Allosteric regulation of [3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine.
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 Bmax of 24 pmol/mg of protein and KD 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 (Ki, 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 P-glycoprotein, 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.
Ferry
D R
DR
University of Birmingham CRC Institute for Cancer Studies, Queen Elizabeth Hospital, Edgbaston, U.K.
Malkhandi
P J
PJ
Russell
M A
MA
Kerr
D J
DJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000494:Allosteric Regulation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004095:Dihydropyridines / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D004357:Drug Synergism; D006801:Humans; D007477:Ions; D007700:Kinetics; D009711:Nucleotides; D011485:Protein Binding; D014316:Tritium; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004095:Dihydropyridines; D007477:Ions; D009711:Nucleotides; D014316:Tritium; D014747:Vinblastine; C054074:niguldipine