Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ADM cells.
The calcium channel blocker verapamil has been shown to reverse multidrug resistance (T. Tsuruo et al., Cancer Res. 41: 1967-1972, 1981), but the mechanism of action of this agent has not been fully elucidated. A radioactive photoactive analogue of verapamil, N-[benzoyl-3,5-3H-(+/-)-5-[(3,4-dimethoxyphenetyl)methylamino]-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylpentylamine, was used to label the plasma membranes of a human myelogenous leukemia cell line (K562), a multidrug-resistant subline selected for resistance to Adriamycin (K562/ADM) and its revertant cell (R1-3). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled Mr 170,000-180,000 protein in the membranes from K562/ADM but not from the drug-sensitive parental K562 and revertant R1-3 cells. The Mr 170,000-180,000 verapamil acceptor was immunoprecipitated by monoclonal antibody MRK16 specific for P-glycoprotein associated with multidrug resistance, indicating that P-glycoprotein in the plasma membrane is a major target of verapamil in K562/ADM cells. The photolabeling of P-glycoprotein with N-[benzoyl-3,5-3H]-(+/-)-5-[(3,4-dimethoxyphenetyl)methylamino]-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylphentylamine was significantly blocked by other calcium channel blockers, nicardipine and diltiazem, that have been shown to overcome multidrug resistance. In addition, the photolabeling was partially blocked by Adriamycin, vincristine, and colchicine, suggesting that the specific binding sites for verapamil on P-glycoprotein are closely related to the binding sites for these calcium channel blockers and antitumor agents. To determine whether verapamil could be a substrate for P-glycoprotein, the cellular accumulation of [3H]verapamil into K562 and K562/ADM was evaluated. The accumulation of [3H]verapamil in the multidrug-resistant cells was 30% of K562 cells and increased when K562/ADM cells were treated with vincristine and nicardipine at 5 microM, indicating that the P-glycoprotein transports verapamil as well as other antitumor agents in the multidrug-resistant cells. These results suggest that verapamil enhances antitumor agent retention through competition for closely related binding sites on P-glycoprotein.
Yusa
K
K
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo.
Tsuruo
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001692:Biological Transport; D001798:Blood Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004351:Drug Resistance / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D011485:Protein Binding; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001798:Blood Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C060845:(N-benzoyl)-5-((3,4-dimethoxyphenethyl)methylamino)-2-(3,4-dimethoxyphenyl)-2-isopropyl-N-4-azidobenzoylpentylamine; D014700:Verapamil