Role of Na^+/H^+ exchanger isoform-1 in doxorubicin-induced multidrug-resistance HL-60 cell line

Objective: To explore the roles of intracellular pH value (pHi) and sodium-hydrogen exchanger isoform-1 (NHE-1) in the mechanism of multidrug resistance of leukemia cells. Methods: Multidrug resistant cell line HL-60 induced by doxorubicin(DOX) (called as HL-60/DOX cells) and their parent cell line HL-60 were employed as experiment group and control group. The proliferation and chemosensitivity of the cells were studied by MTT assay, and the expression of multidrug resistance protein (MRP) was detected by immol/Lunocytochemistry. Meanwhile, pHi was measured by spectrofluorometery with a fluorescence dye BCECF-AM. Based on the pHi recovery curve after intracellular acid loading, the activity of NHE-1 was analyzed. The expression of NHE-1 mRNA and MRP mRNA were determined by semi-quantitative RT-PCR. Cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and apoptotic DNA was extracted and electrophoresed. Results: ① The IC 50 values for DOX, MTZ, VCR and homoharringtonine(HT), in HL-60/DOX cells were significantly higher than those in HL-60 cells (P<0.01). HL-60/DOX cells expressed abundant MRP, but HL-60 cells did not. ② pHi of HL-60/DOX cells were significantly higher than that of HL-60 cells(P<0.001). The expression and activity of NHE-1 in HL-60/DOX cells were significantly stronger than those of HL-60 cells. ③After administration of the specific NHE-1 inhibitor dimethyl amiloride (DMA) at a certain range of concentrations, compared with HL-60 cells, the rate of growth inhibition of HL-60/DOX cells increased significantly (P<0.05), the drug-sensitivity of HL-60/DOX cells was significantly sensitive (P<0.01), the expression of MRP and MRP mRNA decreased significantly (P<0.01), the apoptosis rate increased significantly (P<0.01). Conclusion: NHE-1 is involved in the drug-resistant mechanisms of multidrug-resistant HL-60 cells induced by DOX. The specific NHE-1 inhibitor DMA can partly reverse the multidrug resistance of HL-60 cells induced by DOX.

Proteomic Analysis for Finding Serum Pathogenic Factors and Potential Biomarkers in Multiple Myeloma

Background： Multiple myeloma （MM） is a malignant tumor, which takes the second place in malignant blood disease. The clinical syrnptoms are complicated that make more difficult to diagnose and therapy. Lots of researches locus on the proteins about MM in order to solve those problems. We used proteomic methods to find potential biomarkers in MM patients. Methods： We applied the peptide ligand library beads （PLLBs） to deplete high abundance proteins in serum for finding potential pathogenic factors and biomarkers of MM. Using 1D-Gel-liquid chromatography-tandem mass spectrometry （LC-MS/MS）, we identified 789 and 849 unique serum proteins in MM patients and in healthy controls, respectively. Results： Twenty-two proteins were found differentially expressed between the two groups including sernm anayloid A protein, vitamin D-binding protein isoform-1 precursor, plasma kallikrein, and apolipoprotein A-I. Changes of integrin alpha-11 and isoform-1 of multimerin-l were validated with Western blotting. The linkage of the differentially expressed proteins and the pathogenesis pathways of MM were discussed, Conclusions： PLLB combined with 1D-gel-LC-MS/MS analysis is an efficient method to identity differentially expressed proteins in serum from patients with MM.