Imatinib Mesylate (Gleevec, formely STI571 from NOVARTIS) is a highly selective drug, capable of inhibiting the BCR-ABL tyrosine kinase which is responsible for CML. However, resistance to this drug has been documented, mainly due to an overexpression of the BCR-ABL protein, to mutations in the tyrosine-kinase domain of BCR-ABL itself or to an overexpression of the multidrug resistance protein 1 (P-gp). In the current study we investigated if downregulation of P-gp expression with siRNAs, in a cell line resistant to Imatinib which overexpressed P-gp (K562Dox), would allow to overcome resistance to this drug. In order to optimise transfection of the siRNAs, we used a fluorochrome labelled control-siRNA and fluorescence microscopy to evaluate the % of uptake of the siRNA under different transfection conditions. The optimised conditions (200 nM siRNA using the jetSi™ transfection reagent) allowed us to obtain uptake of siRNAs in 53% of cells. In order to confirm if the siRNAs were capable of downregulating the expression of P-gp we carried out, 24h after transfection, Real-Time Quantitative PCR studies and Western Blots of cells: i) treated with medium only (Blank), ii) transfected with the siRNAs suspension buffer, iii) transfected with a control siRNA (CRNAi), iv) transfected with two different siRNAs for P-gp (MDR-FE or MDR-CR). We verified that in cells treated with the MDR-CR siRNA there was a reduction in the P-gp mRNA expression of 26%, when compared to the CRNAi. Furthermore, when cells were treated with the MDR-FE or MDR-CR siRNAs, protein levels were also reduced by 33% and 57% respectively, when compared to the CRNAi. It was then verified if this downregulation of MDR expression caused sensitization of cells to Imatinib, in comparison to control cells, using both viability (Trypan Blue) and apoptosis (TUNEL) assays. For this purpose, Imatinib Mesylate was added to the cells previously transfected with siRNAs (for P-gp or control siRNAs) and to cells in exponential growth (Blank cells). Results showed that there was an enhancement of the effect of Imatinib in cells previously transfected with both P-gp siRNAs (MDR-FE or MDR-CR). This increase in the effect of Imatinib was due to an increase in cellular apoptosis. Indeed, the % of apoptotic cells increased from 3 % in cells treated with solvent to 7 % in cells treated with 1 μM Imatinib. It was concluded that downregulation of P-gp protein expression by RNAi technology permitted sensitisation of CML cells to Imatinib, suggesting that P-gp inhibition may be considered as an adjuvant therapy in cases of Imatinib resistance mediated by this mechanism.

This work was financed by NOVARTIS PHARMA AG.

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