Reversal of P-glycoprotein-mediated multidrug resistance by siRNA in chemoresistant osteosarcoma cell lines Free

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Overexpression in osteosarcoma cells of the P-glycoprotein (P-gp) encoded by the MDR1 gene results in multidrug resistance (MDR) to many chemotherapeutic drugs, a major cause for treatment failures and relapses in osteosarcoma patients. We evaluated the in vitro inhibition of P-gp expression and reversal of the MDR phenotype in two chemoresistant osteosarcoma cell lines by RNA interference (RNAi) targeting the MDR1 gene. Four different siRNA constructs per cell line were liposome-transfected at 3 different concentrations (50, 100 and 150 nM) into the chemoresistant human osteosarcoma cell line 1547 and the rat osteosarcoma cell line 1661. Effect of RNAi on P-gp expression was evaluated by Western-Blot performed on total cell proteins extracted 24 to 96 hours after transfection. The reversion of the MDR phenotype was evaluated by cell cytotoxicity and cell proliferation assays over the same period of time. For each cell line, two of the 4 siRNA constructs transfected at concentrations of 100 nM led to efficient down-regulation of the MDR1 expression with less than 20% of the total P-gp expression remaining as early as 24 hours after cell transfection. More importantly, the down regulation of P-gp expression in vitro induced a 24-hour reversal of the MDR phenotype associated with a significant increase in chemosensitivity to methotrexate and cisplatinum (40% increase compared to controls), and to a lesser extent, to doxorubicin and vincristin (25% increase). Our results show that the MDR phenotype is reversible in vitro in chemoresistant osteosarcoma cell lines using siRNA-mediated MDR1 suppression. We are currently evaluating the potency of combination of different siRNAs in vitro and in vivo in an orthotopic osteosarcoma model in rats using a clinical-grade, tumor-targetted nanomolecular gene delivery system.