Allosteric regulation of [ 3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine

Abstract

Plasma membranes were prepared from the P-glycoprotein expressing human breast cancer cell line MCF-7 ADR. [ 3H]Vinblastine bound to these membranes saturably with a B max of 24 pmol/ mg of protein and K D of 23 nM. In contrast, membranes from the parent cells MCF-7 WT, which do not express P-glycoprotein, did not bind [ 3H]vinblastine with high affinity. Cytotoxics known to be transported by P-glycoprotein inhibited the binding of [ 3H]vinblastine, as did multidrug reversing agents including the 1,4-dihydropyridine, dexniguldipine-HCl ( K 19, 15 nM). In dissociation kinetic experiments, dexniguldipine-HCl accelerated the dissociation of [ 3H]vinblastine from P-glycoprotein, indicating a negative heterotropic allosteric mechanism of action through a drug binding site distinct from that of vinblastine. Other 1,4-dihydropyridines tested also accelerated [ 3H]vinblastine dissociation from Pglycoprotein, however, multidrug reversing drugs of different chemical classes, including quinidine, verapamil and cyclosporin A did not. These results suggest that P-glycoprotein of MCF-7 ADR cell membranes possesses at least two drug acceptor sites which are allosterically coupled: receptor site-1 which binds vinca alkaloids, and receptor site-2 which binds 1,4-dihydropyridines such as dexniguldipine-HCl, which had the highest affinity of the tested derivatives.