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Liarozole Acts Synergistically with 1,25-Dihydroxyvitamin D₃ to Inhibit Growth of DU 145 Human Prostate Cancer Cells by Blocking 24-Hydroxylase Activity¹

Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: David Feldman, M.D., Stanford University School of Medicine, Stanford, California 94305-5103. E-mail: feldman{at}cmgm.stanford.edu

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] inhibits the proliferation of many cancer cells in culture, but not the aggressive human prostate cancer cell line DU 145. We postulated that the 1,25-(OH)₂D₃-resistant phenotype 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)₂D₃. As this P450 enzyme initiates 1,25-(OH)₂D₃ catabolism, we presumed that a high level of enzyme induction could limit the effectiveness of the 1,25-(OH)₂D₃ antiproliferative action. To examine this hypothesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As imidizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)₂D₃ half-life, thereby enhancing 1,25-(OH)₂D₃ antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells using the MTS assay. When used alone, neither 1,25-(OH)₂D₃ (1–10 nM) nor liarozole (1–10 µM) inhibited DU 145 cell growth. However, when added together, 1,25-(OH)₂D₃ (10 nM)/liarozole (1 µM) inhibited growth 65% after 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1–100 µM) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significant increase in 1,25-(OH)₂D₃ half-life from 11 to 31 h. In addition, 1,25-(OH)₂D₃ can cause homologous up-regulation of the vitamin D receptor (VDR), and in the presence of liarozole, this effect was amplified, thus enhancing 1,25-(OH)₂D₃ activity. Western blot analyses demonstrated that DU 145 cells treated with 1,25-(OH)₂D₃/liarozole showed greater VDR up-regulation than cells treated with either drug alone. In summary, our data demonstrate that liarozole augments the ability of 1,25-(OH)₂D₃ to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydroxylase activity, leading to increased 1,25-(OH)₂D₃ half-life and augmentation of homologous up-regulation of VDR. We raise the possibility that combination therapy using 1,25-(OH)₂D₃ and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatment for prostate cancer.

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