Mechanistic studies of the effects of the retinoid n-(4- hydroxyphenyl)retinamide on prostate cancer cell growth and apoptosis

To explore the mechanisms underlying the chemopreventive effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in prostate cancer, we evaluated the anti-proliferative and apoptosis-inducing effects of 4-HPR in the androgen-sensitive human prostate cancer cell line LNCaR 4-HPR decreased the number of viable LNCaP cells (as measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in a dose- dependent manner. Although 4-HPR exerted a modest G₁ cell-cycle block (as determined by flow cytometry), its effect on reduced cell number appeared to result primarily from induction of apoptosis (as measured by an enzyme- linked immunosorbent assay and flow-cytometric assays). The mitogenic effects of R1881, a non-metabolizable androgen that potently induces LNCaP cell proliferation, was completely blocked by greater than 0.5 μM 4-HPR. Furthermore, increasing the R1881 concentration in the presence of 2.0 μM 4- HPR increased apoptotic cell death. 4-HPR decreased prostate-specific antigen (PSA) protein levels in conditioned medium and decreased PSA mRNA expression. 4-HPR also decreased the ratio of bcl-2 to bax mRNA expression in LNCaP cells by approximately 45%, indicating that the apoptotic effects of 4-HPR may be mediated, at least in part, by alterations in the bcl-2/bax-regulated apoptotic pathway. N-acetylcysteine (4 mM) completely blocked the anti- proliferative and apoptotic-inducing effects of 4-HPR, suggesting that an oxidative mechanism may be involved. We concluded that (i) 4-HPR exerts growth-suppressive and apoptotic effects on LNCaP cells, (ii) 4-HPR can interact with androgen to suppress proliferation and induce apoptosis, (iii) the apoptotic effects of 4-HPR may be mediated in part by the bcl-2/bax pathway, and (iv) a pro-oxidant mechanism may contribute to the anti- proliferative and apoptotic-inducing effects of 4-HPR.