The result indicated that the expression of survivin in HCT116p53+/+ cells is much lower than in HCT116p53-/- cells (Fig. 3A), suggesting the high expression of survivin in HCT116p53-/-

cells may act as a contributing factor to Caspase inhibitor clinical trial bortezomib resistance. Similar results were obtained in other cancer cell lines with different p53 status (Fig. 3B). Consistently, MDA-MB-231 has much higher tumorigenic ability than MCF-7 in mouse xenograft models. Figure 3 Survivin Expression in wild type vs. p53 null cancer cell sublines. A. HCT116 and HCT116p53-/-. B. MCF-7 with wild type HDAC inhibitor p53 and MDA-MB-231 with mutant p53. C. Kms11 with wild type p53 and RPMI-8226 with mutant p53. Sub-confluent cells were lysed,

and the cell lysates were used to determine survivin expression by western blots. Actin is the internal control for total protein loading. The expression of survivin in wild type p53 cells was set at 1 and relative survivin expression is shown after normalization with the actin internal control. Bortezomib induces survivin expression in HCT116p53-/- cells but shows no significant effect on survivin expression in HCT116p53+/+ cells We then tested whether bortezomib could differentially modulate survivin Wnt inhibitors clinical trials expression between HCT116p53+/+ cells and HCT116p53-/- cells. Consistent with the fact that HCT116p53-/- cells are resistant to bortezomib-induced growth inhibition and apoptosis induction, bortezomib appears to significantly induce survivin expression in HCT116p53-/- cells, while it shows minimal induction of survivin in HCT116p53+/+ cells (Fig. 4A). Similar results were also obtained in other cancer cell lines (Fig. 4B), indicating a general principle of this phenomenon. Figure 4 Differential effects of bortezomib on survivin in HCT116p53 -/- cells versus HCT116

cells. A. HCT116 and HCT116p53-/-. B. LNCap with wild type p53 and PC-3 with null p53. Sub-confluent cells were treated with and without bortezomib for 48 hours. Cells were then collected and lysed for western Phosphoglycerate kinase blots to determine survivin expression. Actin was used as the internal control for total lysate protein loading. The expression of survivin in wild type p53 cells was set at 1 and relative survivin expression is shown after normalization with actin. Silencing of survivin expression in HCT116p53-/- cells by survivin mRNA-specific siRNA sensitizes bortezomib-induced growth inhibition To test whether survivin expression indeed plays a role in bortezomib resistance, we employed survivin mRNA-specific siRNA approach [35] to silence survivin expression in HCT116p53-/- cells, which highly expresses survivin. Significantly, we noted that silencing of the expression of survivin (Fig. 5A) reverses bortezomib resistance to growth inhibition (Fig. 5B) and cell death induction (Fig.