GS-X) pump family members: Comparison between multidrug resistance-associated protein and a novel transporter expressed on a cisplatin-resistant cell line (KCP-4)

Abstract

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB-3-1 derived cell lines, C-A500 and KCP-4 was examined; the former expresses multidrug resistance-associated protein (MRP1), whereas the latter is resistant to cis-diamminedichloroplatinum dichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P-glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was overexpressed on KCP-4. Membrane vesicles isolated from C-A500 and KCP-4, but not from KB-3-1, exhibited the ATP-dependent uptake of glutathione conjugates (GS-X) such as leukotriene C4 and 2,3-dinitrophenyl-S-glutathione (DNP-SG), indicating the presence of GS-X pumps on these cells. The uptake of these GS-X by membrane vesicles from C-A500 was approximately twice that in the case of KCP-4. Kinetic analysis indicated that the K(m) and V(max) values for DNP-SG uptake were 2.56 and 1.43 μM, and 570 and 160 pmol/min/mg protein for C-A500 and KCP-4, respectively. In marked contrast, significant ATP-dependent uptake of glutathione-platinum complex was observed only in membrane vesicles from KCP-4, but not those from KB-3-1 and C-A500. The transport properties of estradiol-17β-D-glucuronide (E217βG) were also different between the two cell lines. This was reflected in the findings that the ATP-dependent uptake of this conjugated metabolite in membrane vesicles from C-A500 (K(m) = 2.33 μM, V(max) = 34 pmol/min/mg protein) was much more extensive than that in the case of KCP-4 (K(m) = 5.5 μM, V(max) = 35 pmol/min/mg protein), and that comparable uptake was observed between KCP-4 and KB-3-1. Overall, a clear difference in substrate specificity among GS-X pump family members expressed on resistant tumor cells was demonstrated.