Kinetics of Anthracycline Efflux from Multidrug Resistance Protein-Expressing Cancer Cells Compared with P-Glycoprotein-Expressing Cancer Cells

Abstract

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.

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

“…As can be seen, for the same extracellular drug concentration, the fluorescence signal is higher in sensitive cells than in resistant cells. Similar experiments were performed in energy-depleted cells [10] and the values of F cyto obtained were similar to that determined for sensitive cells. This allowed us to say that the parameter A value was the same for both sensitive and resistant cells.…”

“…Recently, we have performed several studies using K562 intact cells to describe the kinetics of anthracycline transport in MDR cells in order to predict how modifications in the anthracycline molecule affect its transport characteristics [10][11][12][13]. In the present paper we have used the same cell line to characterize the transport of several rhodamines.…”

“…P-gp can transport neutral and positively charged molecules but not negatively charged ones. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane [5][6][7][8][9].Recently, we have performed several studies using K562 intact cells to describe the kinetics of anthracycline transport in MDR cells in order to predict how modifications in the anthracycline molecule affect its transport characteristics [10][11][12][13]. In the present paper we have used the same cell line to characterize the transport of several rhodamines.…”

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

“…It has also been presented that in the case of nonactivated DOX, the kinetics of cellular uptake was comparable with the rate of the efflux by MDR exporting pumps (P-glycoprotein and MRP1). Consequently, a drastic decrease in the cellular accumulation of the drug determining its cytotoxic activity was observed (Mankhetkorn et al, 1996;Marbeuf-Gueye et al, 1998, 1999. Furthermore, there is an increasing number of data demonstrating that the enzymatic activation of DOX results in the formation of reactive intermediates capable of alkylation or crosslinking binding with DNA (Cummings et al, 1991;Cullinane et al, 1994;Skladanowski and Konopa, 1994).…”

Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia

“…[10][11][12] The transport kinetics of anthracyclines by P-gp and MRP1 are rather similar. 13 An important difference between P-gp and MRP1 is that MRP1 transports cationic and neutral compounds only in the presence of glutathione (GSH). 14,15 Recently six additional MRP family members, MRP2 to MRP7, have been identified.…”