The absence of stereoselective P-glycoprotein- and multidrug resistance-associated protein-mediated transport of daunorubicin.

Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. Thus, both P-gp and MRP1 proteins are able to transport anthracycline but the role of chirality has not, up to now, been addressed. In this study, we compared the P-gp- and MRP1-mediated efflux of daunorubicin and its enantiomer WP900 in multidrug-resistant cells overexpressing either P-gp (K562/ADR cells) or MRP1 (GLC4/ADR cells). Using fluorescence techniques, we showed that in both cell lines the presence of the pump yielded a gradient of drug concentration: the intracellular free drug concentration in the cytosol was lower than the extracellular free drug concentration. Our data showed that the gradient of concentration generated by the pump was the same whether DNR or WP900 was used. This means that P-gp on the one hand and MRP1 on the other recognise WP900 as well as DNR and that the chirality of the molecule plays no role.

Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells.

Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs. In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR. However, the nature and role of intracellular vesicles in MDR are unclear. We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug-resistant counterpart K562/ADR that overexpresses P-glycoprotein. We used four anthracyclines (all of which are P-glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pKa and lipophilicity as daunorubicin. The intrinsic fluorescence of anthracyclines allowed us to use macro- and micro-spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells. We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3'-amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug-resistant cells. These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrug-resistant K562 cells.

Analysis of drug transport kinetics in multidrug-resistant cells: implications for drug action.

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins--the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1)--that pump drugs out of MDR cells. The intracellular level of a drug, which influences the drug's cytotoxic effect, is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). One possible pharmacological approach to overcoming drug resistance is the use of specific inhibitors that enhance the cytotoxicity of known antineoplastic agents. Many compounds have been proven to be very efficient in inhibiting P-gp activity, but only some of them can inhibit MRP1. However, the clinical results obtained so far by this approach have been rather disappointing. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs whose physicochemical properties favor the uptake of such drug by resistant cells. Our recent studies have shown that whereas the P-gp- and MRP1-mediated efflux of different anthracycline-based drugs may not differ considerably, their kinetics of uptake do. Thus, the high uptake of drug by cells may lead to concentrations at the cellular target site high enough to achieve the needed cytotoxicity against MDR cells. Therefore, increased drug lipophilicity might be one factor in improving drug cytotoxicity in MDR cells. In vitro studies have shown that idarubicin, an analogue of daunorub cin, is more effective than daunorubicin and doxorubicin against MDR tumor cell lines and that this increased effectiveness is related in part to the increased lipophilicity of idarubicin. Other studies have also confirmed the strong impact of lipophilicity on the uptake and retention of anthracyclines in MDR cells.