Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising candidate for treatment of cancer, and displays variable cytotoxicity in cell lines. The mechanisms of sensitivity and resistance have not been fully elucidated, but both AKT and NF-κB pathways have been shown to modulate cytotoxic responses. We analyzed the effects of hsp90 inhibition by 17-AAG, which has been shown inhibit signaling through AKT and NF- κB, on cytotoxicity of TRAIL in the resistant colon cancer cell lines, HT29 and RKO. IC50 values for a 72-hour exposure to TRAIL in these lines were 2000 and 3000 ng/ml, respectively. Cytotoxicity assays demonstrated synergism of the TRAIL/17-AAG combination, with combination indices at IC50 of 0.68 for RKO cell line and 0.77 for HT29 cells. In these cell lines, the combination of 17-AAG and TRAIL resulted in activation of both extrinsic and intrinsic apoptotic pathways, though with quantitative differences between HT29 and RKO cells. Inhibition of AKT by 17-AAG was more profound in RKO cells, whereas HT29 cells demonstrated greater inhibition of NF-κB activation by 17-AAG. Analyzing these pathways pharmacologically, we found greater synergism of TRAIL with inhibitors of AKT in the RKO cell line, while combinations with NF-κB inhibitors were more effective in HT29 cells. Thus, despite equivalent baseline activation status of NF-κB and AKT in the two cell lines, the apoptotic signal induced by the TRAIL/17-AAG combination resulted from selective inhibition by 17-AAG of primarily NF-κB in HT29 or AKT in RKO cell lines. Despite these observed differences between the two cell lines, the combination ultimately led to release of apoptotic factors from mitochondria, down-regulation of X-linked inhibitor of apoptosis protein (XIAP) and enhanced activation of effector caspases in both cell lines. We conclude that both pathways mediate resistance to TRAIL, and that the ability of 17-AAG to target multiple putative determinants of TRAIL sensitivity warrants their further investigation in combination.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]