| Objective: The development of multidrug resistance (MDR) of leukemia cells is the main cause of the chemotherapy failure and leukemia recurrence. At present, all the drugs for MDR reversion are not very effective. The reason is that mechanisms of MDR are complicated and different mechanisms often exist at the same time. Recently it was found that the intracellular pH (pHi) of MDR leukemia cells is higher than that of sensitive cells. It suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp, MRP, LRP and BCRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance.Sodium hydrogen exchanger isoform-1 (NHE-1) is the main ionic channel protein to regulate pHi of leukemia cells by 1:1 efflux of H+ and influx of Na+ ions. The expression and activity of NHE-1 directly regulate pHi of leukemia cells. It has been confirmed that chemosensitivity of leukemia cells decreases when its pHi increases. So, NHE-1 may be related with the development of MDR of leukemia cells, and even may be the "common pathway" of different mechanisms of MDR. This study was designed to research the role of NHE-1 in MDR mechanisms of leukemia cells and search for new theoretical support and method for MDR reversion.Methods: HL-60/ADM cells with multidrug resistance induced by adriamycin (ADM) were experimental group, and the sensitive HL-60 cells were used as control group. (1) The proliferation and the chemosensitivity of cells were studied by MTT assay method. (2) The expression of multidrugresistance protein (MRP) on the membrane of cells was detected by immunocytochemistry. (3) pHi was measured using fluorescence dye BCECF-AM. (4) The pHi recovery curve was drawn after intracellular acid loading to show the activity of NHE-1. (5) The expression of NHE-1mRNA and MRP mRNA were determined by semi-quantitive RT-PCR method. (6) Cell apoptosis was observed with terminal deoxynucleotidyl transferase-medited dUTP nick end labeling (TUNEL) and apoptotic DNA was extracted and electrophoresed. (7) The above indexes were measured after administration of the speacific NHE-1 inhibitor DMA.Results: (1) The IC50 values, for ADM, MTZ, VCR and H, in HL-60/ADM cells were higher than those in HL-60 cells significantly (P<0.01), and the values of resistance fold were all more than 20 times. HL-60/ADM cells expressed abundant MRP, but HL-60 cells did not express. (2) pHi value of HL-60/ADM cells increased significantly than that of HL-60 cells(P<0.001). The expression and activity of NHE-1 in HL-60/ADM cells increased significantly than those of HL-60 cells. (3) After Administration of the specific NHE-1 inhibitor DMA within a certain range of concentrations, the rate of growth inhibition of HL-60/ADM cells increased significantly than that of HL-60 cells(P<0.05). (4) After Administration of DMA, the sensitivities of HL-60/ADM cells to ADM, MTZ, VCR and H increased significantly than those of HL-60 cells (P<0.01), and the expression of MRP and MRP mRNA of HL-60/ADM cells decreased significantly than those in HL-60 cells (P<0.01). Meanwhile, the apoptosis rate of HL-60/ADM cells increased significantly than that of HL-60 cells(P<0.01) and the DNA ladders of HL-60/ADM cells were more typical than that of HL-60 cells after Administration of DMA.Conclusions:(1) Expressing MRP on the membrane, HL-60/ADM cell line showed resistance to multiple drugs with different chemical structure and different mechanisms of drug resistance, so it could be used for MDRresearch in vitro. (2) It is the increase of expression and activity of NHE-1 in HL-60/ADM cells that play an important role in that pHi value of HL-60/ADM cells is higher significantly than that of HL-60 cells. (3) The growth inhibition rate of HL-60/ADM cells was higher significantly than that of HL-60 cells after Administration of DMA within a certain range of concentrations. (4) The NHE-1 inhibitor could reverse MDR of HL-60/ADM cells, and the mechanisms were associated with decrease of MRP exp...
|
PDF Full Text Request