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 pro tein that helps other proteins avoid misfolding pathways that produce inact ive or aggregated states), leads to depletion of important oncogenic protei ns, including Raf-1 and mutant p53 (also known as TP53), Given its ansamyci n benzoquinone structure, we questioned whether the antitumor activity of 1 7AAG was affected by expression of the NQO1 gene, which encodes the quinone -metabolizing enzyme DT-diaphorase. Methods: The antitumor activity of 17AA G and other Hsp90 inhibitors was determined by use of a sulforhodamine B-ba sed 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 exp ression 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 inc rease in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG i n the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibit ion mechanism was maintained in cells with high and low levels of DT-diapho rase. 17AAG was shown to be a substrate for purified human DT-diaphorase. C onclusion: These results suggest that the antitumor activity and possibly t he toxicologic properties of 17AAG in humans may be influenced by the expre ssion of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the le vel of tumor DT-diaphorase activity is therefore recommended in clinical tr ials with 17AAG.