Evaluation of XIAP as a Molecular Target To Increase Sensitivity to Imatinib.

Imatinib, a potent and selective inhibitor of BCR-ABL, has emerged as an effective drug for targeted therapy of CML. However, resistance to this drug has been arising, mainly due to an overexpression of BCR-ABL, to mutations in the tyrosine-kinase domain of BCR-ABL or to an overexpression of the multidrug resistance protein (Pgp). Cytotoxic drugs induce cellular apoptosis and resistance to these drugs is often due to resistance to apoptosis. Cancer cells may escape apoptosis due to an overexpression of anti-apoptotic proteins such as XIAP, which binds caspases 3, 7 and 9 and inhibits their action.

Downregulation of XIAP expression has been documented to increase sensitivity of several cell lines to some conventional drugs. In the current study we investigated if downregulation of XIAP expression, following treatment with XIAP-siRNAs, would increase sensitivity to Imatinib in: i) a sensitive CML cell line (K562), when compared to treatment with control siRNAs and ii) a resistant cell line which overexpresses Pgp (K562Dox), when compared to treatment with Pgp-siRNAs or with control siRNAs. To confirm if the siRNAs were capable of downregulating the expression of their targets, Western Blots were carried out at 24, 48 and 72h after transfection. The cells had been: i) treated with medium only (Blank), ii) transfected with a control-siRNAs, iii) transfected with siRNAs for XIAP and iv) tranfected with siRNAs for Pgp (in the K562Dox cell line only). It was verified that the XIAP-siRNAs decreased XIAP protein levels in both cell lines 48h following transfection and Pgp-siRNAs decreased Pgp protein levels in the resistant cell line (K562Dox) 24h following transfection, when compared to the control-siRNA treatments. To further investigate if the downregulation of XIAP expression (and Pgp in the resistant cell line) caused sensitization of cells to1μM Imatinib, Imatinib was added to the cells previously transfected with siRNAs (for P-gp or XIAP or control siRNAs) and to cells in exponential growth (Blank cells). Viable cell number was counted by the Trypan Blue exclusion assay and apoptosis was verified with the TUNEL assay. Results of viable cell numbers from several experiments indicate that Imatinib treatment on its own, as expected, decreased more the viable cell number of the sensitive cell line than of the resistant cell line (from 100% in the Blank cells to 63 % in the treated K562 cells and to 86 % in the treated K562Dox cells). Furthermore, downregulation of XIAP expression on its own decreased the viable cell number of both cell lines (from 100% in the Blank cells to 62% in the K562 XIAP-siRNA treated cells and to 80% in the K562Dox XIAP-siRNA treated cells). Concomitant treatment with both XIAP-siRNAs and Imatinib further reduced the number of viable cells to 36% in the K562 cells and to 57% in the K562Dox cells. In the resistant cell line, downregulation of Pgp expression on its own reduced the viable cell number from 100% in Blank cells to 84% in the Pgp-siRNA treated cells. Concomitant treatment with Pgp-siRNAs and Imatinib further reduced the viable cell number to 60%. The reduction of viable cell number coincided with an increase in apoptosis. It was concluded that downregulation of XIAP expression may be a good approach to enhance sensitivity to Imatinib, even in the case of existing Pgp overexpression.