Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells.

We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks' balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0 degrees C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Transport mechanism of anthracycline derivatives in rat polymorphonuclear leukocytes: effect of sodium fluoride on pirarubicin uptake.

We previously revealed that pirarubicin (THP) was actively taken up by rat polymorphonuclear leukocytes via a carrier-mediated transport system. In the experiment on the effects of the metabolic inhibitors, rotenone, 2,4-dinitrophenol and sodium cyanide significantly decreased the THP transport. However, sodium fluoride (NaF) significantly increased the uptake, and this result is different from that in some reports. Therefore, we examined the action of NaF on THP uptake by the leukocytes to clarify the discrepancy in the effect of NaF on drug transport. The accelerating effect of 30 mM NaF on the THP uptake by the cells had an optimum period of action (15-20 min), and was concentration-dependent (5-30 mM). Thirty mM potassium fluoride, as well as NaF, increased the uptake amount. On the other hand, NaF (5-30 mM) dose-dependently decreased the ATP content in these cells. Additionally, the viable cells in the reaction suspension decreased by about 40% after incubation with 30 mM NaF for 15 min. Observing these leukocytes treated with NaF by optical microscopy, swelling of the cell and an alteration of the nuclei form occurred. On the basis of these results, we speculated that the increased THP transport in polymorphonuclear leukocytes by NaF, probably F-, might be due, at least in part, to an alteration of the morphological form.