Characterization of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine at androgen receptor: mechanistic support for its role in prostate cancer

Abstract

2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) is a dietary mutagenic carcinogen that has been shown not only to induce the formation of DNA adducts, but is capable of inducing tumors in the colon, mammary, and prostate glands. The normal development and maturation of the prostate gland, as well as early progression of prostate cancer, is dependent on androgens acting on the androgen receptor (AR). The actual mechanism by which PhIP interacts with our biological system and its potential interaction at the AR has yet to be fully defined. Here, we describe our work in evaluating the molecular events associated with PhIP-mediated disruption of AR function in LNCaP human prostate cancer cells. We demonstrate, by molecular docking simulation, that PhIP and its metabolite can bind to the ligand-binding domain (LBD). The binding competes with dihydrotestosterone (DHT) in the native AR binding cavity of the receptor. In vitro assays show that PhIP increase AR protein expression in LNCaP cells and alters its responsiveness through PSA protein and mRNA expression. We propose that the mechanism for the tissue-specific carcinogenicity seen in the rat prostate tumors and the presumptive human prostate cancer associated with the consumption of well-done meat may be mediated by this receptor activation. Our results indicate that PhIP may play an important role in modifications of AR function.

Keywords: 2-amino-1-methyl-6-phenylimidazo[4; 5b]pyridine (PhIP); dietary carcinogenesis; molecular modeling; prostate cancer.

Figure 1

Molecular visualization of PhIP in the active site of the androgen receptor.

Figure 3

Molecular visualization of N-OH-PhIP in the active site of the androgen receptor.

Figure 2

Molecular visualization of 4-OH-PhIP in the active site of the androgen receptor.

Figure 4

Effect of PhIP and N²-OH-PhIP on protein levels of AR. LNCaP cells were treated with 20 or 100 μM PhIP and 1 or 6 μM N²-OH-PhIP for (A) 24, (B) 48 or (C) 72 hr. Whole cell lysates were analyzed by Western blot analysis as described in section 4.2.5. Antibodies were used to detect the AR proteins. GAPDH served as a loading control. Significant effect compared to control is indicated with an asterisk (** indicates P < 0.01; ***P < 0.001).

Figure 5

Effect of PhIP and N²-OH-PhIP on protein levels of PSA. LNCaP cells were treated with 20 or 100 μM PhIP and 1 or 6 μM N²-OH-PhIP for (A) 24, (B) 48 or (C) 72 hr. Whole cell lysates were analyzed by Western blot analysis as described in section 4.2.5. Antibodies were used to detect the PSA proteins. GAPDH served as a loading control. Significant effect compared to control is indicated with an asterisk (* indicates P < 0.05; ***P < 0.001).

Figure 6

Compilation of all time-points and mRNA expression mRNA expression of PSA in LNCaP cells following treatment of PhIP and N²-OH-PhIP following 24, 48 and 72 hr. Significant effect compared to control are indicated with an asterisk (* indicates P < 0.05; ***P < 0.001).