title: Role for NAD(P)H:quinone oxidoreductase 1 and manganese-dependent superoxide dismutase in 17-(allylamino)-17-demethoxygeldanamycin-induced heat shock protein 90 inhibition in pancreatic cancer cells.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado-Denver, Aurora, CO 80045, USA. david.siegel@ucdenver.edu
authors.forename: David
authors.initials: D
authors.lastname: Siegel
authors.forename: Biehuoy
authors.initials: B
authors.lastname: Shieh
authors.forename: Chao
authors.initials: C
authors.lastname: Yan
authors.forename: Jadwiga K
authors.initials: JK
authors.lastname: Kepa
authors.forename: David
authors.initials: D
authors.lastname: Ross
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology*; D045744:Cell Line, Tumor; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D013482:Superoxide Dismutase / Q000502:physiology*
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C112765:tanespimycin; D013482:Superoxide Dismutase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human