Relation between MDR1 mRNA levels, resistance factor, and the efficiency of P-glycoprotein-mediated efflux of pirarubicin in multidrug-resistant K562 sublines.

In this work, we sought to investigate the relation existing between MDR1 mRNA levels, the resistance factor (RF), and the efficiency of efflux of pirarubicin (THP) mediated by P-glycoprotein (P-gp) in multidrug-resistant (MDR) K562 sublines. The MDR K562 sublines were selected from K562/adr cells by exposure to different adriamycin concentrations: 300 nM (K562/300), 1,000 nM (K562/1,000), and 10,000 nM (K562/10,000), yielding RF values of 23.2, 26.5, and 39.6, respectively. The analysis of the P-gp encoding MDR1 gene overexpression by reverse transcriptase - polymerase chain reaction provided evidence of increased MDR1 mRNA levels when the adriamycin concentration used for the MDR cell selection increased. We used spectrofluorometric methods to determine the kinetics of the uptake and P-gp-mediated efflux of THP in the different selected MDR K562 sublines. Our data showed that (i) the maximal rate of P-gp-mediated efflux of THP, Vmax, increased with increasing RF; (ii) the observed Michaelis constant, Km, had the same value for all selected sublines, thus leading to an overall increase in the ratio Vmax/Km (5.1 x 10(-3), 6.2 x 10(-3), 6.8 x 10(-3), and 9.3 x 10(-3) s(-1) for K562/adr, K562/300, K562/1,000, and K562/10,000 cells, respectively), and (iii) the determination of the Hill coefficient (nH) gave values close to 2, which suggested a positive cooperative transport of THP with the expelling of two molecules of THP per turnover of P-gp. This study demonstrated that, in the K562/adr sublines used in our experiments, P-gp played a major role in conferring the MDR phenotype. Moreover, under our experimental conditions, intracellular acidic organelles were shown to contribute to decreased drug-target interaction and, thereby, decreased cytotoxicity. The variation of the concentrations of THP accumulated in the acidic organelles as a function of the total TFP concentration added to the cells was the same, within the limits of experimental errors, whatever the degree of resistance of the studied MDR K562 sublines. Finally, this study suggested that, in the selected MDR K562 sublines, the K+/H+ antiporter exchanger could be activated by the pirarubicin transport, leading to a probable acidification of intracellular pH. The P-gp-mediated efflux of THP and an accumulation of THP in acidic organelles confer an advantage for MDR cells in surviving prolonged exposure to cytotoxic agents and giving rise to high degrees of resistance.