Upregulation of mdr1 gene is related to activation of the MAPK/ERK signal transduction pathway and YB-1 nuclear translocation in B-cell lymphoma

Abstract

Objective: Multidrug resistance (MDR) in human B-cell lymphoma constitutes a major obstacle to the effectiveness of chemotherapy. The aim of this study was to investigate the molecular mechanism of MDR in B-cell lymphoma.

Materials and methods: The B-cell lymphoma MDR sublines were developed by exposing the parental Daudi cells to stepwise increasing concentrations of doxorubicin. Interaction of Y-box binding protein-1 (YB-1) with the Y-box motif of the mdr1 gene promoters was studied by electrophoretic mobility shift assay. The effects of YB-1 on mdr1 promoter activity were examined by luciferase assay. After silencing of YB-1 gene by shRNA, the role of YB-1 nuclear translocation in the formation of induced MDR was examined. Expression of mdr1 and YB-1 was examined further after Daudi cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitor PD98059 for 1 hour.

Results: Doxorubicin-resistant sublines was generated from the Daudi cell line by stepwise selection in doxorubicin. We found that acquisition of MDR is associated with enhanced YB-1 nuclear translocation and MAPK/extracellular signal-regulated kinase (ERK) activity. Electrophoretic mobility shift assay revealed that doxorubicin increased binding of YB-1 to the Y-box of mdr1 promoter. Luciferase reporter assays demonstrated that the Y-box region is essential for YB-1 regulation of mdr1 expression. The introduction of exogenous YB-1 shRNA into Daudi cells resulted in decreased levels of the expression of mdr1 gene and P-glycoprotein induced by doxorubicin. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 and the expression of mdr1 gene.

Conclusion: Doxorubicin can increase expression of mdr1/P-glycoprotein through activating MAPK/ERK transduction pathway, then increasing expression of YB-1, inducing YB-1 nuclear translocation, and enhancing DNA-binding activity of YB-1.