Targeting XIAP for Chemosensitization Purposes: Different Effect with Different Cytotoxic Drugs.

Defects in apoptosis have been implicated in the resistance of cancer cells to a wide variety of anticancer drugs. XIAP, an inhibitor of both intrinsic and extrinsic apoptotic pathways by inhibiting caspases-3, -7 and -9, has been proposed as a good molecular target for enhancing the effects of cytotoxic drugs since:

• it is widely expressed in human cancer cell lines and human cancer tissues;

• downregulation of XIAP expression enhanced the effects of chemotherapeutic agents in various cancer cell line models;

• XIAP expression correlates with prognosis in some cancers, including in acute myeloid leukemia.

Small molecules able to inhibit XIAP have been developed. Furthermore, antisense oligonucleotide inhibitors of XIAP (AEG35156) are already in clinical trials. However, the potential chemosensitization effect of XIAP has not been thoroughly explored, both concerning different tumor models and drugs. The purpose of the current study was to investigate if downregulation of XIAP enhances the effects of doxorubicin and of cytarabine, drugs used in the treatment of acute leukemias, in an in vitro model of blastic phase Chronic Myeloid Leukemia, the K562 cell line. The approach was to downregulate XIAP in K562 cells by using:

• transient transfection with siRNAs and

• stable transfection with shRNAs cloned into an appropriate vector.

Two different siRNAs targeting XIAP were tested, using two different transfection conditions for siRNA delivery. Relatively modest downregulation of XIAP was observed by Western Blot, and the more potent siRNA sequence and transfection conditions were chosen for the chemosensitization studies. Our results show that downregulation of XIAP expression:

• reduced cellular viability but this effect was not significant;

• sensitized cells to doxorubicin (IC50 decreased from 78nM to 55nM) but not to cytarabine.

To confirm these results, stable cell lines were established by transfection of shRNAs cloned into an appropriate vector. Modest downregulation of XIAP was achieved in the stable cell line when compared to the control shRNA cell line. In these cell lines the results confirmed that downregulation of XIAP sensitizes to doxorubicin (IC50 decreased from 68nM to 50nM) but not to cytarabine. Further analysis of the cells following treatment with the cytotoxic drugs allowed to confirm that both drugs induced programmed cell death by apoptosis (with cleavage of pro-caspase 3) and further reduction of XIAP protein levels, which is probably due to the apoptotic process itself. However, the shRNAs did not provide a better model for studying chemosensitization than the siRNAs. This fact, together with the modest levels of XIAP downregulation, probably reflects that the shRNA sequence corresponds to the siRNA sequence previously used. In conclusion, downregulation of XIAP sensitized K562 cells to doxorubicin but not to cytarabine. This may be partially due to the level of XIAP downregulation obtained but may also be due to the different mechanisms of action of these drugs, indicating that this should be taken into account when considering targeting XIAP for achieving sensitization to cytotoxic drugs.