Decreased Expression of Nucleophosmin/B23 Increases Drug Sensitivity of Adriamycin-Resistant Lymphoblastic Leukemia Molt-4 Cells through mdr-1 Regulation

Nucleophosmin /B23 (NPM, also known as B23, numatrin, or N038) is regularly identified as multifunctional nuclear protein, not only an important player

in ribosome biogenesis but also a potential regulator for cell proliferation. Aberrant expression of NPM, such as mutation, deletion, over-expression or rearrangement, could lead to malignant transformation in tumor cells. It is reported that over-expression of NPM in solid tumor had been detected as a poor prognostic factor and related to drug-resistance development. In adult de novo acute myeloid leukemia (AML), mutated NPM without FLT3-ITD showed a good response to chemotherapy in AML. Although a great deal of knowledge of NPM has been accumulated, little information on the role of NPM in drug resistance in leukemia is available. According to our previous investigation, NPM levels are up regulated in drug-resistant cell lines and primary leukemic cells. High expressions of NPM could be observed in refractory or relapsed acute leukemia patients, which significantly

correlated with poor prognosis. In this study, we wanted to evaluate whether NPM is involved in drug resistance in lymphocytic leukemia cells. We generated an adriamycin (ADM)-resistant lymphoblastic cell line Molt-4/ADR (MAR). Cell proliferation, sensitivity to chemotherapy agents and expressions of drug resistance related molecules were assessed. The IC50 of Molt-4 cells were 0.584±0.113umol/L and of MAR cells were 22.56±1.94 umol/L, meaning MAR cells were 38.63 fold resistant to ADM. The confocal microscopy assay showed that MAR cells had very low levels of ADM fluorescence,

however, when treated with the P-gp inhibitor Verapamil Hydrochloride, ADM accumulation in MAR cells increased greatly. These cells were not only resistant to other MDR agents like daunorubicin and vincristine, but also slightly resistant to a panel of non-MDR agents, such as VP-16 and cyclophosphamide. Furthermore, MAR cells gained an expression of mdr-1(P-gp) and a higher expression of NPM compared to Molt-4 cells. Knockdown of NPM by RNA interference (RNAi) suppressed the viability of both Molt-4 and MAR cells. In both Molt-4 and MAR cells, IC 50 of NPM knockdown groups decreased sharply. IC50 of Molt-4 cells was reduced from 0.582±0.112 μmol/L before NPM RNAi to 0.186±0.019μmol/L after NPM RNAi. IC50 of MAR was reduced from 22.56±1.94μmol/L before NPM RNAi to 3.83±0.381μmol/L after NPM RNAi. Knocking down NPM not only increased Molt-4 cells’ drug sensitivity but also reverse resistance of MAR towards Adriamycin. With an increase of drug sensitivity, we found the down-regulation of P-gp and Akt /mTOR signaling in MAR cells after NPM RNAi. To further understanding the relationship between NPM and mdr-1, we applied RNAi to down-regulate mdr-1 in MAR cells. Knockdown of mdr-1 could also reverse the drug resistance. NPM expression revealed no significantly fluctuation after mdr-1 silencing . It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating P-gp and Akt /mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance.