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Endocrinology Vol. 140, No. 5 2071-2076
Copyright © 1999 by The Endocrine Society

ARTICLES

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

Lan H. Ly, Xiao-Yan Zhao, Leah Holloway and David Feldman

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1{alpha},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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJOR biological action of 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3], the active metabolite of vitamin D, is to maintain calcium homeostasis in the body (1). Recent findings indicate that 1,25-(OH)2D3 is also involved in regulating cellular proliferation and differentiation in various target tissues that possess vitamin D receptors (VDR) (1, 2, 3, 4). 1,25-(OH)2D3 and less calcemic analogs have been shown to inhibit cell growth in both human prostate carcinoma cell lines (5, 6, 7, 8, 9, 10, 11) and primary cultures of normal and prostate cancers (12). However, 1,25-(OH)2D3 showed only minimal inhibition of cell proliferation of DU 145, a human prostate cancer cell line derived from a brain metastasis, despite the presence of substantial amounts of VDR in this cell type (5, 7). The mechanism for the relative unresponsiveness of DU 145 to the antiproliferative action of 1,25-(OH)2D3 is not known.

DU 145 cells have been shown to express high levels of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) after treatment with 1,25-(OH)2D3 (5, 7). LNCaP cells can be induced to express low levels of 24-hydroxylase activity [12.6 ± 3.1 x 10-9 µmol/2 x 106 cells·30 min of 24,25(OH)2D3 produced] and are substantially growth inhibited by 1,25-(OH)2D3, whereas DU 145 cells can be induced to express very high levels of 24-hydroxylase activity (96.7 ± 39.5 x 10-9 µmol/2 x 106 cells·30 min) and are minimally growth inhibited (5, 7). As this P450 enzyme initiates the 1,25-(OH)2D3 inactivation pathway (1), we (8) and others (7, 13) have considered the possibility that rapid breakdown of 1,25-(OH)2D3 by 24-hydroxylase might be the cause of the resistant phenotype in DU 145 cells. In this study, we examine the premise that combination treatment with 1,25-(OH)2D3 and an inhibitor of 24-hydroxylase might render DU 145 cells more sensitive to the antiproliferative action of 1,25-(OH)2D3.

Combination therapy is often used to enhance the anticancer activity of various agents. Ketoconazole, liarozole, and other inhibitors of P450 enzymes may exhibit anticancer properties via several pathways, including actions on critical enzyme pathways (13, 14). In this study, we examined the possibility that combination treatment with 1,25-(OH)2D3 and liarozole, an imidazole derivative with antiprostate cancer properties (15, 16), might result in enhanced growth inhibition of DU 145 cells. Liarozole is known to inhibit several cytochrome P-450 enzymes, including retinoic acid 4-hydroxylase and aromatase (15, 17, 18). It is suspected that the former activity prolongs the half-life of retinoic acid and thereby increases the antiproliferative activity of endogenous retinoic acid when liarozole is administered to patients with prostate cancer (15, 16, 18). Here we show that 1,25-(OH)2D3 and liarozole interact synergistically to inhibit DU 145 cell growth. Our data demonstrate, for the first time, the ability of liarozole to directly inhibit 24-hydroxylase activity. The mechanism of liarozole action on DU 145 cells appears to be via inhibition of 24-hydroxylase, which causes a dual effect to prolong 1,25-(OH)2D3 half-life and to enhance up-regulation of VDR levels. Additional mechanisms may also play a role.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
25-Hydroxy-[3H]vitamin D3 (SA, 12.7 Ci/mmol) was obtained from Amersham Chemical Co. (Arlington Heights, IL). Liarozole fumarate (5-[(3-chlorophenyl)(1H-imidazol-1-yl)methyl]1H-benzimidazole fumarate) was a gift from Dr. C. Bowden (Janssen Research Foundation, Spring House, PA), and 1,25-(OH)2D3 was a gift from Dr. M. Uskokovic (Hoffmann-LaRoche, Inc., Nutley, NJ). Aprotinin, pepstatin, and soybean trypsin inhibitor were purchased from Boehringer Mannheim (Indianapolis, IN). Tissue culture media were purchased from Mediatech (Herndon, VA). FBS was obtained from Life Technologies (Gaithersburg, MD). CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS reagent) was purchased from Promega Corp. (Madison, WI). Silica gel TLC plates were purchased from E. M. Science (Darmstadt, Germany). All other reagents, except where indicated, were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture
The DU 145 human prostate carcinoma cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were routinely cultured in RPMI 1640 medium supplemented with 5% FBS and antibiotics at 37 C in a humidified atmosphere of 5% CO2.

Assay of cell growth
Cell growth was assessed by measurement of viable cells using the MTS assay. DU 145 cells were trypsinized and seeded at a density of approximately 2,000 cells/well in 96-well tissue culture plates (Falcon, Lincoln Park, NJ) in 200 µl culture medium. The cells were allowed to attach for 24 h, and the medium was replaced with fresh medium containing 5% FBS. Cells were then treated with vehicle (ethanol), 1,25-(OH)2D3, and/or liarozole. Triplicate wells were used for each experimental condition. The medium containing vehicle or test compounds was renewed every 2 days during the course of the experiment. After the appropriate incubation period, the cells were processed by replacing them with fresh RPMI 1640 medium containing MTS reagent (100 µl medium plus 20 µl MTS reagent/well). The plates were incubated at 37 C in a humidified atmosphere of 5% CO2 for approximately 3–4 h. The absorbance at 490 nm was read using an automatic plate reader (Emax Precision Microplate Reader, Molecular Devices, Menlo Park, CA) and was linear up to the highest cell concentration tested (40,000 cells/well).

Induction of 24-hydroxylase activity
24-Hydroxylase enzyme activity was assayed in a cell suspension system slightly modified from the method previously described (19). A 100-mm2 confluent DU 145 culture, growing under standard conditions, was treated for various times (0.5, 3, 6, 16, and 20 h) with either vehicle (ethanol) or 10 nM 1,25-(OH)2D3. Cells were then rinsed with 10 ml PBS and incubated with 10 ml culture medium at 37 C in a humidified atmosphere of 5% CO2 for approximately 30 min to remove 1,25-(OH)2D3. Cells were then trypsinized and resuspended at 106 cells/200 µl RPMI 1640 containing 10 mM HEPES with 1% FBS. The cells were incubated for 30 min at 37 C with 1.0 nM [3H]25-OHD3 and 1.0 µM 25-(OH)D3. The reaction was terminated by the addition of 750 µl methanol-chloroform (2:1) and 20 µl 24,25-(OH)2D3. The metabolites were extracted three times with 200 µl chloroform. The organic extracts were combined, dried with a Speed-Vac (Savant Instruments, Farmingdale, NY) and dissolved in a 90:10 mixture of hexane-isopropanol. The production of [3H]24,25-(OH)2D3 was quantitated by TLC on silica gel/aluminum foil plates developed in methylene chloride-ethyl acetate (1:1) run with authentic standards. The TLC strips were cut into 14 fractions and placed individually in minicounting vials. This TLC system produced good separation of [3H]25-(OH)2D3 from [3H]24,25-(OH)2D3.

Inhibition of 24-hydroxylase activity
Time-course studies indicate that induction of 24-hydroxylase activity could be detected at 3 h by 10 nM 1,25-(OH)2D3 treatment with a plateau at approximately 20 h. Therefore, the conditions selected for studying the inhibition of 24-hydroxylase activity by liarozole were 20-h induction, 106 cells, 1 nM [3H]25-(OH)D3, and 30-min incubation with various concentrations of liarozole (1, 10, 50, and 100 µM).

Determination of 1,25-(OH)2D3 half-life
The half-life of 1,25-(OH)2D3 in DU 145 cells was determined by measuring the residual unmetabolized [3H]1,25-(OH)2D3 in the conditioned medium at various time points after addition. Confluent DU 145 cells were treated for various times with [3H]1,25-(OH)2D3 in the presence or absence of liarozole. Two hundred microliters of conditioned medium were mixed with 750 µl methanol-chloroform (2:1), and the metabolites were extracted with 200 µl chloroform. Chloroform extraction was repeated three times. The organic extracts were dried with a Speed-Vac and dissolved in a 90:10 mixture of hexane-isopropanol. The disappearance of [3H]1,25-(OH)2D3 and the production of [3H]1,24,25-(OH)3D3 were quantitated by a TLC system using methylene chloride-ethyl acetate (1:3). After 145 min of development, the TLC strips were dried and fractionated by cutting regions identified as 1,25-(OH)2D3 and 1,24,25-(OH)3D3 by comigration of authentic standards. This TLC system gave good separation between [3H]1,25-(OH)2D3 and [3H]1,24,25-(OH)3D3. The Rf value for 1,25-(OH)2D3 was 0.667, and that for 1,24,25-(OH)3D3 was 0.333.

Western blot analysis of vitamin D receptor (VDR)
Cell monolayers grown in RPMI 1640 supplemented with 5% charcoal-stripped serum in 100-mm dishes were incubated with ethanol vehicle, 1,25-(OH)2D3 (0.1, 1, and 10 nM), and/or liarozole (10 µM) for 4 days. After 4 days of incubation, cells were harvested, and Western blot analysis was performed as described previously using anti-VDR monoclonal antibody (9A7) (20). The experiment was repeated twice with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Combination effect of 1,25-(OH)2D3 and liarozole on DU 145 cell growth
DU 145 cells are only minimally responsive to the antiproliferative effect of 1,25-(OH)2D3 (5, 7, 8). In our current studies, DU 145 cells were treated with increasing concentrations of 1,25-(OH)2D3 (1, 10, and 100 nM) over a time course of 2, 4, and 6 days (Fig. 1Go, A and B). The growth of DU 145 cells was not significantly inhibited by the lower concentrations of 1 and 10 nM; however, at the highest concentration (100 nM), there was a slight growth inhibition of approximately 20% on day 6. Similarly, as shown in Fig. 1Go, C and D, liarozole failed to inhibit the proliferation of DU 145 cells at 1 and 10 µM, but 100 µM resulted in 50% growth inhibition at 4 days and 60% growth inhibition at 6 days. However, 100 µM liarozole is a toxic dose and when administered to patients at these concentrations it causes hypervitaminosis A. Neither 10 nM 1,25-(OH)2D3 nor 1 µM liarozole had any antiproliferative effect when used alone. However as shown in Fig. 1Go, E and F, the combination treatment caused 60% growth inhibition. These data indicate that 1,25-(OH)2D3 and liarozole interact synergistically to inhibit DU 145 cell growth.



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  		Figure 1. Dose-response effect of 1,25-(OH)2D3, liarozole, and the combination on DU 145 cell growth over a time course of 6 days. Cells were plated at approximately 2000 cells/well in 96-well tissue culture plates in 200 µl medium with the indicated concentrations of hormone. Media were changed every 2 days. Cell proliferation was estimated using the MTS assay. Data are expressed as the mean ± SD (n = 3) in the left panels. The right panels show a single representative experiment comparing treatment to vehicle and expressed in absorbance units. *, Significant changes (P < 0.05) compared with the ethanol control. A and B, Treatment with 1,25-(OH)2D3. C and D, Treatment with liarozole. E and F, Treatment with a combination of 1,25-(OH)2D3 (10 nM) and liarozole (1 µM).

 
Inhibition of 24-hydroxylase activity by liarozole
We next investigated the possible mechanisms by which liarozole enhanced the ability of 1,25-(OH)2D3 to inhibit DU 145 cell proliferation. As shown in many other cell culture systems, we found here that 1,25-(OH)2D3 induced 24-hydroxylase activity in DU 145 cells in a time-dependent manner (Fig. 2Go). The level of 24-hydroxylase activity in DU 145 cells is much higher than that in other cell types, particularly compared with LNCaP cells, which are substantially inhibited by 1,25-(OH)2D3 alone (5, 7). Liarozole had no intrinsic ability to induce 24-hydroxylase activity. However, liarozole can inhibit 24-hydroxylase activity. After treating cells with 10 nM 1,25-(OH)2D3 for 20 h to induce 24-hydroxylase activity, we examined the abilities of various concentrations of liarozole to inhibit enzyme activity by blocking the conversion of [3H]25-OHD3 to 24,25-(OH)2D3. As shown in Fig. 3Go, liarozole (1, 10, 50, and 100 µM) was able to directly inhibit 24-hydroxylase activity in a dose-dependent manner, such that 10 µM liarozole resulted in approximately 80% inhibition of enzyme activity compared with the activity of the induced cells in the absence of liarozole.



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  		Figure 2. Time course of the effect of 1,25-(OH)2D3 or liarozole on 24-hydroxylase activity in DU 145 cells. Cells were treated with 10 nM 1,25-(OH)2D3, 10 µM liarozole, or ethanol vehicle, and enzyme activity was measured at 0.5, 3, 6, 16, and 20 h. At 20 h, 10 nM 1,25-(OH)2D3 induced a 27-fold rise in 24-hydroxylase activity compared with the effect of vehicle. This is a representative experiment that was performed twice with similar results.

 


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  		Figure 3. Dose-dependent effect of liarozole on 24-hydroxylase activity in DU 145 cells. Cells were treated with 10 nM 1,25-(OH)2D3 for 20 h. Treated cells were subsequently incubated with liarozole at various concentrations (0, 1, 10, 50, and 100 µM) for 30 min before enzyme activity was measured. This is a representative experiment that was performed three times with similar results.

 
Effect of liarozole on 1,25-(OH)2D3 half-life
As our data indicated that liarozole was capable of directly inhibiting 24-hydroxylase activity, the enzyme involved in the first step of 1,25-(OH)2D3 inactivation, we next investigated the effect of liarozole on the 1,25-(OH)2D3 half-life. We treated two group of cells at various times, one with the single addition of 10 nM 1,25-(OH)2D3 and the other with a combination of 10 nM 1,25-(OH)2D3 and 1 µM liarozole. As anticipated, in cultures treated with 1,25-(OH)2D3 alone, the half-life of 1,25-(OH)2D3 was shorter (~10 h) compared with that of cells treated with the combination (~30 h; Fig. 4Go). Therefore, these data are consistent with the observation that liarozole directly inhibits 24-hydroxylase activity, thereby prolonging 1,25-(OH)2D3 half-life. This finding provides one possible mechanism for the synergistic growth inhibitory effect of combination therapy with 1,25-(OH)2D3 and liarozole.



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  		Figure 4. Effect of liarozole on 1,25-(OH)2D3 half-life. DU 145 cells were incubated with [3H]1,25-(OH)2D3 (0.5 nM) plus unlabeled 1,25-(OH)2D3 (10 nM) in the absence or presence of 10 µM liarozole. Conditioned media were collected at various time points (0, 4, 24, 32, and 48 h), and the residual amount of unmetabolized [3H]1,25-(OH)2D3 was determined by TLC. This is a representative experiment performed twice with similar results.

 
Effect of 1,25-(OH)2D3 and liarozole on VDR protein level
As previously shown in other cell culture systems, 1,25-(OH)2D3 and other vitamin D analogs induce homologous up-regulation of the VDR (21, 22). We next investigated whether liarozole, by inhibiting 24-hydroxylase activity and prolonging 1,25-(OH)2D3 half-life, is also capable of augmenting VDR up-regulation. Cells were treated with various concentrations of 1,25-(OH)2D3 (0.1, 1, and 10 nM) with and without the addition of 10 µM liarozole over a time course of 4 days. Protein extracts were made from these treated cells and were subjected to Western blot analysis to evaluate VDR content. Using the monoclonal antibody 9A7, the 50-kDa VDR protein was visualized. As shown in Fig. 5AGo, 1,25-(OH)2D3 alone resulted in a slight up-regulation of the VDR protein level (2-fold). Liarozole alone had no significant effect on VDR abundance. However, the combination of 1,25-(OH)2D3 and liarozole resulted in a 1,25-(OH)2D3 dose-dependent increase in the VDR protein level. This effect was observed most profoundly after a combination treatment with 10 nM 1,25-(OH)2D3 and 10 µM liarozole. This combination, as shown in Fig. 5BGo, resulted in a 5-fold increase in VDR abundance. Our data suggest that an increase in the VDR protein level may serve as a second and related mechanism, added to the prolonged 1,25-(OH)2D3 half-life, that contributes to the synergistic effect of 1,25-(OH)2D3 and liarozole inhibition of DU 145 cell growth.



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  		Figure 5. A, Western blot analysis of VDR levels in DU 145 cells in response to various treatments. Cells were treated with ethanol or increasing concentrations of 1,25-(OH)2D3, liarozole, or 1,25-(OH)2D3 and liarozole for 4 days. High salt extracts were prepared, and 100 µg protein were loaded onto an 8% SDS-PAGE. After gel transfer, the blot was probed with anti-VDR monoclonal antibody 9A7, and the signal was detected using the enhanced chemiluminescence method. B, Densitometric analysis of Western blot. The pixel intensities of VDR bands were quantitated using a laser densitometer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study was designed to investigate the combination of 1,25-(OH)2D3 and liarozole as a possible treatment for prostate cancer. We carried out our studies using an aggressive human prostate cancer cell line, DU 145, because of its resistance to the growth inhibitory effects of 1,25-(OH)2D3. We successfully inhibited DU 145 cell growth by 60% using the combination treatment of 1,25-(OH)2D3 and liarozole. In addition, we gained insight into the mechanism of the DU 145 cell unresponsiveness and the possibility of reversing the resistance with combination therapy.

As discussed earlier, at concentrations that are nontoxic, neither liarozole nor 1,25-(OH)2D3 alone substantially inhibited cell growth. Yet the combination of 1 µM liarozole and 10 nM 1,25-(OH)2D3 resulted in significant synergistic antiproliferative effects. Furthermore, this synergy is observed at a pharmacologically relevant concentration for both compounds. Based on this observation, we explored the possible mechanisms behind this synergy. We discovered that liarozole directly inhibited 24-hydroxylase activity in addition to inhibiting the already known P-450 enzymes, such as 4-hydroxylase and aromatase (15, 17, 18). As 24-hydroxylase is the initial enzyme for inactivating 1,25-(OH)2D3, we measured 1,25-(OH)2D3 half-life, and indeed, it was prolonged from 11 to 31 h. Therefore, by preventing rapid inactivation of 1,25-(OH)2D3 and prolonging the exposure time of cells to active hormone, DU 145 cells were able to respond to its antiproliferative effect. This provided the first possible mechanism for the synergistic activity of the 1,25-(OH)2D3/liarozole combination.

It is known that receptor regulation is an important mechanism for modulating target cell responsiveness to hormone (20, 23, 24). We explored the possibility that homologous up-regulation of the VDR would be enhanced in the presence of liarozole. Western blot analysis demonstrated a 5-fold increase in VDR protein level when cells were treated with 10 nM 1,25-(OH)2D3 and 10 µM liarozole compared with that after treatment with 1,25-(OH)2D3 alone. DU 145 cells treated with 1,25-(OH)2D3 alone only demonstrated a slight up-regulation of the VDR protein level. The possible explanation for this observation is that 1,25-(OH)2D3 is a potent stimulus of 24-hydroxylase activity; therefore, this would induce rapid degradation of 1,25-(OH)2D3, causing only a transient homologous up-regulation of VDR in DU 145 cells. Augmentation of VDR up-regulation has previously been reported using ketoconazole to inhibit 24-hydroxylase in a similar manner (25, 26). Enhanced VDR up-regulation is the second contributing mechanism explaining the liarozole synergistic interaction with 1,25-(OH)2D3. The increase in both ligand and receptor is a plausible mechanism for the enhanced antiproliferative activity of the 1,25-(OH)2D3/liarozole combination therapy in DU 145 cells. It is of interest that analogs of 1,25-(OH)2D3 designed to prevent 24-hydroxylation, such as 19-nor-25,26-hexafluoro-1,25-(OH)2D3, inhibit DU145 cell proliferation (9).

Our data suggest that DU 145 cells are more responsive to the antiproliferative effect of 1,25-(OH)2D3 when both its hormone and receptor are increased. Although the presence of VDR is essential for 1,25-(OH)2D3 activity (27), the level of VDR abundance in different prostate cancer cell lines by itself is not necessarily predictive of the amplitude of hormonal response (5, 28). However, in a given cell, increased abundance of receptor does appear to predict the extent of hormonal responsiveness, and increased or decreased receptor levels are usually correlated with increased and decreased responsiveness, respectively (20, 23, 24).

It should be noted that Zhao et al. have shown that combination therapy with either ketoconazole or liarozole and 1,25-(OH)2D3 or its analogs is cell type specific (13). That finding supports the concept that differences in cellular metabolism can at least partially explain the different potencies of various vitamin D analogs and differences in antiproliferative activity between different cancer cells. The fact that some cells are substantially growth inhibited by 1,25-(OH)2D3 alone (LNCaP and primary cultures) and other cells are not (DU 145) depends on a combination of factors, including, but not limited to, VDR abundance and inducible 24-hydroxylase activity (5, 7, 28). Liarozole in combination with 1,25-(OH)2D3 improves both parameters; by increasing VDR abundance and inhibiting 24-hydroxylase activity, it allows the otherwise resistant DU 145 cell to be growth arrested by 1,25-(OH)2D3. In preliminary experiments, liarozole also augmented the growth inhibitory activity of 1,25-(OH)2D3 in PC-3 and LNCaP cells, but to a much lesser extent (data not shown) than shown here for DU145 cells. The smaller augmentation was probably due to the greater antiproliferative activity of 1,25-(OH)2D3 alone in these cells (5) as well as the lesser induction of 24-hydroxylase in these cell lines (7), making the liarozole action to inhibit 24-hydroxylase less essential for 1,25-(OH)2D3-mediated growth inhibition.

Another possible mechanism for the enhanced antiproliferative effect in the presence of liarozole is its ability to inhibit retinoid metabolism, leading to increased retinoid levels (15, 18, 29). Retinoids have been shown to inhibit various cancer cell lines, including prostate cancer (30), and to be synergistic with 1,25-(OH)2D3 in inhibiting prostate cancer cell growth (10, 31). In fact, the beneficial effect of liarozole in patients with prostate cancer is attributed to this activity (15, 18, 29). We investigated the possibility that this activity might be contributing to the growth inhibition in our experiments. We treated DU 145 cells with a combination of 1,25-(OH)2D3 and increasing concentrations of retinoic acid to mimic the liarozole effect. We observed only a slight enhancement of growth inhibition (data not shown). As liarozole alone had no antiproliferative activity, and retinoids were not added in our standard combination experiments, we believe that the inhibition of retinoid metabolism does not substantially contribute to the effects that we have seen in cultured cells. However, in patients, the ability of liarozole to inhibit retinoid metabolism would be expected to further enhance the synergistic activity that we have demonstrated.

The mechanism(s) by which 1,25-(OH)2D3 inhibits the growth of prostate cancer cells is complex, multifactorial, and different in different cell lines. Several investigators have reported that treatment with 1,25-(OH)2D3 causes LNCaP cells to accumulate in the G1 phase of the cell cycle (10, 11). 1,25-(OH)2D3 also elicits a reduction of cyclin-dependent kinase 2 activity and an increase in the level of hypophosphorylated retinoblastoma (Rb) protein, which is a critical regulator of the G1/S checkpoint (11). Interestingly, DU 145 cells lack functional Rb protein. However, ectopic expression of functional Rb in DU 145 cells was not sufficient to restore the growth response to 1,25-(OH)2D3 (32). Others have found that growth inhibition of prostate cancer cells by a potent vitamin D analog involves the induction of p21waf1, p27kip1, and E-cadherin (9). In addition, we have demonstrated that 1,25-(OH)2D3 significantly regulates androgen receptor gene expression, which contributes to the regulation of LNCaP cell growth (33). Therefore, the mechanism by which 1,25-(OH)2D3 inhibits cell proliferation involves multiple signaling pathways and differs in various prostate cancer cell lines.

In summary, our data suggest that liarozole directly inhibits 24-hydroxylase activity, thereby effectively prolonging the 1,25-(OH)2D3 half-life. The increase in the 1,25-(OH)2D3 half-life resulted in enhanced up-regulation of VDR protein levels. We believe that this combination of increased 1,25-(OH)2D3 hormone levels as well as augmented VDR abundance represents the principal mechanism for the synergistic effect of 1,25-(OH)2D3 and liarozole in our experiments. However, additional mechanisms may play a role in the synergistic effect of this combination in DU 145 cells. In conclusion, the novel combination of liarozole and 1,25-(OH)2D3 therapy may serve as an effective treatment regimen for prostate cancer patients.


   Acknowledgments
 
We thank Dr. J. W. Pike for the anti-VDR monoclonal antibody (9A7); Dr. M. Uskokovic, Hoffmann-LaRoche, Inc. (Nutley, NJ), for providing 1,25-(OH)2D3; and Dr. C. Bowden, Janssen Research Foundation (Spring House, PA) for providing liarozole.


   Footnotes
 
1 This work was supported by Stanford Medical Scholar Program (Resident & Pfeiffer), NIH Grant DK-42482, and a grant from the American Institute for Cancer Research. Portions of this work Were presented at the American Federation for Medical Research, Carmel, California, February 1998. Back

Received June 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Feldman D, Malloy PJ, Gross C 1996 Vitamin D: metabolism and action. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, New York, pp 205–235
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