The proteasome inhibitor bortezomib is emerging as a potent anti-cancer agent. 
Still, recent clinical trials have revealed a significant secondary toxicity of 
bortezomib. Consequently, there is much interest in dissecting the mechanism of 
action of this compound to rationally improve its therapeutic index. The 
cytotoxic effect of bortezomib is frequently characterized by interfering with 
downstream events derived from the accumulation of proteasomal targets. Here we 
identify the first chemical agent able to act upstream of the proteasome to 
prevent cell killing by bortezomib. Specifically, we show that the polyhydroxyl 
compound Tiron can function as a competitive inhibitor of bortezomib. This 
effect of Tiron was surprising, since it is a classical radical spin trap and 
was expected to scavenge reactive oxygen species produced as a consequence of 
bortezomib action. The inhibitory effect of Tiron against bortezomib was 
selective, since it was not shared by other antioxidants, such as vitamin E, 
MnTBAP, L-N-acetyl-cysteine, and FK-506. Comparative analyses with nonboronated 
proteasome inhibitors (i.e. MG132) revealed a specificity of Tiron for 
bortezomib. We exploited this novel feature of Tiron to define the "point of no 
return" of proteasome inhibition in melanoma cells and to block cell death in a 
three-dimensional model of human skin. Cells from T-cell lymphoma, breast 
carcinoma, and non-small cell lung cancer were also responsive to Tiron, 
suggesting a broad impact of this agent as a bortezomib blocker. These results 
may have important implications for the analysis of bortezomib in vivo and for 
the design of drug mixtures containing proteasome inhibitors.