RESUMEN
The multidrug resistance protein (MRP) has been shown to mediate ATP-dependent efflux of anticancer agents of diverse structure, such as daunorubicin (DNR), vincristine and etoposide. Thus, this protein does confer a multidrug resistant phenotype to cancer cells, similar to P-glycoprotein (Pgp). The substrate specificity of both transporter proteins is partly overlapping but is otherwise very distinct; because MRP is a multiple organic anion transporter, it transports certain glutathione conjugates and may be partly dependent on intracellular glutathione levels for the transport of anthracyclines. We have studied the transport kinetics of a series of anthracyclines in MRP and Pgp that overexpress tumor cell lines to obtain information on the substrate specificity of these proteins. The anthracyclines have modifications in the sugar moiety. The mean active efflux coefficient Ka, used to characterize the efficiency of the active efflux, was very similar for DNR and one of its 4'-deoxy-derivatives (eso-DNR) for MRP and Pgp [10-20 x 10(-10)/sec/(cells/ml)]. The permanently neutral derivatives 3'-deamino-3'-hydroxy-doxorubicin (OH-DOX) and 3'-deamino-3'-hydroxy-daunorubicin (OH-DNR) were effluxed by both proteins but had a lower Ka [2 x 10(-10) and 6 x 10(-10)/sec/(cells/ml) (OH-DOX)] and 2 x 10(-10) and 5 x 10(-10)/sec/(cells/ml) (OH-DNR)] for MRP and Pgp. Two anthracyclines, the doxorubicin derivative pirarubicin and 2'-bromo-4'-epi-DNR seemed to have a slightly higher Ka value for Pgp than for MRP. The apparent Michaelis-Menten constants (K(m)) and maximal efflux rates (VM) for the active transport were within a narrow range for both transporters, except for OH-DOX and OH-DNR, which had a lower VM in the case of MRP-mediated transport, suggesting a role of the amino group in the interaction with glutathione. Determination of the Hill coefficient (nH) of the MRP-mediated efflux gave most values close to 2, which suggests cooperativity of the transport of anthracyclines as reported before for Pgp. In conclusion, the transport kinetics of anthracyclines by MRP and Pgp are very similar.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Carcinoma de Células Pequeñas/metabolismo , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacocinética , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Humanos , Cinética , Leucemia Eritroblástica Aguda/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Resistencia a Múltiples MedicamentosRESUMEN
Multidrug resistance (MDR) is frequently associated with decreased cellular drug accumulation resulting from enhanced drug efflux. This is correlated with the presence of a membrane protein, the P-glycoprotein, which pumps a wide variety of drugs out of cells, reducing their intracellular concentration and thus their toxicity. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the P-glycoprotein-medicated efflux. Four anthracycline derivatives, doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin, which have the same amino sugar, were used to analyze the respective contribution of the kinetics of uptake and the P-glycoprotein-mediated efflux in their impaired accumulation in MDR cells. The kinetics of uptake of the four drugs vary over a very large range: the kinetics of uptake of daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin are 16, 200, and 400 times higher than that of doxorubicin, respectively. However, the four drugs are extruded by P-glycoprotein at comparable rates. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic drug concentrations at half-maximal velocity for the cell lines used, were approximately 2.2 microM for daunorubicin and and approximately 1 microM for idarubicin and 8-(S)-fluoroidarubicin.