The Effect Of Intracellular Acidification On The P-glycoprotein In The Multidrug Resistant Leukemia Cells

Background:As the major obstacle of chemotherapy,multidrug resistance(MDR) leads to the chemoresistance and the decreased survival in leukemias.The tumor MDR is induced by one drug and results in the tumor resistance to other drugs and the failure of chemical therapy.The mechanism of MDR is complicate,and one of the most important reasons is the expression of P-gp.The P-gp is encoded by the MDR1 gene,and could transport the lipophilia drugs out of the tumor cells,which results in the decreased therapeutic effect and the redistribution of the anticancer drugs.With the development of P-gp modulators to the third generation,the regulation of pogp is still unsatisfactory.In recent years,it has been reported that cancer cells show a strong tendency towards an alkaline deviation of the entire homeostasis when they are resistant to therapeutic intervention.In order to clarify the internal correlation between pH_i and P-gp in MDR leukemia cells,we tested a potential role for intracellular acidification in reversing of MDR through P-gp regulation.Objective:The aim of the study was to investigate the effect of intracellular acidification on the P-gp in K562/A02 cells,clarify the relation between intracellular acidification and the P-gp,and provide new strategy to reverse the MDR.Method:Confocal laser microscope was used to determine the pH_i of the cells,and MTT assay was for determining the cytotoxicity of intracellular acidification on K562 and K562/A02 cells.By using real-time RT-PCR and western blot,the influences of intracellular acidification on P-gp expression at mRNA and protein levels were determined.Flow cytometry was applied to detect the influence of intracellular acidification on the activity of P-gp.Confocal laser microscope was also used to determine the accumulation and distribution of doxorubicin in K562 and K562/A02 cells. Western blot assay was used to detect the effect of intracellular acidification on the expression of P-gp,the phosphorylation level of p38 mitogen-activated protein kinase (MAPK),the extracellular signal regulated kinase(ERK) and the c-Jun amino-terminal kinase(JNK).Results:The results indicated that intracellular acidification had no obvious cytotoxicity on K562 and K562/A02 cells,and it reduced the level of P-gp in a pH_i and time-dependent manner in K562/A02 cells.This occurred primarily at the mRNA level, which was revealed by real-time RT-PCR analysis.The P-gp expression was also down-regulated by intracellular acidification that revealed by western blot assay.The decrease of P-gp level was associated with a significant increased accumulation, decreased efflux of Rh123 and the increased accumulation of doxorubicin in the K562/A02 cells induced by the intracellular acidification.The phosphorylation level of p38 MAPK was decreased and the phosphorylation level of ERK and JNK was increased by the intracellular acidification.When the inhibitor of ERK was used,the phosphorylation level of p38 MAPK was increased significantly,and the JNK was not affected by the inhibitor.Conclusions:The data indicated that the intracellular acidification could reverse the MDR of K562/A02 cells through down-regulation of P-gp activity and expression.The intracellular acidification might down-regulated the P-gp expression through MAPK signal transportation pathway.