Reversal Of Drug-resistance Of Leukemia K562/ADM Cells To Chemotherapeutical Agents By RNA Interference

ObjectiveTo investigate the effect of small interfering RNA (siRNA) on the expression of mdr1 and P-glycoprotein (P-gp) and the reversal of drug-resistance of multidrug-resistant leukemia K562/ADM cells, and to explore the molecular mecanism of siRNA reversing the drug-resistance in leukemia cells.MethodsHuman multidrug-resistant leukemia cell line K562/ADM overexpressing mdr1 gene was used as the target cells. Four siRNAs (si-mdr1/333,si-mdr1/764, si-mdr1/ 1074 and si-mdr1/2832) speciafically targeting mdr1 mRNA were chemically synthesized and transfected into K562/ADM cells with RNAi-Mate. The silencing efficiency of siRNAs were evaluated by the inhibition of mdr1 mRNA expression measured with RT-PCR and Real-time PCR. The expression of caspase-9 and caspase-3 mRNA was examined with RT-PCR. P-glycoprotein (P-gp) expression and its function, mitochondrial inner membrane potential (Δψm) and active Caspase-3 were measured by flow cytometry (FCM). Double staining of FITC-Annexin V and propidium iodide (PI) were used to detect apoptosis in K562/ADM cells. The sensitivity of the cells to Adriamycin (ADM),Daunorubicin (DNR) and Etoposide (Vp16) was detected with a MTT colorimetric assay.ResultsThe siRNA target to the site 333 of mdr1 mRNA (si-mdr1/333) possessed the best silencing efficiency to the given gene. After transfected with si-mdr1/333, the expression of mdr1 mRNA was reduced by 69% and 63% detected with RT-PCR and Real-time PCR, respectively. The P-gp positive rate declined from 95.6% to 62.3% after transfecting with si-mdr1/333 for 72h, the function of P-gp was restrained so that the content of ADM was increased from control 50.6% to 93.6%. si-mdr1/333 treated for 48h, the expression caspase-9 and caspase-3 mRNA was not raised, but Caspase-3 activity was markedly enhanced, the activeCaspase-3 increased from 0.84% to 14.5%. There was no evident change in mitochondial inner membrane potential treated with si-mdr1/333 for 48h. si-mdr1/333 could significantly enhance the sensitivity of K562/ADM cells to ADM, DNR and Vp16, the reversal efficiency was 3.45-folds, 1.70-folds and 2.91-folds, respectively. The Vp16-induced apoptosis of K562/ADM cells was greatly augmented, the apoptotic rate increased from 2.32% (control) to 14.42%( si-mdr1/333 + Vp16) after exposure with 40μmol/L Vp16 for 24 hours.ConclusionsiRNA can speciafically down-regulate the expression of mdr1 gene and its product P-gp in multidrug-resistant K562/ADM cells and improve the sensitivity to general chemotherapeutical agents. The possible reversing mechanism is that siRNA blocks the expression of mdr1 and its product P-gp, and weakens the suppression of P-gp on Caspase-3 activity to overcome the P-gp-mediated multidrug-resistance and apoptosis resistance in drug-resistant K562/ADM cells.