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This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.

Multidrug resistance (MDR) is a major problem facing patients with cancer. Although Neutrophil gelatinase-associated lipocalin (NGAL) is highly expressed in various cancers, the possible role of NGAL in MDR is still obscure. In this article, we evaluated the effect of NGAL on Rh123 accumulation in cancer cells. NGAL was first down-regulated by short hairpin RNA-mediated interference. In correlation with the reduced NGAL expression, intracellular Rh123 accumulation was significantly decreased. We finally observed that inhibiting both of the ERK1/2 and p38 MAPK could seriously down-regulate NGAL expression and also decrease the intracellular accumulation of Rh123, indicating that NGAL-mediated Rh123 accumulation is regulated by the phosphorylation of ERK1/2 and p38 MAPK. Pretreatment of MDA-MB-231 with NGAL recombinant protein and antibody had significant effects on the intracellular accumulation of Rh123, whereas little effect was observed in K562 cells treated with the same method, suggesting that NGAL was involved in the regulation of Rh123 accumulation in these two types of cancers, although different pathways. Here we provide new evidence that directly shows the possibility of small chemical substances Rh123 intracellular accumulation that is regulated by NGAL. These results suggest the possibility of NGAL involvement in drug transportation and cancer MDR formation, and indicate the potential of NGAL in cancer therapy., (© 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

The aim of this study was to investigate the effect of intracellular acidification on accumulation of rhodamine 123 (rh123) in non-mature cells with none or low expression of multidrug resistance MDR1. The expression of MDR1 mRNA was detected by real-time quantitative RT-PCR. Confocal laser microscopy was used to determine the calibration curve of intracellular acidification (pHi). MTT assay was used to detect the cytotoxicity of intracellular acidification on HL-60, MSC and CD34(+) cells from umbilical cord blood. Flow cytometry was applied to measure the influence of intracellular acidification. The results indicated that the intracellular acidification had no obvious cytotoxicity on HL-60, MSC and CD34(+) cells. The acidification resulted in the increased rhodamine 123 accumulation in HL-60, MSC and CD34(+) cells without P-gp activity. Moreover, the more primitive cells, the less accumulation of intracellular Rh123 were observed. It is concluded that the intracellular acidification can reverse the MDR of HL-60, MSC and CD34(+) cells.