Previous work demonstrated that
NAD(P)H:
quinone oxidoreductase 1 (NQO1) metabolized the
heat shock protein 90 (Hsp90) inhibitor
17-(allylamino)-17-demethoxygeldanamycin (
17AAG) to the corresponding
hydroquinone (17AAGH₂). The formation of 17AAGH₂ by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent
quinone.
17AAG induced substantial growth inhibition in human
pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by
17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-
indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1.
After treatment with
17AAG,
biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of
17AAG and 17AAGH₂ were measured in human
pancreatic cancer cells, and it was observed that larger amounts of
17AAG and 17AAGH₂ could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH₂), reduction of
17AAG to 17AAGH₂ by NQO1 leads to substantially greater intracellular concentrations of
17AAG and 17AAGH₂. In addition, oxidation of 17AAGH₂ could be prevented by
superoxide dismutase (SOD), demonstrating that 17AAGH₂ was sensitive to oxidation by
superoxide. Stable transfection of
manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH₂ with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to
17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH₂, the active Hsp90 inhibitor derived from
17AAG.