ATP-dependent uptake of anti-neoplastic agents by acidic organelles

Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I. (1986) Cancer Res. 46, 5941-5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H(+)-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+. Quinidine (5 microM), verapamil (5 microM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H(+)-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A1 or concanamycin A, or by increasing the internal pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide dissipated the delta pH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the delta pH established by vacuolar-type ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.