Study Of NF-κB In The Regulation Of Multidrug Resistance Gene Transcription In Acute Myeloid Leukemia

Objective Multidrug resistance (MDR) was a major problem in treatment failure of human acute leukemia and numerous mechanisms had been known. One of the most important mechanisms was the overexpression of mdr-1 gene and its product, P-glycoprotein (P-gp). NF-κB was a family of ubiquitous transcription factors, which played an important role in inflammation responses and stress responses. Recent publications indicated that NF-κB played an important role in the regulation of mdr-1 expression in various cell lines. Until now the study on the role for NF-κB in the regulation of mdr-1 expression in primary tumor cells had not been reported. We screened primary drug-resistant acute myeloid leukemia (AML) in newly diagnosed AML and demonstrated the role of NF-κB in the regulation of the mdr-1 gene expression in human primary drug-resistant AML.Methods Mononuclear cells from peripheral blood or bone marrow taken from newly diagnosed AML were separated through a Ficoll-Conray density gradient. P-gp expression was assessed by immunocytochemisty dyeing with P-gp antibody. P-gp function was assessed by flow cytometry using Rho123 (rhodamine123) efflux test. Two methods were used to screen primary drug-resistant AML cases. Cells were divided into two groups. One was an experimental group with MG-132 1μmol/L and the other was a control group. Cells were stained with NF-κB P65 antibody after being cultured for 6 hours, then laser confocal microscope and real-time PCR were used to evaluate the change of NF-κB activity and mdr-1 RNA level. Apoptosis of primary AML cells was detected by flow cytometry after MG-132 treatment for 6 hours and 12 hours marked with Annexin V and 7AAD.Results 11 of 36(30.6%) newly diagnosed AML cases were immunocytochemistry positive and Rho123 efflux test positive. MG-132 1μmol/L for 6 hours obviously reduced the distribution of NF-κB in the cell nuclear and the activity of NF-κB was inhibited. Mdr-1 transcription was decreased largely and mRNA level was decreased by 4.3±2.3 folds. MG-132 1μmol/L for 12 hours synergized strongly to induce robust apoptosis, mean viability of 12-hour group cells was significantly lower than that of control group cells (49±9% versus 79±3%, P<0.01); MG-132 1μmol/L for 6 hours was only mildly toxic to cells and mean viability of cells was not significantly different with control group cells (82±5%versus 86±5%, P>0.05); mean viability of cells of 12-hour group was significantly lower than that of 6-hour group cells (49±9% versus 82±5%, P<0.01).Conclusion Primary drug-resistant cases were immunocytochemistry positive and Rho123 efflux test positive. NF-κB positively regulates the transcription of mdr-1 in primary drug-resistant AML. The inhibition of NF-κB activity not only decreases the mdr-1 expression but also increases cell apoptosis in primary drug-resistant AML.