Kinetic analysis in living cells of the inhibition of the P-glycoprotein-mediated efflux of anthracyclines by vinca alkaloids

Abstract

Cells that overexpress the mdr 1 gene have decreased steady-state accumulation and increased efflux of many anticancer drugs including anthracyclines and vinca alkaloids. The mechanism(s) of P-glycoprotein-mediated efflux of drugs is (are) still poorly understood. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, the cellular accumulation has been examined of pirarubicin, doxorubicin and idarubicin alone and in conjunction with four vinca alkaloid derivatives--vinblastine, navelbine, vindesine and vincristine. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. It has been shown that the P-glycoprotein-mediated efflux of these three anthracyclines can be inhibited by vinca alkaloids derivatives. At pH 7.2, 50% of the P-glycoprotein-mediated efflux of daunorubicin and idarubicin was inhibited by about 40 +/- 10 microM vinblastine and that of pirarubicin by 10 +/- 2 microM vinblastine. The vinblastine concentration required to inhibit 50% of the active efflux of these anthracyclines did not depend on the anthracycline concentrations used, indicating that the inhibition was non competitive. The ability of navelbine, vincristine and vindesine to inhibit the active efflux of pirarubicin was also checked; 15 +/- 3 microM navelbine are required to inhibit 50% of the active efflux but at concentrations lower than 100 microM, neither vincristine nor vindesine were able to inhibit this efflux, indicating that the vinca alkaloids compounds which are the most efficient are the most lipophilic. For the four vinca alkaloids, the concentration required to inhibit 50% of the efflux was lower as the pH was higher. A detailed kinetics analysis of the P-glycoprotein-mediated efflux of pirarubicin in the presence of vinblastine indicates a non competitive inhibition with K(I) = 12 +/- 2 microM.