Flutamide ameliorates oxidative DNA damage caused by dihydrotestosterone (DHT) in human prostate cancer cells

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Reactive oxygen species (ROS) are mechanistically implicated in the development and progression of prostate cancer. They may cause peroxidation of membrane lipids and oxidative DNA damage, resulting in the formation of DNA adducts such as 8-oxo-deoxyguanosine (8-oxo-dG) and the pyrimidopurinone adduct of deoxyguanosine (M₁G), both of which have been associated with carcinogenesis. It has been shown that DHT increases ROS levels in androgen-sensitive human prostate cancer cells (Ripple et al, J Natl Cancer Inst. 89: 40-48, 1997). We determined the influence of DHT on levels of oxidative DNA adducts in human prostate cancer cell lines and tested the hypothesis that flutamide, a competitive anti-androgen, inhibits DHT-induced changes. Human prostate cancer cells LNCaP (androgen-sensitive), PC3 and DU145 cells (both androgen-insensitive) were treated with DHT (100 nM) daily for 7 days. Levels of M₁G and 8-oxo-dG adducts in extracted genomic DNA were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry, respectively. In LNCaP cells, the effect of DHT at 2.5, 10, 25 and 50 nM was also studied. To exclude any interference by endogenous androgens, cells were cultured without serum when the effect of 2.5 nM DHT was studied. The flutamide concentrations used were 5 μM and 10 μM. In the following, mean M₁G and 8-oxo-dG levels are expressed as adducts per 10⁷ and 10⁶ nucleotides, respectively. M₁G levels in control DU145, PC3 and LNCaP cells were 7.5±5.6, 6.9±4.7, and 12.6±5.6, respectively. In DU145 and PC3 cells treated with 100 nM DHT, M₁G levels were not significantly different from control levels. In LNCaP cells exposed to 100 nM DHT, M₁G levels were 17.2±8.6, thus significantly different from untreated cells (p<0.05). M₁G levels in LNCaP cells treated with 2.5 nM DHT were also significantly increased over controls. Flutamide prevented the increase in M₁G levels induced by DHT at 2.5 nM and 100 nM DHT from 5.0±2.3 to 10.9±2.6 and from 6.6±1.6 to 12.7±3.7, respectively. The changes in oxidative DNA adduct levels in LNCaP cells treated with DHT and flutamide suggest that androgens increase oxidative DNA damage in androgen-sensitive prostate cells, and that this response can be pharmacologically prevented. Overall the results intimate that oxidative DNA adducts may serve as biomarkers of efficacy in chemoprevention trials of anti-androgens.