Masako Nomiyama

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

Abstract 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°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.

Ticlopidine inhibits activation of mitogen-activated protein kinase by platelet-derived growth factor in cultured rat renal mesangial cells

BackgroundWe previously found that ticlopidine inhibits the proliferation of cultured rat mesangial cells that is induced by fetal bovine serum. This study was designed to examine the effects of ticlopidine on platelet-derived growth factor (PDGF)-induced DNA synthesis and mitogen-activated protein (MAP) kinase activation in such mesangial cells to clarify the mechanism of the antiproliferative action.MethodsGlomerular mesangial cells were isolated from rat kidneys, and cells were incubated with various combinations of ticlopidine, PDGF, epidermal growth factor, phorbol 12-acetate 13-myristate (PMA), cilostazol (a phosphodiesterase inhibitor), and H-89 (a cAMP-dependent protein kinase A [PKA] inhibitor). A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell count involving the trypan blue exclusion test were performed for determination of cell viability. DNA synthesis and MAP kinase activity were assessed by means of a tritiated thymidine ([3H]thymidine) incorporation assay and the BiotrakTM MAP kinase assay system, respectively.ResultsTiclopidine (1 μmol/L) significantly inhibited both PDGF-induced DNA synthesis and MAP kinase activation. Also, 1 μmol/L ticlopidine substantially blocked PMA-induced MAP kinase activation. Pretreatment with H-89 did not abolish the ability of ticlopidine to inhibit PDGF-induced MAP kinase activation, while H-89 pretreatment significantly reserved the inhibitory action of cilostazol on PDGF-induced MAP kinase activation.ConclusionThese results suggest that ticlopidine might inhibit PDGF-induced DNA synthesis after MAP kinase activation by intercepting the signal transduction from c-Raf-1 to MAP kinase, independent of the cAMP-PKA pathway.