Induction of androgen receptor by 1α,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells

We have recently shown that 1 alpha,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)(2)D-3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)(2)D-3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)(2)D-3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)(2)D-3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)(2)D-3 exhibited delayed kinetics. In response to 1,25-(OH)(2)D-3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)(2)D-3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)(2)D-3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)(2)D-3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)(2)D-3-treated cells. This increased expression of AR was followed by 1,25-(OH)(2)D-3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)(2)D-3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)(2)D-3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)(2)D-3 and B-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)(2)D-3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.