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CELLULAR AND MOLECULAR

A 24-Phenylsulfone Analog of Vitamin D Inhibits 1,25-Dihydroxyvitamin D₃ Degradation in Vitamin D Metabolism-Competent Cells

Department of Pathophysiology, Medical University of Vienna, Austria (D.L., T.M., E.B., H.S.C.); and Department of Chemistry, Johns Hopkins University, Baltimore, Maryland (G.H.P.).

Received for publication October 13, 2006
Accepted December 19, 2006.

	Abstract

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The antimitotic, prodifferentiating, and proapoptotic steroid hormone, 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], at supraphysiological levels has potential for tumor therapy. However, epithelial cells from tumor-prone organs such as colon, prostate, and breast express not only the vitamin D receptor, but also vitamin D hydroxylases. In contrast to normal cells, malignant cells have high basal levels of the hydroxylase 25-hydroxyvitamin D₃-24-hydroxylase (CYP24) and, in addition, have the potential to induce CYP24 in response to 1,25-(OH)₂D₃. Because 24-hydroxylation by CYP24 would rapidly degrade the steroid hormone in the course of therapy, the enzyme activity in tumor cells should be inhibited. We demonstrate that a 24-phenylsulfone analog of 1,25-(OH)₂D₃, KRC-24SO₂Ph-1 (S-4a), rapidly and potently inhibits 24-hydroxylase activity in human tumor cells derived from colon, prostate, and mammary gland. Although enzymatic inhibition is a consequence of direct interaction, S-4a as a vitamin D analog apparently binds to the vitamin D receptor and induces CYP24 mRNA, which, however, is not translated into increased enzymatic activity. 25-Hydroxyvitamin D₃-1-hydroxylase expression is not affected at all by S-4a. When both 1,25-(OH)₂D₃ and S-4a are added to the cell culture, transcription of CYP24 is increased, possibly because of an increase in the half-life of the hormone. The colon cell line COGA-13 has very high levels of CYP24 and is, therefore, resistant to the action of vitamin D. Yet, S-4a imparts antimitotic activity to 1,25-(OH)₂D₃ and may therefore constitute a therapeutic to stimulate the antiproliferative potential of vitamin D-based antitumor activity.

The discovery that epithelial cells from organs prone to sporadic cancer, such as colon (Cross et al., 1997; Bareis et al., 2001), prostate (Schwartz et al., 1998), and mammary gland (Townsend et al., 2005), efficiently synthesize 1,25-(OH)₂D₃ from 25-OH-D₃ has opened a new avenue for the treatment and prevention of many malignant diseases. In the colon, CYP27B1 is primarily expressed in regions that contain differentiated tumor cells, its concentration rises during early tumor progression, and it is frequently found in cells that express the vitamin D receptor (Bises et al., 2004). Conceivably, by autocrine/paracrine action, 1,25-(OH)₂D₃ synthesized in colon cells could slow or even stop disease progression. However, this system seems to fail in late-stage, high-grade undifferentiated tumors (Cross et al., 2001). One reason may be that an aberrantly expressed 25-hydroxyvitamin D₃-24-hydroxylase (CYP24) at the tumor site prevents biosynthesis of 1,25-(OH)₂D₃ (Bareis et al., 2001). CYP24 mRNA expression is increased as cell differentiation decreases from high to low (Cross et al., 2005). In prostatic tumor-derived cell cultures, high expression of CYP24 prevents the regulatory effect of added 1,25-(OH)₂D₃ (Ly et al., 1999).

Advanced malignancies are characterized by high expression of the potential oncogene CYP24 (Albertson et al., 2000). Accumulation of 1,25-(OH)₂D₃ in extrarenal tissues, similar to that in renal cells, may alter CYP27B1 and CYP24 expression, with an increase in CYP24 activity curtailing the effectiveness of vitamin D compounds in cancer therapy (for a graphic explanation, see Fig. 1A). It is, therefore, of interest to be able to inhibit catabolic activity without interfering in 1,25-(OH)₂D₃ synthesis. Posner et al. (2004) have shown that a 24-phenylsulfone analog of 1,25-(OH)₂D₃ selectively inhibits CYP24 activity. We now demonstrate, utilizing cell lines derived from human colon, prostate, and mammary gland, that the phenyl analog rapidly and dose-dependently inhibits CYP24, but not CYP27B1. Preventing vitamin D degradation without reduction of CYP27B1 activity may therefore potentiate the control of cell proliferation by 1,25-(OH)₂D₃ in cancer cells that metabolize vitamin D and express the vitamin D receptor.

View larger version (37K): [in this window] [in a new window]   Fig. 1. A, vitamin D metabolism in renal and nonrenal cells. The active 1,25-(OH)2D3 is synthesized from its serum precursor 25-OH-D3 by CYP27B1. Sufficient 1,25-(OH)2D3 will induce CYP24 expression. Both 1,25-(OH)2D3 and 25-OH-D3 are converted to less active metabolites by CYP24. B, RT-PCR analysis of basal expression of CYP27B1, CYP24, and VDR mRNA in four cell lines derived from colon (Caco-2, COGA-13), prostate (DU-145), and breast (MCF-7). -Actin served as loading control. C, HPLC analysis of vitamin D metabolism in untreated cells. 1-Hydroxylated metabolites, 1,25-dihydroxy-3-epi-vitamin D3 (1,25-epi-D) and 1,25-(OH)2D3 (1,25-D), are eluted after 23 and 27 min, respectively. CYP27B1 activity is high in Caco-2, low in DU-145 and MCF-7, and nil in COGA-13. 24-Hydroxylated metabolites (CYP24 metabolites) identified between 4 and 9 min are abundant in MCF-7, DU-145, and particularly in COGA-13 cells, but almost absent in Caco-2.

	Materials and Methods

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Culture Conditions. Cells were routinely cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The media used for the colon cell lines and MCF-7 were Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 medium for DU-145. Media were supplemented with 10% fetal calf serum, 100 IU of penicillin, 100 µg/ml streptomycin, and 4 mM glutamine (all from Invitrogen, Lofer, Austria). In addition, RPMI 1640 medium was supplemented with 1 mM sodium pyruvate (Sigma, Vienna, Austria) and DMEM with 10 mM HEPES. Media were changed every other day. For evaluation of [³H]thymidine incorporation into DNA, cells were seeded in 24-well plates and otherwise in six-well plates. For high-pressure liquid chromatography (HPLC) studies, fetal calf serum was replaced with 10 µg/ml transferrin (Sigma) and 5 ng/ml sodium selenite (Merck, Darmstadt, Germany).

Treatments. 1,25-(OH)₂D₃ was kindly provided by Hoffmann-La Roche (Basel, Switzerland). 25-OH-D₃ was purchased from Sigma. The 1,25-(OH)₂D₃ analog KRC-24SO₂Ph-1 (S-4a; kindly provided by Cytochroma Inc., Markham, Canada) was synthesized as published by Posner et al. (2004). All compounds were dissolved in ethanol and were diluted in DMEM for experimentation.

HPLC. [³H]25-OH-D₃ (0.5 µCi/ml; specific activity, 30 Ci/mmol; purchased from GE Healthcare, Buckinghamshire, UK) as tracer was added to each well containing 1 ml of serum-free medium to reach a final concentration of 16.6 nM 25-OH-D₃. Experiments were terminated by the addition of 1 ml of methanol. For lipid extraction, cells were scraped off, and 1 nM 25-OH-D₃ and 1 nM 1,25-(OH)₂D₃ were added as internal standards to evaluate efficiency of extraction. After three extractions with dichloromethane (CH₂Cl₂), the lower phase was collected and dried gently at 55°C under nitrogen. After reconstitution in the mobile phase (94% n-hexane, 6% isopropanol), extracts were analyzed by HPLC.

HPLC was performed with the following components of the system: a pump (model 515; Waters, Vienna, Austria) operating with an isocratic flow rate of 2 ml/min, an Ultrasphere Silica column (5 µm, 4.6 x 250 mm; Beckman Instruments, Fullerton, CA) to separate the vitamin D metabolites, and a photodiode array detector (model SPD-M10Avp; Shimadzu, Kyoto, Japan) to monitor UV absorption of the standards. Tritium-labeled metabolites were detected by flow radiochromatography (model C505TR; Packard Bioscience, Groningen, The Netherlands) after addition of Ultima Flow M scintillation fluid (Packard Bioscience) with a flow rate of 2 ml/min. Counts per minute were measured, and enzymatic activity was evaluated from the areas under the curve.

Eluted metabolites of [³H]25-OH-D₃ were identified by comigration with known standards. [³H]25-OH-D₃ is detectable as the first large peak after approximately 3 min. CYP24 activity leads to hydroxylation of [³H]25-OH-D₃, which gives rise to a variety of previously identified metabolites that are eluted between approximately 4 and 9 min (Makin et al., 1989; Dilworth et al., 1995; Bischof et al., 1998; Weinstein et al., 1999; Bareis et al., 2001). CYP27B1 transforms [³H]25-OH-D₃ into labeled 1,25-(OH)₂D₃, which emerges as a peak after approximately 27 min. This is subsequently transformed into labeled 1,25-dihydroxy-3-epi-vitamin D₃ (Bischof et al., 1998; Astecker et al., 2000), which emerges as a peak after approximately 23 min. [³H]25-OH-D₃ was added for the last 6 h before termination of experiments. Elution of metabolites was monitored for a total time of 60 min. However, no major metabolites were eluted after the first 30 min, which indicates that no significant amounts of added precursor are lost due to transformation to more hydrophilic metabolites. Frequent comparison of total counts per minute of [³H]25-OH-D₃-metabolites recovered after incubation of cells, with counts per minute measured for identical amounts of pure stock solution of [³H]25-OH-D₃, showed that our conditions (lipid extraction and chromatography) allow recovery of radioactivity above 90%.

RNA Isolation and cDNA Synthesis. Total RNA was prepared with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Two micrograms of RNA was reverse-transcribed (SuperScript II; Invitrogen) with the aid of an oligo(dT) primer [for semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR)] and a random hexamer primer (for real-time RT-PCR), respectively.

RT-PCR. cDNA strands were used for PCR (TaqPCR Core Kit; QIAGEN, West Sussex, UK). To amplify a 440-bp segment of CYP27B1 cDNA, primers used were: 5'-CAG AGG CAG CCA TGA GGA AC-3' (sense) and 5'-GGG TCC CTT GAA GTG GCA TAG-3' (antisense), using the GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT). To identify a 507-bp fragment of CYP24, the following primers were used: 5'-CCC ACT AGC ACC TCG TAC CAA C-3' (sense) and 5'-CGT AGC CTT CTT TGC GGT AGT C-3' (antisense). Expression of VDR mRNA was determined by amplification of a 421-bp segment from the region coding for the hormone binding domain using the sense sequence 5'-CGC TCC AAT GAG TCC TTC ACC-3' and the antisense sequence 5'-GCT TCA TGC TGC ACT CAG GC-3'. The following primers were used to evaluate -actin mRNAs: 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' (sense) and 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3'. PCR products were loaded onto a 2% agarose gel that contains ethidium bromide and were separated at 80 V and 250 mA. Bands were analyzed under UV light with the aid of a video camera imaging system (Herolab, Wiesloch, Germany).

Quantitative Real-Time RT-PCR. CYP27B1 mRNA levels were also quantified by the comparative C_T method. Reliability of data was improved by including an invariant endogenous control, 18S rRNA, to correct for sample-to-sample variations in RT-PCR efficiency and for errors in sample quantification. Relative abundance values were then calculated for 18S rRNA as well as for the experimental CYP27B1 sequence. The relative abundance value for each sample was normalized to the value derived from the control sequence (18S rRNA). The normalized values of different samples were compared directly. Real-time PCR was carried out in triplicate with an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and analyzed with 5-carboxyfluorescein-labeled Assays-on-Demand Gene Expression products for 18S rRNA and CYP27B1 (Applied Biosystems).

Thymidine Incorporation. DNA synthesis was assessed by [³H]thymidine incorporation into DNA. Cells were incubated with 4 µCi/ml [³H]thymidine (70 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) for 6 h and were precipitated twice with 5% trichloroacetic acid. After solubilization in 1 ml of 0.1 M NaOH, extracts were counted in a Wallac 1410 Liquid Scintillation Counter (GE Healthcare). Total protein content of the samples was determined with the BCA kit (Sigma). Results were expressed as counts per minute per microgram of protein and averaged on the basis of three experiments, with n = 6 per group.

Western Blotting. For total protein preparation, cells were rinsed and lysed with boiling lysis buffer (1% SDS in 10 mM Tris, pH 7.4). After homogenization (Polytron PT1200; Kinematica, Basel, Switzerland) and subsequent boiling, lysates were centrifuged, and supernatants were stored at -80°C. Protein content was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL). Total protein extracts were separated by electrophoresis on a 12% SDS-polyacrylamide gel and blotted to a nitrocellulose membrane at 4°C overnight. Unspecific binding was blocked in 3% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20 (Sigma). Following incubation with primary antibodies (monoclonal mouse anti-CK8, Chemicon International, Temecula, CA; polyclonal rabbit anti-cyclin D1, Neomarker, Fremont, CA), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and subsequently exposed to the SuperSignal West Pico Chemiluminescence Substrate system (Pierce). Bands were evaluated with a video camera imaging system (Herolab).

Statistical Analysis. Data are presented as mean ± S.D. from triplicate experiments. Where indicated, results were expressed as percentage of controls to correct for interassay differences. Statistical significance between more than two groups was determined by one-way ANOVA followed by Tukey multiple comparisons test using the GraphPad InStat software (version 3.06 for Windows). For comparison of two groups (Figs. 4D and 6B), two-tailed Student's t test was applied. Differences with P < 0.05 were considered significant.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effects of S-4a and 1,25-(OH)2D3 (1,25-D) on CYP24 mRNA expression in selected cancer cell lines. -Actin mRNA served as loading control. In MCF-7 (A) and DU-145 (C) cells, S-4a caused a dose-dependent increase of CYP24 mRNA transcripts (both P < 0.0001), but the dose required was at least 100-fold higher than with 1,25-D. B, high basal amounts of CYP24 mRNA in COGA-13 cannot be increased further by addition of either S-4a (P = 0.963) or 1,25-D (P = 0.976). In A to C, S-4a was administered for 4 h. ANOVA followed by post hoc analysis was used for multiple group analysis. ***, P < 0.001; and **, P < 0.01 indicate significant differences between treatment groups and the respective control group (Co). D, induction of CYP24 with 100 nM S-4a in DU-145 cells was weaker and occurred later than with 100 nM 1,25-D. Two-tail Student's t test was used for statistical analysis (***, P < 0.001; **, P < 0.01).

View larger version (24K): [in this window] [in a new window]   Fig. 6. A, analysis of CYP27B1 mRNA expression by quantitative RT-PCR. S-4a (1 µM), administered for 4, 8, 12, and 24 h, had no significant effect in MCF-7 breast cancer cells (ANOVA: P = 0.369). B, [H3]thymidine incorporation into DNA of subconfluent COGA-13 colon cancer cells. Proliferation was significantly reduced when both 10 nM 1,25-(OH)2D3 (1,25-D) and 1 µM S-4a were added to the cell culture for 48 h. C, S-4a (1 µM), administered for 48 h, significantly down-regulated cyclin-D1 protein expression in COGA-13 cells. Two-tailed Student's t test was used for statistical analysis (B and C; **, P < 0.01).

	Results

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Concentration- and Time-Dependent CYP24 Inhibition by S-4a in COGA-13 Cells. COGA-13 cells that display only CYP24 activity were used to investigate the time- and concentration-dependent action of S-4a, the 1,25-(OH)₂D₃ analog. Figure 2A shows that addition of 0.01 µM S-4a for 4 h resulted in a 20% reduction of 24-hydroxylated metabolites, whereas 1 µM S-4a for 4 h inhibited generation of 24-hydroxylated metabolites almost completely (P < 0.001). Using 1 µM S-4a, we then studied the kinetics of the inhibition. As shown in Fig. 2B, CYP24 activity was reduced by 20% after 30 min, by 70% after 4 h, by 80% after 24 h, and by 90% after 36-h exposure. The very rapid response after 30 min suggests a direct interaction of the inhibitor with the hydroxylase.

View larger version (19K): [in this window] [in a new window]   Fig. 2. HPLC analysis of effects of the vitamin D analog S-4a on vitamin D metabolism in COGA-13 cells. A, after 4 h, S-4a dose-dependently inhibited CYP24 activity (P < 0.0001), which was almost abolished when 10 µM S-4a was administered. B, S-4a (1 µM) decreased CYP24 activity in a time-dependent manner (P < 0.0001). Effects occurred as early as 30 min after addition of S-4a. ANOVA and post hoc analysis were used for multiple group analysis: ***, P < 0.001; and **, P < 0.01 indicate significant differences between the respective treatment group and the control group (Co). C, HPLC traces demonstrating the impact of 1 µM S-4a on vitamin D metabolism after 30 min (top) and 24 h (bottom), respectively. For a comparison with untreated COGA-13 cells, see Fig. 1C.

Effect of S-4a on Vitamin D-Modulated CYP24 and CYP27B1 Activity in MCF-7 Cells. CYP24 activity can be induced by 1,25-(OH)₂D₃ in many vitamin D metabolism-competent cells. However, this is not true for COGA-13 cells, which exhibit maximum expression in the unstimulated state. Therefore, to test the action of the S-4a on vitamin D-stimulated CYP24 activity, we chose MCF-7 cells whose CYP24 activity is relatively low. When MCF-7 cells were treated with 10 nM 1,25-(OH)₂D₃ for 12 h, CYP24 activity increased more than 5-fold above control levels demonstrated in Fig. 1C. Cotreatment with 1 µM S-4a reduced the amount of 24-hydroxylated metabolites by more than 80% (Fig. 3A). It should be noted that S-4a significantly elevated the very low amounts of 1-hydroxylated metabolites normally found in these breast cancer cells (Fig. 3A, comparison of top panel with bottom panel). To identify a possible mechanism, MCF-7 cells were subjected for 12 h to 10 nM 1,25-(OH)₂D₃, with the addition of 1 µM S-4a for 0.5 to 36 h. Figure 3B shows that 24-hydroxylation by 1,25-(OH)₂D₃ was induced over a period of 36 h and that the time-dependent reduction of CYP24 activity by S-4a was similar to that found in unstimulated COGA-13 cells, i.e., 20% after 0.5 h and approximately 80% after 4 h of treatment, maintained for at least 36 h. 1-Hydroxylated metabolites were markedly reduced by the addition of 1,25-(OH)₂D₃ over the same time frame of 36 h. However, treatment with S-4a time-dependently counteracted this reduction (Fig. 3C).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Coincubation of MCF-7 cells with 1 µM S-4a (added for 0.5–36 h) and 0.01 µM1,25-(OH)2D3 (1,25-D; added for 12 h). For controls, see Fig. 1C and hatched columns in B and C. Addition of 1,25-D caused marked stimulation of CYP24 (3A, top; white columns in B), whereas levels of CYP27B1 metabolites (1,25-D and 1,25-epi-D) decreased (white columns in C). S-4a (1 µM) counteracted both effects in a time-dependent manner (3A, bottom; black columns in B and C; both P < 0.0001). ANOVA followed by Tukey multiple comparisons test was used for group analysis: ***, P < 0.001; and **, P < 0.01 indicate significant differences between the respective treatment group (1,25-D + S-4a) and groups treated with 1,25-D alone.

Comodulation of CYP24 mRNA by 1,25-(OH)₂D₃ and S-4a. To test whether the CYP24 inhibitor S-4a would prevent degradation of 1,25-(OH)₂D₃ by 24-hydroxylase-competent cells, MCF-7 cells were cultured in the presence of 10 nM 1,25-(OH)₂D₃ for 5 h to stimulate CYP24 mRNA expression (see also Fig. 4A). Coincubation with 1 µM S-4a from 2 to 12 h resulted in a progressive increase of CYP24 mRNA, with CYP24 at least doubled after 4 h (Fig. 5A). Caco-2 cells, however, responded quite differently. Although 10 nM 1,25-(OH)₂D₃ induced CYP24 mRNA expression at least 5-fold, there was no further induction by the addition of 1 µM S-4a (Fig. 5B).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effects of 1 µM S-4a and 10 nM 1,25-(OH)2D3 (1,25-D; administered for 12 h) on CYP24 mRNA levels. A, in MCF-7 cells, S-4a time-dependently increased CYP24 mRNA levels (P = 0.015) beyond those induced by 1,25-D. B, no additive effects were seen in Caco-2 cells (P = 0.941). Depicted blots are representative for 8-h experiments. ANOVA and post hoc analysis were used for statistical multiple group analysis: **, P < 0.01; and *, P < 0.05 indicate significant differences between the respective treatment group and the control (Co).

Growth Response of COGA-13 Cells to 1,25-(OH)₂D₃ with and without S-4a Cotreatment. When measuring [³H]thymidine incorporation into DNA, neither treatment with 10 nM 1,25-(OH)₂D₃ nor with 1 µM S-4a alone affected proliferation of COGA-13 cells. However, cotreatment of cells with both 1,25-(OH)₂D₃ and S-4a inhibited the DNA synthesis by almost 50% (Fig. 6B).

Cyclin-D1 Is Down-Regulated by S-4a. Western blot analysis of cyclin-D1 protein expression demonstrated that 1 µM S-4a, added to COGA-13 cells for 48 h, significantly down-regulated this important cell-cycle regulator (Fig. 6C).

	Discussion

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To investigate the therapeutic properties of S-4a, the novel vitamin D 24-phenylsulfone analog, we chose cell lines derived from three organs that are particularly affected by age-related sporadic cancers: colon, mammary gland, and prostate. Cell lines were also chosen for their differential expression of vitamin D hydroxylases (Fig. 1). The two colon cell lines were the highly differentiated Caco-2 (low CYP24, high CYP27B1) and the undifferentiated COGA-13 cells (extremely high CYP24, no CYP27B1 expression). Mammary gland-derived cells were MCF-7, and the prostate-derived cells were DU-145. Both of these cell lines express CYP24 and CYP27B1 to about the same extent.

S-4a inhibited CYP24 activity rapidly (within 30 min or less) and dose-dependently, the effect lasting at least 36 h in all tumor cell lines tested (Figs. 2 and 3). This is most probably due to a direct interaction of the 1,25-(OH)₂D₃ analog with the enzyme, although this remains to be proven experimentally. On the other hand, at the mRNA level, S-4a seems to bind to the VDR and to act as a transcription factor (Posner et al., 2004), raising CYP24 mRNA levels within 4 h to those due to 1,25-(OH)₂D₃. However, the vitamin D metabolite is effective at a concentration 2 orders of magnitude lower than is S-4a (Fig. 4). Even after 48-h exposure, both the 24-phenylsulfone analog and 1 µM1,25-(OH)₂D₃ still elevate CYP24 mRNA expression beyond control levels in DU-145 cells (Fig. 4D). COGA-13 cells are an exception because their already high levels of CYP24 cannot be increased by either 1,25-(OH)₂D₃ or S-4a (Fig. 4B).

Although 1,25-(OH)₂D₃ significantly increases 24-hydroxylase activity, leading to enhanced production of 24-hydroxylated metabolites (Fig. 3B), S-4a does not do so. Rather, it causes CYP24 activity to drop rapidly and for a long period (Figs. 2 and 3). At the same time, 1-hydroxylated metabolites are elevated to the same degree by which 24-hydroxylated metabolites are reduced (Fig. 3). This suggests that as a result of the reduction in CYP24 activity, more precursor 25-OH-D₃ becomes available for 1-hydroxylation. Alternatively, newly synthesized 1-hydroxylated metabolites may be subject to less rapid degradation.

As shown in Fig. 5A, S-4a, when added to the MCF-7 culture, increased CYP24 mRNA beyond that due to 1,25-(OH)₂D₃ treatment alone. This may be the result of an uncoupling of the effects of the two compounds or of a prolongation of the half-life of 1,25-(OH)₂D₃ due to inhibition of 24-hydroxylase activity. However, Caco-2 cells responded differently in that S-4a did not further raise 1,25-(OH)₂D₃-induced CYP24 mRNA levels (Fig. 5B). Because the 1,25-(OH)₂D₃-induced increase in CYP24 mRNA did not lead, for presently unknown reasons, to higher CYP24 activity in Caco-2 cells, the effect of S-4a in MCF-7 cells is likely to involve a prolongation of the vitamin D effect.

In view of the potential importance of vitamin D treatment for cancer patients, attempts have been made to inhibit degradation of the active vitamin D metabolite (Masuda and Jones, 2006). Ketoconazole inhibits CYP24 expression (Peehl et al., 2002) but also affects CYP27B1 expression negatively inasmuch as it is a broad-spectrum P450 inhibitor. This, in the case of prostate cells, gives ketoconazole an additional advantage because it apparently prevents androgen biosynthesis (Blagosklonny et al., 2000). Liarozole has similar action on vitamin D metabolism (Ly et al., 1999) but is very toxic. Genistein inhibits CYP24 mRNA expression, and this phytoestrogen also directly inhibits 24-hydroxylase activity within minutes in prostate cells. On the other hand, daidzein, another isoflavone, does not have the inhibitory action of genistein (Farhan et al., 2002, 2003). Genistein inhibits CYP24 also in breast- and colon-derived cells (Lechner et al., 2006a) as well as in vivo in mouse colon mucosal cells (Cross et al., 2003, 2004).

Consumption of genistein in soy-consuming countries, by inhibiting vitamin D degradation, may be responsible for the reduced incidence of prostate, mammary, and colon cancer, but it is unlikely to constitute a therapeutic agent in the treatment of cancer. Although a tetralone derivative has been shown to improve the potency of 1,25-(OH)₂D₃ in DU-145 prostate cancer cells (DU-145), it is not active in PC-3, another prostate cell line (Yee et al., 2006). This raises the question of what properties of cancer cells make this treatment possible and what potential it has for treating tumor patients.

The 1,25-(OH)₂D₃ analog S-4a used in the present study has several remarkable properties that make it potentially more useful than other CYP24 inhibitors. For one, it inhibits only CYP24 enzymatic activity, but not that of CYP27B1. Because cancer patients may be also deficient in the precursor 25-OH-D₃, treatment with a broad-spectrum CYP inhibitor like the potent antimycotic ketoconazole could result in hypovitaminosis D and toxicity at higher concentrations.

Inhibition of solely 24-hydroxylase activity with a vitamin D analog that is nontoxic at high concentrations could protect from degradation of both vitamin D and vitamin D analogs, a property important for tumor therapy especially when considering that analogs may have enhanced antimalignant potential due to altered side-chain structure (Hofer et al., 1999). Such altered side-chain structure could slow degradative processes, but it could also enhance transcriptional activity (Posner et al., 2000). S-4a is at least 10 times more potent than the commonly used CYP inhibitor ketoconazole. It is nontoxic in rats, even at the high concentration of 10 µg/kg body weight, and does not increase urinary calcium levels, whereas these are raised highly significantly already with 0.5 µg/kg body weight calcitriol. In our in vitro assays, we have achieved almost 90% inhibition of 24-hydroxylase activity with 1000 nM S-4a.

For another, 1000 nM S-4a may exert direct, inhibitory effects on the cell cycle by significantly down-regulating cyclin D1 protein expression (Fig. 6C). Although the binding affinity of S-4a for the VDR is approximately 4% of that of 1,25-(OH)₂D₃, and the ED₅₀ for the transcriptional activity of S-4a is 1000 nM (Posner et al., 2004), S-4a as a vitamin D analog can apparently still exert genomic activity via binding to the VDR, and an additional direct antiproliferative potential is therefore not surprising. Although no vitamin D-responsive elements are known in its promoter, cyclin D1 has been demonstrated previously to be a vitamin D target gene (Verlinden et al., 1998; Tong et al., 1999). At the low genomic activity of S-4a, it is also not surprising that much higher concentrations than that of calcitriol are needed to stimulate CYP24 mRNA expression. We suggest that it is exactly such a high concentration of S-4a that, by binding and neutralizing CYP24 activity, leads to inhibition of 1,25-(OH)₂D₃ degradation.

	Acknowledgements

 
We thank Dr. Felix Bronner (University of Connecticut Health Center, Farmington, CT) for critical reading of the manuscript and Martin Petkovich (Cytochroma Inc.) for providing KRC-24SO₂Ph-1.

	Footnotes

 
This work was supported by the Hans and Blanca Moser Stiftung (Austria), by a grant from the American Institute of Cancer Research, and by Project 9850 from the Austrian National Bank (Vienna, Austria).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.115451.

ABBREVIATIONS: 1,25-(OH)₂D₃,1,25-dihydroxyvitamin D₃; 25-OH-D₃, 25-hydroxyvitamin D₃; CYP27B1, 25-hydroxyvitamin D₃-1-hydroxylase; CYP24, 25-hydroxyvitamin D₃-24-hydroxylase; DMEM, Dulbecco's modified Eagle's medium; HPLC, high-pressure liquid chromatography; S-4a, KRC-24SO₂Ph-1; RT, reverse transcriptase; PCR, polymerase chain reaction; VDR, vitamin D receptor; ANOVA, analysis of variance.

Address correspondence to: Heide S. Cross, Department of Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: heide.cross{at}meduniwien.ac.at

	References

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