Liarozole acts synergistically with 1 alpha,25-dihydroxyvitamin D-3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity

1 alpha,25-Dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] inhibits the proliferatio n of many cancer cells in culture, but not the aggressive human prostate ca ncer cell line DU 145. We postulated that the 1,25(OH)(2)D-3-resistant phen otype in DU 145 cells might result from the high levels of expression of 25 -hydroxyvitamin D-24-hydroxylase (24-hydroxylase) induced by treatment with 1,25-(OH)(2)D-3. As this P450 enzyme initiates 1,25-(OH)(2)D-3 catabolism, we presumed that a high level of enzyme induction could limit the effectiv eness of the 1,25(OH)(2)D-3 antiproliferative action. To examine this hypot hesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As i midizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)(2 )D-3 half-life, thereby enhancing 1,25-(OH)(2)D-3 antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells u sing the MTS assay. When used alone, neither 1,25-(OH)(2)D-3 (1-10 nM) nor liarozole (1-10 mu M) inhibited DU 145 cell growth. However, when added tog ether, 1,25-(OH)(2)D-3 (10 nM)/liarozole (1 mu M) inhibited growth 65% afte r 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1-100 mu M) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significan t increase in 1,25-(OH)(2)D-3 half-life from 11 to 31h. In addition, 1,25-( OH)(2)D-3 can cause homologous up-regulation of the vitamin D receptor (VDR ), and in the presence of liarozole, this effect was amplified, thus enhanc ing 1,25-(OH)(2)D-3 activity. Western blot analyses demonstrated that DU 14 5 cells treated with 1,25-(OH)(2)D-3/liarozole showed greater VDR up-regula tion than cells treated with either drug alone. In summary, our data demons trate that liarozole augments the ability of 1,25-(OH)(2)D-3 to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydrox ylase activity, leading to increased 1,25-(OH)(2)D-3 half-life and augmenta tion of homologous up-regulation of VDR. We raise the possibility that comb ination therapy using 1,25-(OH)(2)D-3 and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatme nt for prostate cancer.