Gene Silence Of Nucleophosmin (NPM) Increased The Drug Sensitivity On Acute Lymphoblastic Leukemia Cells CA46/Jurkat

Objective According to our previous investigation, 2-DIGE(fluorescence two-dimensional differential in gel electrophoresis)-based proteomic approach identified nucleophosmin(NPM, B23) as over-expressed proteins in relapsed/refractory acute leukemia patients. High expression of NPM may be involved in the mechanism of drug resistance.In this study, we investigated the biological changes of CA46 and Jurkat lymphoblastic leukemia cells, including cell proliferation, apoptosis and drug sensitivity, explored the potential mechanism of increased drug sensitivity by knockdown of NPM in cell lines.Methods 1. Plasmid encoding short hairpin RNAs(sh RNA) were designed against NPM. Lentiviral-based vectors were packaged and infected CA46/Jurkat cells respectively. The efficiency of RNA interference was detected by western blot and real-time quantitative PCR. 2. MTT and colony formation assays were applied to access the cell growth inhibition and to evaluate the effect on cell proliferation by knockdown of NPM in CA46/Jurkat cell lines. 3. Annexin V-FITC single staining FACS cytometry analysis was performed to acess apoptosis. Proteins related to apoptosis were detected by western blot. 4. MTT assay was used to evaluate the drug sensitivity in CA46/Jurkat cell lines by inhibiting the NPM expression. Proteins and m RNA related resistance were detected by western blot and real-time quantitative PCR. 5. Western blot was also performed to study the change of related signaling pathways.Results 1. Lentivirus vetors(NPM-RNAi-LV) targeted NPM gene silencing were successfully constructed. Lentivirus mediated transfection successfully knocked down the expression of NPM. 2. Knockdown of NPM in CA46/Jurkat cells via RNAi resulted in significant inhibition of cell proliferation and colony formation ability in CA46/Jurkat cells. 3. Inhibition of NPM expression could markedly induce CA46/Jurkat cells to apoptosis. In this study, we found that caspase family was activated, cleaved PARP was up-regulated and anti-apoptotic protein Bcl-2 was down-regulated. 4. NPM gene silencing could partially increase the sensitivity to adriamycin(ADM) in CA46 and Jurkat cells. The IC50 of CA46 and Jurkat cells were reduced from 0.526±0.138 ?g/ml and 0.528±0.098 ?g/ml(before NPM RNAi) to 0.099±0.025 ?g/ml and 0.223 ± 0.031 ?g/ml, after RNA interference to silence NPM expression, respectively. 5. NPM gene silencing could downregulate the expression of BCRP/LRP, suppress the activation of AKT/m TOR signaling pathway and inhibit the expression of c-Myc in both CA46 and Jurkat cells. Besides, the p-ERK was down-regulated in Jurkat.Conclusion RNA interference could inhibit the expression of NPM in CA46 and Jurkat cells. Cell proliferation was suppressed and apoptosis was increased by knockdown of NPM. The possible mechanism was associated with caspase activation, cleaved PARP up-regulation and Bcl-2 family changes. At the same time, knockdown of NPM could partially enhance the sensitivity to adriamycin in CA46 and Jurkat cells, with dowm-regulation of BCRP, LRP, c-Myc, GST- ? and AKT/m TOR signaling.In addition, it may be related to p-ERK signaling in Jurkat.