DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90.
BACKGROUND
To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase.


METHODS
The antitumor activity of 17AAG and other Hsp90 inhibitors was determined by use of a sulforhodamine B-based cell growth inhibition assay in culture and by the arrest of xenograft tumor growth in nude mice. DT-diaphorase activity was determined by use of a spectrophotometric assay, and protein expression was determined by means of western immunoblotting.


RESULTS
In two independent in vitro human tumor cell panels, we observed a positive relationship between DT-diaphorase expression level and growth inhibition by 17AAG. Stable, high-level expression of the active NQO1 gene transfected into the DT-diaphorase-deficient (by NQO1 mutation) BE human colon carcinoma cell line resulted in a 32-fold increase in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG in the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibition mechanism was maintained in cells with high and low levels of DT-diaphorase. 17AAG was shown to be a substrate for purified human DT-diaphorase.


CONCLUSIONS
These results suggest that the antitumor activity and possibly the toxicologic properties of 17AAG in humans may be influenced by the expression of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the level of tumor DT-diaphorase activity is therefore recommended in clinical trials with 17AAG.
Kelland
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L. R. Kelland, S. Y. Sharp, P. M. Rogers, P. Workman, Cancer Research Campaign Centre for Cancer Therapeutics, The Institute of Cancer Research, Surrey, U.K.
Sharp
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Rogers
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PM
Myers
T G
TG
Workman
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P
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000201:enzymology*; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D011809:Quinones; D017828:Rifabutin; C112765:tanespimycin; D008058:Dihydrolipoamide Dehydrogenase; C035359:monorden; C001277:geldanamycin