Reversal Of P-Glycoprotein-Mediated Multidrug Resistance With Small Interference RNA (SIRNA) In Leukemia Cells

A major cause of treatment failure in hematological malignancies is the emergence of drug resistant cells, either at diagnosis or at the time of relapse. The typical MDR phenotype is due to the overexpression of membrane proteins among which the main representative is P- glycoprotein (P-gp) encoded by the MDR1 gene. Many attempts to modulate MDR by chemosensitizers have been unsuccessful in human therapy due to their intrinsic toxic effects.The present study was designed to determine if siRNA against MDR1 gene can effectively inhibit expression of MDR1 gene product, P-gp. We first designed 3 different siRNAs targeting MDR1 gene and tested their effectiveness in silencing MDR1 mRNA expression. The results showed that transfection of one siRNA duplex (si-MDR1) caused a significant inhibition of MDR1 mRNA expression in human MDR cell line K562/A02. The reduction of P-gp expression was then detected in K562/A02 cells by Western blot and flow cytometry analysis. A significant suppression of P-gp expression by si-MDR1 was observed. We then investigated whether the treatment with si-RNA could not only reduce P-gp expression level, but also recover the sensitivity to the P-gp transportable cytotoxic drugs, such as doxorubicin(ADM), vincristine(VCR) and etoposide (VP-16), in K562/A02 cells. The IC50 values that obtained in K562/A02 cells after exposure to cytotoxic agents were found to be decreased only in the si-MDR1-treated K562/A02 cells. The si-MDR1 caused about 4-, 11-, 17- fold reversal of cellular resistance to ADM, VCR and VP-16, respectively. DNR retention is known as one of the most sensitive assays for P-gp function (6). An increase of intracellular DNR retention in si-MDR1-treated cells was observed, confirming the inhibition of P-gp function by siRNA targeted MDR1. One base pare mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression. In conclusion, the present work demonstrates that introduction of an MDR1-targeted small interfering RNA duplex into drug-resistant leukemia cells can effectively inhibit the expression of MDR1 mRNA and P-gp, resulting in an increase in the intracellular accumulation of P-gp transportable chemotherapeutic drugs and in the sensitivity restoration of leukemia cells to the chemotherapeutic drugs. This approach may be applicable to leukemia or other cancer as a specific means to reverse tumor cells with a P-gp-dependent MDR phenotype back to a drug-sensitive one.