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Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells
Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 228–235
Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells Sook Wah Yee a , Moray J. Campbell b,∗ , Claire Simons a,∗∗ b
a Division of Medicinal Chemistry, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK Division of Medical Sciences, Institute of Biomedical Research, Birmingham University, Birmingham B12 2TT, UK
Accepted 8 November 2005
Abstract Induction of growth arrest and differentiation by 1␣,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the antiproliferative action of 1,25-(OH)2 D3 . This appears to be due to increased 1,25-(OH)2 D3 -metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D3 metabolising enzymes (IC50 3.5 M), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC50 20 M). The combination of the inhibitor and 1,25(OH)2 D3 caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)2 D3 plus inhibitor of Vitamin D3 metabolising enzymes co-ordinately upregulated CYP24, p21waf1/cip1 and GADD45␣. © 2006 Elsevier Ltd. All rights reserved. Keywords: Vitamin D3 metabolising enzymes; Tetralone; Prostate cancer cells
1. Introduction The majority of the prostate cancer patients demonstrate good initial responses to surgical castration and/or hormonal therapy [1]. Unfortunately, hormonal therapy is not capable of producing durable responses in the majority of the patients who subsequently develop androgen-independent prostate cancer (AIPC). New effective therapies are needed in the management of AIPC patients. One potential therapeutic strategy is to employ a differentiating agent to restore the normal balance of proliferation and differentiation, such as with the biological active metabolite of Vitamin D3 , 1␣,25dihydroxyvitamin D3 (1,25-(OH)2 D3 ). Encouragingly 1,25(OH)2 D3 exerts some pro-differentiating actions and inhibits proliferation of prostate cancer cells in vitro and in vivo [2–4]. ∗
Corresponding author. Tel.: +44 121 4158713; fax: +44 121 4158712. Corresponding author. Tel.: +44 29 20876307, fax: +44 29 20874149. E-mail addresses: [email protected] (M.J. Campbell), [email protected] (C. Simons). ∗∗
0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.11.004
In parallel to the well established endocrine synthesis of 1,25-(OH)2 D3 via sequential hydroxylation steps in the liver and kidney, it has become apparent there is local autocrine/paracrine synthesis of 1,25-(OH)2 D3 . The principle enzymes in this process are the cytochrome P450 enzymes, CYP27B1 (1␣-OHase) and CYP24 (24-OHase). It has been shown that CYP24 and CYP27B1 are also expressed in many target tissues, including prostate-epithelial cells [5,6], supporting the role for local 1,25-(OH)2 D3 synthesis in the prostate. One well established VDR target gene is the CYP24, which is highly inducible by 1,25-(OH)2 D3 , resulting in an increase in the metabolism of 1,25-(OH)2 D3 . Prostate cancer cells expressed high level of 1,25-(OH)2 D3 -induced CYP24 activity, which is inversely proportional to growth inhibition [7]. It has been suggested that in cancer cells the rapid breakdown of 1,25-(OH)2 D3 by over-active CYP24 might be the cause of resistance to 1,25-(OH)2 D3 . Reflecting this, P450 inhibitors which are able to inhibit the activities of CYP24, e.g. ketoconazole (1), genistein (2), and liarozole
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Fig. 1. Inhibitors of Vitamin D3 metabolising enzymes–ketoconazole, 1; genistein, 2; liarozole, 3; the tetralone derivative, 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one, 4.
(3), result in increased 1,25-(OH)2 D3 half-life and antiproliferative effect in DU-145 cells [8–11]. We have demonstrated that 2-substituted-3,4-dihydro-2H-naphthalen-1-one (tetralone) derivatives inhibit the activities of Vitamin D3 and/or retinoic acid metabolising enzymes [13,14]. Among the tetralone derivatives, 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) (Fig. 1) showed inhibition against the Vitamin D3 metabolising enzymes in the rat kidney mitochondria in vitro assay (IC50 = 3.5 M, compared to ketoconazole, IC50 = 20 M) [13]. In the current study, we have examined the antiproliferative effects of the inhibitors, ketoconazole (1) and the tetralone derivative (4), both alone and in combination with 1,25-(OH)2 D3 in DU-145 and PC-3 cells. We also examined the effect of the inhibitors alone and in combination with 1,25-(OH)2 D3 on the regulation of VDR target genes, CYP24, p21waf1/cip1 and GADD45␣.
2. Materials and methods 2.1. Chemicals 1␣,25-Dihydroxyvitamin D3 (1,25-(OH)2 D3 ) was a kind gift from Dr. Milan R. Uskokovic (Hoffman-La Roche, Nutley, NJ). 25-Hydroxyvitamin D3 (25-(OH)D3 ) (Fluka chemicals, Dorset, UK) and 1,25-(OH)2 D3 compounds were stored at 1 mM in ethanol at −20 ◦ C in the dark. 25-Hydroxy-[26,27-methyl-3 H]-vitamin D3 (30 Ci/mmol) was purchased from Amersham Biosciences (Buckinghamshire, UK). 2-(4-Hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) was chemically synthesised in the laboratory as described previously [13] and ketoconazole (Sigma, Poole, UK) stored as a 1 mM stock solution in phosphate buffered saline (PBS) pH 7.5 at 4 ◦ C. (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) for cell-proliferation assay was purchased from Sigma (Poole, UK). All solvents used for HPLC were of
HPLC grade and were purchased from Fisher Scientific (Leicestershire, UK). 2.2. Cell culture The androgen-independent prostate cancer cell lines PC3 and DU-145 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained in RPMI 1640 medium (GibcoBRL, Paisley), supplemented with 10% fetal calf serum (Gibco-BRL), 100 units/mL penicillin and 100 g/mL streptomycin. The cells were passaged by trypsinising with 0.25% trypsin–EDTA (Gibco-BRL). The cells were grown at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. 2.3. Preparation of rat kidney mitochondria Mitochondria were isolated from male Wistar rats. Three Wistar rats (250 g each) were fed for 2 weeks with calcium and Vitamin D3 replete diet (calcium carbonate was added to the feed to achieve a 1% calcium level and Vitamin D3 was added to achieve 2200 i.u./kg in the feed). The isolated kidneys were washed with ice-cold phosphate buffer (50 mM, pH 7.4) containing 0.25 M sucrose, then resuspended in icecold Tris–acetate buffer (15 mM, pH 7.4) containing 0.25 M sucrose. The kidney was cut into smaller pieces using scissors and the tissues were homogenised using an Elvejhm-Potter homogeniser. The nuclei and unbroken cells were pelleted by centrifugation for 20 min at 600 × g at 4 ◦ C. The above supernatant was then centrifuged for 20 min at 12 000 × g at 4 ◦ C. The pellet containing the mitochondria was washed with the Tris–acetate buffer and resuspended in ice-cold 20% glycerol and 15 mM Tris–acetate pH 7.4, containing 0.6% sodium cholate. This 20% (w/w) homogenate was stirred on ice for 1 h and the homogenate was centrifuged at 12 000 × g for 1 h to disrupt the mitochondria pellet. The suspension was then distributed into 1.5 mL capped microcentrifuge tubes, frozen in liquid N2 and stored at −80 ◦ C until needed.
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2.4. High performance liquid chromatography (HPLC) analysis of 25-hydroxyvitamin D3 metabolism The assay performed was based on a modification of the general procedure previously described [12,15]. Tubes in duplicate, with a total volume of 500 L, containing 25-hydroxy-[26,27-methyl-3 H]-vitamin D3 (0.05 Ci as a tracer) with unlabelled 25-(OH)D3 (25 M) (10 L), inhibitor (10 L), NADPH (4 mM, 50 L), phosphate buffer (50 mM, pH 7.4, 400 L) and rat kidney mitochondria (30 L) were incubated in a shaking water bath for 30 min at 37 ◦ C. The reaction was terminated by the addition of acetic acid (1% (v/v), 100 L). The Vitamin D3 metabolites were obtained by extraction with ethyl acetate (2 mL) containing 0.02% butylated hydroxyl anisole. After evaporation of the organic solvent, the residue was analysed by HPLC system connected to a -RAM online scintillation detector, connected to a Compaq PC running Laura data acquisition and analysis software (LabLogic Ltd.). The separated [3 H]-vitamin D3 metabolites were separated on the 5 M ODS EXSIL® 4.6 mm × 200 mm column using an isocratic method: 750 mL acetonitrile/250 mL 1% (w/v) ammonium acetate/1 mL acetic acid at a flow rate of 1.5 mL/min. 25-(OH)D3 metabolites were identified by matching their elution rates to the retention times of the known standards, 1,25-(OH)2 D3 , 24,25-(OH)2 D3 and 1,24,25-(OH)3 D3 (generously supplied by Dr. Lise Binderup, Leo Pharma, Denmark) by using a photodiode array detector to monitor UV absorption at 265 nm. The separated [3 H]-vitamin D3 metabolites were quantitatively calculated from the areas under the curves. Using a control with solvent (DMSO) instead of inhibitor, these results were expressed as ‘percentage inhibition relative to control’ (%) = 100[metabolites (control) − metabolites (inhibitor)/metabolites (control)]. 2.5. Inhibition of CYP24 enzyme activity in DU-145 cells A confluent DU-145 cell culture from a 75 cm2 sterile flask was treated with 10 nM 1,25-(OH)2 D3 for 24 h. Cells were then rinsed with 10 mL PBS and incubated with culture medium at 37 ◦ C in a humidified atmosphere of 5% CO2 for approximately 30 min to remove 1,25-(OH)2 D3 . Cells were detached by trypsinization and 2 × 106 cells were assayed in duplicate. Cells were incubated in 200 L medium (RPMI 1640 with 1% FBS) for 30 min at 37 ◦ C in a shaking water bath with 25-hydroxy-[26,27-methyl-3 H]-vitamin D3 (0.05 Ci/tube as tracer), 1 M 25-(OH)D3 and with ethanol or various concentrations of inhibitor (1, 10, 20 and 50 M). The reaction was terminated by the addition of 620 L methanol–chloroform (2:1). The tubes were then centrifuged at 500 × g for 10 min. The supernatant was then transferred into respective glass tubes containing 200 L of chloroform and 100 L of distilled water. After vortexing, the tubes were centrifuged at 500 × g for 15 min. The
lower organic phase was transferred into another clean glass tube, whereas the water phase was re-extracted with 200 L chloroform. The combined organic phases in the tubes were placed in a rotating evaporator for 20–30 min. The production of [3 H]-24,25-(OH)2 D3 was quantitated by HPLC using the mobile phase and HPLC column described above (Section 2.4). 2.6. Cell proliferation assays Cells were plated in 96-well plates (Appleton Woods, Birmingham, UK). Both the DU-145 and PC-3 cells were seeded at 2 × 103 cells/well. Cells were allowed to attach (left at least 7 h), and treated with growth media containing varying concentrations of the inhibitor alone, varying concentrations of the inhibitor with 10 nM 1,25-(OH)2 D3 , or 10 nM 1,25-(OH)2 D3 alone, resulting in a final volume of 100 L/well. The plates were incubated for 96 h, with re-dosing after 48 h. After 93 h incubation, 20 L of MTT solution (5 mg/mL in distilled water and filtered) was added to each well and the cells were left for 3 h in the incubator (37 ◦ C and 5% CO2 ). The 120 L of MTT-containing cells and medium were removed without disturbing the blue precipitate attached to the well. One hundred microliters of DMSO (Sigma–Aldrich, Dorset, UK) were added into each well and incubated for 15 min at room temperature. Finally, the absorption was measured at 550 nm using the absorbance microplate reader (EMax® , Molecular Devices Corporation). The growth inhibition was expressed as a percentage of control. 2.7. Extraction of RNA and reverse transcription DU-145 and PC-3 prostate cancer cells were both seeded at 3.5 × 105 cells/well in a six-well plate. Cells were dosed as indicated and total RNA was extracted using Tri Reagent (Sigma–Aldrich) following the manufacturer’s instructions. Total RNA amounts were quantified by measuring absorbance at 260 nm. The A260 /A280 nm absorption ratio was greater than 1.7. For real-time RT-PCR, cDNA was prepared from 2 g of total RNA by reverse transcription with M-MLV reverse transcriptase (SuperscriptTM II Reverse Transcriptase, Invitrogen, UK) and performed in a thermocycler (Applied Biosystems Gene Amp PCR System 9700). 2.8. Real-time quantitative RT-PCR The expression of the specific mRNAs (i.e. CYP24, p21waf1/cip1 and GADD45␣) was quantitated using the ABI PRISM 7700 Sequence Detection System. DU-145 cells were treated with DMSO vehicle, 10 nM 1,25-(OH)2 D3 for 7 h, inhibitor (10 M tetralone derivative (4) or 7.5 M ketoconazole) for 7 h, or pre-treatment with the inhibitor overnight prior to the addition of 10 nM 1,25-(OH)2 D3 for 7 h. Total RNA was isolated as described above. Each sample was amplified in triplicate wells in 20 L volumes
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Table 1 The primers and probe sequences Primers and probe
Sequence
CYP24 forward primer CYP24 reverse primer CYP24 probe p21waf1/cip1 forward primer p21waf1/cip1 reverse primer p21waf1/cip1 probe GADD45␣ forward primer GADD45␣ reverse primer GADD45␣ probe
5 -CAAACCGTGGAAGGCCTATC-3 5 -AGTCTTCCCCTTCCAGGATCA-3 5 -ACTACCGCAAAGAAGGCTACGGGCTGT-3 5 -GCAGACCAGCATGACAGATTTC-3 5 -GGATTAGGGCTTCCTCTTGGA-3 5 -CCACTCCAAACGCCGGCTGATCTTT-3 5 -AAGACCGAAAGGATGGATAAGGT-3 5 -GTGATCGTGCGCTGACTCA-3 5 -TGCTGAGCACTTCCTCCAGGGCAT-3
containing 2× qPCRTM QuickGoldStar Master Mix (5 mM MgCl2 , dNTPs, QuickGoldStar DNA polymerase, UracilN-glycosylase), 2.5 pmol FAM-labelled TaqMan probe and 18 pmol primers. All-reactions were multiplexed with preoptimised control primers and VIC labelled probe for 18S ribosomal RNA (TaqMan® Ribosomal RNA control reagents, Applied Biosystems, Warrington, UK). Primer and probe sequences are given in Table 1. Reactions were cycled as follows: 50 ◦ C for 2 min, 95 ◦ C for 10 min (for activation of the QuickGoldStar DNA polymerase); then 44 cycles for 95 ◦ C for 15 s and 60 ◦ C for 1 min. Data were expressed as Ct values and used to determine ␦Ct values. ␦Ct = Ct of the target gene minus Ct of the housekeeping gene. The housekeeping gene is the 18S ribosomal RNA. The data was transformed through the equation 2−␦␦Ct to give fold changes in gene expression. ␦␦Ct = ␦Ct of the target gene from treated cells minus ␦Ct of the target gene from non-treated (control) cells. To exclude potential bias due to averaging of data all statistics were performed with ␦Ct values. Measurements were carried out at least two times each in triplicate wells for each condition. 2.9. Statistical analyses The data were obtained from at least two different experiments and are presented as mean ± S.E.M. All statistical analyses were performed using the Student’s t-test. P < 0.05 was considered statistically significant.
3. Results 3.1. Inhibition of Vitamin D3 metabolising enzymes in rat kidney mitochondria The HPLC analysis identified major polar metabolites of [3 H]-25-(OH)D3 , namely 1,25-(OH)2 D3 and 24,25(OH)2 D3 , and a minor polar metabolite, 1,24,25-(OH)3 D3 , in the organic solvent extracts of rat kidney mitochondria.
Fig. 2. DU-145 cells were treated with 10 nM 1,25-(OH)2 D3 for 24 h. Treated cells were detached by trypsinization and incubated with various concentrations of the inhibitor for 30 min, 37 ◦ C in water bath as described in Section 2.5. The separated [3 H]-25-(OH)D3 and [3 H]-24,25-(OH)2 D3 were quantitatively calculated from the areas under the curves by HPLC as described in Section 2.4. Using DMSO solvent as control instead of inhibitor, the results were expressed as percentage inhibition relative to control. This experiment was performed two times and each time in duplicate. Values represent mean ± S.E.M. (n = 4).
The rat kidney mitochondria express both CYP24 and CYP27B1 enzyme activities. Ketoconazole and tetralone derivative (4) have an inhibitory effect on both enzymes, and inhibit the metabolism of 25-(OH)D3 in a dose-dependent manner (between 1 and 100 M). The IC50 for the inhibition of vitamin D3 metabolising enzymes was 3.5 and 20 M, for tetralone derivative (4) and ketoconazole, respectively, as previously reported [13]. The IC50 values were the mean of two experiments and were determined from a dose–response curve. 3.2. Inhibition of CYP24 enzyme activity in DU-145 cells The CYP24 enzyme activity in DU-145 cells is enhanced by the presence of 10 nM 1,25-(OH)2 D3 for 12–24 h. Whereas, ketoconazole and tetralone derivative (4) alone, demonstrated no intrinsic ability to induce CYP24 enzyme activity. After DU-145 cells were treated with 10 nM 1,25(OH)2 D3 for 24 h to induce CYP24 enzyme activity, various concentrations of the inhibitor (1, 10, 20 and 50 M) were examined for their abilities to inhibit the CYP24 enzyme activity. As illustrated in Fig. 2, ketoconazole and tetralone derivative (4), were able to inhibit directly CYP24 enzyme activity in a dose-dependent manner by blocking the conversion of [3 H]-25-(OH)D3 to [3 H]-24,25-(OH)2 D3 . Ketoconazole and tetralone derivative (4), at 10 M, resulted in approximately 50% and 65% inhibition of CYP24 enzyme activity. 3.3. Effects of Vitamin D3 hydroxylases inhibitor and 1,25-(OH)2 D3 on prostate cancer cell growth DU-145 and PC-3 are relatively insensitive to the antiproliferative effect of 1,25-(OH)2 D3 with ED50 > 100 nM
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Fig. 3. The effect of Vitamin D3 metabolising enzymes inhibitor, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 on the growth of DU-145 (a and b) and PC-3 (c and d) cells. These effects were assessed by MTT assay as described in Section 2. Results are expressed as a mean percentage (±S.E.M.) of untreated control wells (n = 3). Each point represents a mean of three experiments with triplicate dishes. * P < 0.05, ** P < 0.01 when samples were compared to samples treated with the inhibitor alone.
compared to non-malignant prostate epithelial cells, as demonstrated previously by our group [17]. In the current study 10 nM 1,25-(OH)2 D3 resulted in approximately 5% growth inhibition after 96 h. DU-145 cells, treated with either ketoconazole or the tetralone derivative (4) alone showed significant anti-proliferation effect at >10 M (with >20% growth inhibition) (Fig. 3a and b). However, the combination treatment with either 5 M ketoconazole or 10 M of the tetralone derivative (4) in combination with 10 nM 1,25(OH)2 D3 caused more than 30% growth inhibition in DU-145 (Fig. 3a and b), suggesting a co-operative effect. High doses of ketoconazole and the tetralone derivative (4) alone showed significant anti-proliferation effects at, ≥10 and >25 M, respectively in PC-3. In contrast, PC-3 cells were not only recalcitrant to 1,25-(OH)2 D3 (at 10 nM) but also did not show significant anti-proliferation when the inhibitors and 1,25-(OH)2 D3 were used together (Fig. 3c and d).
have significant effects on the CYP24 mRNA level in DU-145 cells. Ketoconazole (100 M), when combined with 1,25(OH)2 D3 (10 nM) reduced the CYP24 mRNA to 362-fold change in DU-145 cells, relative to the cells treated with 10 nM 1,25-(OH)2 D3 alone (P < 0.05). By contrast, 7.5 M ketoconazole when combined with 10 nM 1,25-(OH)2 D3 increased the CYP24 mRNA transcription level strikingly
3.4. Effect of the Vitamin D3 metabolising enzymes inhibitor and 1,25-(OH)2 D3 on CYP24 mRNA expression The untreated DU-145 cells expressed very low level of CYP24 mRNA. However, when the cells were treated with 10 nM 1,25-(OH)2 D3 for 7 h, DU-145 cells showed approximately 2000-fold induction in CYP24 mRNA relative to the untreated cells (Fig. 4). Ketoconazole and the tetralone derivative (4) alone at high and low concentrations did not
Fig. 4. Quantitative real-time RT-PCR determination of CYP24 mRNA levels in DU-145 cells treated with, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 . * P < 0.05, ** P < 0.01 and *** P < 0.001 when samples were compared to cells treated with the 10 nM 1,25-(OH)2 D3 alone or with untreated cells. Values are shown as mean ± S.E.M. (n = 3).
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Fig. 5. Quantitative real-time RT-PCR determination of (a) p21waf1/cip1 and (b) GADD45␣ mRNA levels in DU-145 cells treated with, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 . * P < 0.05. Values are shown as mean ± S.E.M. (n = 3).
(5340-fold change) in DU-145 compared to cells treated with 1,25-(OH)2 D3 alone (2000-fold change) (P < 0.01), as shown in Fig. 4. On the other hand, combination treatment with the tetralone derivative (4) (100 or 10 M) and 10 nM 1,25(OH)2 D3 in DU-145 significantly enhanced the CYP24 mRNA (>10 000-fold change) compared to the cells treated with 1,25-(OH)2 D3 alone (1999-fold change) (P < 0.001). 3.5. Effect of the Vitamin D3 metabolising enzymes inhibitor and 1,25-(OH)2 D3 on p21waf1/cip1 and GADD45α mRNA expression The results here showed that treatment with 10 nM 1,25(OH)2 D3 alone had no significant effect on p21waf1/cip1 and GADD45␣ mRNA expression in DU-145 cells compared to untreated cells (Fig. 5a and b). However, when the cells were treated with the inhibitor alone (at 100 M) or combination with 1,25-(OH)2 D3 (10 nM), the p21waf1/cip1 and GADD45␣ mRNA were enhanced significantly (P < 0.01) (data not shown here). This observation indicated that the inhibitor itself at 100 M has a significant effect on p21waf1/cip1 and GADD45␣ mRNA, and reflects the direct anti-proliferative effect of the (100 M) alone. Lower concentrations of the inhibitor alone (10 M of the tetralone derivative (4)), did not have an effect on p21waf1/cip1 and GADD45␣ mRNA level in DU-145 (Fig. 5a and b). It is interesting to observe the co-operatively enhanced p21waf1/cip1 and GADD45␣ mRNA level by the tetralone derivative (4) (10 M) and 1,25-(OH)2 D3 in DU-145 (Fig. 5a and b) (P < 0.05). However, this co-operative enhancement of p21waf1/cip1 and GADD45␣ mRNA level was not observed in combination treatment with ketoconazole (7.5 M) and 1,25-(OH)2 D3 , as ketoconazole (at 7.5 M) alone still has an enhanced effect on the p21waf1/cip1 and GADD45␣ mRNA compared to untreated cells (P < 0.05) (Fig. 5a and b).
4. Discussion The tetralone derivative (4) displayed potent inhibitory activity for 25-(OH)D3 metabolism, as revealed using the rat kidney mitochondria assay, compared with ketoconazole. This quick and robust in vitro assay is a more rapid and facile assay in identifying potential inhibitors compared with the inhibition studies carried out using DU-145 or other intact cell-lines and also allows screening of large libraries of potential inhibitors. It is known from various studies that ketoconazole inhibits the activity of Vitamin D3 metabolising enzymes, i.e. both CYP27B1 and CYP24 enzymes [18,19] and thus limits the conversion of active metabolite, 1,25-(OH)2 D3 , to inactive metabolites. The rat kidney mitochondria assay described here demonstrated that ketoconazole and the tetralone derivative (4) inhibit the activities of these Vitamin D3 metabolising enzymes. Likewise in the CYP24-induced DU-145 cell assay, ketoconazole and the tetralone derivative (4) both demonstrated CYP24 enzyme inhibition. As a result this probably resulted in the prolonged half-life of 1,25-(OH)2 D3 , which in turn potentiated the anti-proliferative action of 1,25-(OH)2 D3 (Fig. 3). However, this potentiation of the anti-proliferative effect of 1,25-(OH)2 D3 was not significant in PC-3 cells. This demonstrated that the mechanisms of resistance to 1,25(OH)2 D3 are multifactorial [17]. In the real-time quantitative RT-PCR studies in DU-145 cells, two different concentrations of the inhibitors were used, based on the cell proliferation (MTT) assay and the kidney mitochondrial assay. We decided to choose 100 M of the inhibitors, ketoconazole or tetralone derivative (4), as at such concentration, significant inhibition (>70%) of the Vitamin D3 metabolising enzymes were observed. At lower concentration of the inhibitor, 7.5 M ketoconazole or 10 M tetralone derivative (4), it was possible to observe more cooperative effects.
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From the real-time RT-PCR analysis, there was no inhibition of the basal CYP24 mRNA expression with the inhibitor alone. However, we saw an increase in 1,25-(OH)2 D3 induced CYP24 mRNA with either tetralone derivative (4) (10 and 100 M) or ketoconazole (7.5 M) co-treatment. This co-operatively enhanced CYP24 mRNA level observed from the combination treatment could be the result of the prolonged half-life of 1,25-(OH)2 D3 , which in turn enhanced the genomic actions of 1,25-(OH)2 D3 to induce VDR target genes such as CYP24. The multiple VDR response elements in CYP24 made this target gene a highly sensitive indicator of VDR effects. Similarly Cross and co-workers demonstrated that genistein at higher concentration (50 M) reduced the basal CYP24 mRNA and the 1,25-(OH)2 D3 -induced CYP24 mRNA but not when a lower concentration of genistein (5 and 25 M) was used [8,16]. Intracellular metabolic profiles are required to resolve if there is a disparity between the CYP24 mRNA levels and actual enzyme activity in the presence of these inhibitors. Consistent with the cell-proliferation assay, ketoconazole or the tetralone derivative (4) (at 100 M) either alone or co-treated with 1,25-(OH)2 D3 , caused significant antiproliferative effects, which correlated with enhanced levels of p21waf1/cip1 and GADD45␣ mRNA in DU-145 (Fig. 5a and b). The p21waf1/cip1 protein, a cell-cycle regulatory protein, inhibits the activity of cyclin-dependent kinases in the G0 /G1 cell-cycle phase, resulting in cell-cycle arrest. Whereas, induced expression of GADD45␣ inhibits cell proliferation and it is induced by various agents that damage DNA and arrest cell growth [20]. Induction of GADD45␣ contributes to the growth-inhibitory effects of 1,25-(OH)2 D3 in 1,25-(OH)2 D3 -sensitive cell lines [17]. The tetralone derivative (4) (at 10 M) did not alter the p21waf1/cip1 and GADD45␣ mRNA level, however, combination with 1,25-(OH)2 D3 (10 nM) co-operatively increased the p21waf1/cip1 and GADD45␣ mRNA levels in DU-145 (Fig. 5a and b). The increase in the p21waf1/cip1 and GADD45␣ mRNA seen with this combination treatment is reflective of the significant co-operative growth inhibition observed from the cell-proliferation assay (Fig. 3b). In contrast, the combination treatment of ketoconazole (7.5 M) with 1,25-(OH)2 D3 did not enhance the p21waf1/cip1 and GADD45␣ mRNA level in DU-145 despite the enhanced anti-proliferative effect observed with this combination treatment in the cellproliferation assay. This could be due to the less targeted effects of ketoconazole. In summary, the current study demonstrates that the sensitivity of DU-145 cells to the growth inhibitory actions of 1,25-(OH)2 D3 is increased by co-treatment with the inhibitor of Vitamin D3 metabolising enzymes, ketoconazole and the tetralone derivative (4). It is possible that the tetralone derivative (4) enhances the anti-proliferative action of 1,25-(OH)2 D3 in DU-145 by inhibiting the vitamin D3 metabolising enzymes and thus preventing the metabolism of the 1,25-(OH)2 D3 . By impeding the inactivation of 1,25(OH)2 D3 , this could lead to enhanced activation of VDR-
mediated target genes and to render the cells sensitive to the induction of growth inhibition and growth arrest by 1,25(OH)2 D3 . Taken together the use of 1,25-(OH)2 D3 with targeted CYP24 inhibitors may be an attractive combination therapy for patients with androgen-independent prostate cancer. Acknowledgment S.W. Yee would like to acknowledge the ORS Awards Scheme for a United Kingdom Scholarship. References [1] H.J. Smith, P.J. Nicholls, C. Simons, R. LeLain, Inhibitors of steroidogenesis as agents for the treatment of hormone-dependent cancers, Expert Opin. Ther. Patents 11 (2001) 789–824. [2] M.J. Campbell, E. Elstner, S. Holden, M. Uskokovic, H.P. Koeffler, Inhibition of proliferation of prostate cancer cells by a 19nor-hexafluoride vitamin D3 analogue involves the induction of p21(waf1), p27(kip1) and E-cadherin, J. Mol. Endocrinol. 19 (1997) 15–27. [3] T.C. Chen, M.F. Holick, Vitamin D and prostate cancer prevention and treatment, Trends Endocrinol. Metab. 14 (2003) 423–430. [4] R.J. Skowronski, D.M. Peehl, D. Feldman, Vitamin D and prostate cancer–1,25-dihydroxyvitamin D3 receptors and actions in human prostate cancer cell-lines, Endocrinology 132 (1993) 1952–1960. [5] D.M. Peehl, R.J. Skowronski, G.K. Leung, S.T. Wong, T.A. Stamey, D. Feldman, Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells, Cancer Res. 54 (1994) 805–810. [6] G.G. Schwartz, T.A. Oeler, M.R. Uskokovic, R.R. Bahnson, Human prostate-cancer cells: inhibition of proliferation by Vitamin D analogs, Anticancer Res. 14 (1994) 1077–1081. [7] G.J. Miller, G.E. Stapleton, T.E. Hedlund, K.A. Moffatt, Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1alpha,25-dihydroxyvitamin D3 in seven human prostatic carcinoma cell lines, Clin. Cancer Res. 1 (1995) 997–1003. [8] H. Farhan, K. Wahala, H.S. Cross, Genistein inhibits Vitamin D hydroxylase CYP24 and CYP27B1 expression in prostate cells, J. Steroid Biochem. Mol. Biol. 84 (2003) 423–429. [9] L.H. Ly, X.Y. Zhao, L. Holloway, D. Feldman, Liarozole acts synergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU145 human prostate cancer cells by blocking 24-hydroxylase activity, Endocrinology 140 (1999) 2071–2076. [10] D.M. Peehl, E. Seto, J.Y. Hsu, D. Feldman, Preclinical activity of ketoconazole in combination with calcitriol or the Vitamin D analogue EB1089 in prostate cancer cells, J. Urol. 168 (2002) 1583–1588. [11] S. Swami, A.V. Krishnan, D.M. Peehl, D. Feldman, Genistein potentiates the growth inhibitor effects of 1,25-dihyroxyvitamin D3 in DU145 human prostate cancer cells: role of the direct inhibition of CYP24 enzyme activity, Mol. Cell Endorinol. 241 (2005) 49–61. [12] A.J. Kirby, R. LeLain, F. Maharlouie, P. Mason, P.J. Nicholls, H.J. Smith, C. Simons, Inhibition of retinoic acid metabolising enzymes by 2-(4-aminophenylmetyl)-6-hydroxy-3,4-dihydronaphthalene1(2H)-one and related compounds, J. Enzyme Inhib. Med. Chem. 18 (2003) 27–33. [13] S.W. Yee, C. Simons, Synthesis and CYP24 inhibitory activity of 2substituted-3,4-dihydro-2H-naphthalen-1-one (tetralone) derivatives, Bioorg. Med. Chem. Lett. 14 (2004) 5651–5654. [14] S.W. Yee, L. Jarno, M.S. Gomaa, C. Elford, L.L. Ooi, M.P. Coogan, R. McClelland, R.I. Nicholson, B.A. Evans, A. Brancale, C. Simons,
S.W. Yee et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 228–235 Novel tetralone-derived retinoic acid metabolism blocking agents: synthesis and in vitro evaluation with liver microsomal and MCF7 CYP26A1 cell assays, J. Med. Chem. 48 (23) (2005) 7123– 7131. [15] A.J. Kirby, R. LeLain, P. Mason, F. Maharlouie, P.J. Nicholls, H.J. Smith, C. Simons, Some 3-(4-aminophenyl) pyrrolidine-2,5-diones as all-trans-retinoic acid metabolising enzyme inhibitors (RAMBAs), J. Enzyme Inhib. 17 (2002) 321–327. [16] H. Farhan, K. Wahala, H. Adlercreutz, H.S. Cross, Isoflavonoids inhibit catabolism of Vitamin D in prostate cancer cells, J. Chromatogr. B 777 (2002) 261–268. [17] F.L. Khanim, L.M. Gommersall, V.H.J. Wood, K.L. Smith, L. Montalvo, L.P. O’Neill, Y. Xu, D.M. Peehl, P.M. Stewart, B.M. Turner, M.J. Campbell, Altered SMRT levels disrupt Vitamin D3 recep-
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tor signalling in prostate cancer cells, Oncogene 23 (2004) 6712– 6725. [18] S. Kang, X-Y. Li, E.A. Duell, J.J. Voorhees, The retinoid X receptor agonist 9-cis-retinoic acid and the 24-hydroxylase inhibitor ketoconazole increase activity of 1,25-dihydroxyvitamin D3 in human skin in vivo, J. Invest. Dermatol. 108 (1997) 513–518. [19] D.M. Peehl, E. Seto, D. Feldman, Rationale for combination ketoconazole/Vitamin D treatment of prostate cancer, Urology 58 (2001) 123–126. [20] N. Akutsu, R. Lin, Y. Bastien, A. Bestawros, D.J. Enepekides, M. Black, J.H. White, Regulation of gene expression by 1␣,25dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma cells, Mol. Endorinol. 15 (2001) 1127–1139.
Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells Sook Wah Yee a , Moray J. Campbell b,∗ , Claire Simons a,∗∗ b
a Division of Medicinal Chemistry, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK Division of Medical Sciences, Institute of Biomedical Research, Birmingham University, Birmingham B12 2TT, UK
Accepted 8 November 2005
Abstract Induction of growth arrest and differentiation by 1␣,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the antiproliferative action of 1,25-(OH)2 D3 . This appears to be due to increased 1,25-(OH)2 D3 -metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D3 metabolising enzymes (IC50 3.5 M), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC50 20 M). The combination of the inhibitor and 1,25(OH)2 D3 caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)2 D3 plus inhibitor of Vitamin D3 metabolising enzymes co-ordinately upregulated CYP24, p21waf1/cip1 and GADD45␣. © 2006 Elsevier Ltd. All rights reserved. Keywords: Vitamin D3 metabolising enzymes; Tetralone; Prostate cancer cells
1. Introduction The majority of the prostate cancer patients demonstrate good initial responses to surgical castration and/or hormonal therapy [1]. Unfortunately, hormonal therapy is not capable of producing durable responses in the majority of the patients who subsequently develop androgen-independent prostate cancer (AIPC). New effective therapies are needed in the management of AIPC patients. One potential therapeutic strategy is to employ a differentiating agent to restore the normal balance of proliferation and differentiation, such as with the biological active metabolite of Vitamin D3 , 1␣,25dihydroxyvitamin D3 (1,25-(OH)2 D3 ). Encouragingly 1,25(OH)2 D3 exerts some pro-differentiating actions and inhibits proliferation of prostate cancer cells in vitro and in vivo [2–4]. ∗
Corresponding author. Tel.: +44 121 4158713; fax: +44 121 4158712. Corresponding author. Tel.: +44 29 20876307, fax: +44 29 20874149. E-mail addresses: [email protected] (M.J. Campbell), [email protected] (C. Simons). ∗∗
0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.11.004
In parallel to the well established endocrine synthesis of 1,25-(OH)2 D3 via sequential hydroxylation steps in the liver and kidney, it has become apparent there is local autocrine/paracrine synthesis of 1,25-(OH)2 D3 . The principle enzymes in this process are the cytochrome P450 enzymes, CYP27B1 (1␣-OHase) and CYP24 (24-OHase). It has been shown that CYP24 and CYP27B1 are also expressed in many target tissues, including prostate-epithelial cells [5,6], supporting the role for local 1,25-(OH)2 D3 synthesis in the prostate. One well established VDR target gene is the CYP24, which is highly inducible by 1,25-(OH)2 D3 , resulting in an increase in the metabolism of 1,25-(OH)2 D3 . Prostate cancer cells expressed high level of 1,25-(OH)2 D3 -induced CYP24 activity, which is inversely proportional to growth inhibition [7]. It has been suggested that in cancer cells the rapid breakdown of 1,25-(OH)2 D3 by over-active CYP24 might be the cause of resistance to 1,25-(OH)2 D3 . Reflecting this, P450 inhibitors which are able to inhibit the activities of CYP24, e.g. ketoconazole (1), genistein (2), and liarozole
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Fig. 1. Inhibitors of Vitamin D3 metabolising enzymes–ketoconazole, 1; genistein, 2; liarozole, 3; the tetralone derivative, 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one, 4.
(3), result in increased 1,25-(OH)2 D3 half-life and antiproliferative effect in DU-145 cells [8–11]. We have demonstrated that 2-substituted-3,4-dihydro-2H-naphthalen-1-one (tetralone) derivatives inhibit the activities of Vitamin D3 and/or retinoic acid metabolising enzymes [13,14]. Among the tetralone derivatives, 2-(4-hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) (Fig. 1) showed inhibition against the Vitamin D3 metabolising enzymes in the rat kidney mitochondria in vitro assay (IC50 = 3.5 M, compared to ketoconazole, IC50 = 20 M) [13]. In the current study, we have examined the antiproliferative effects of the inhibitors, ketoconazole (1) and the tetralone derivative (4), both alone and in combination with 1,25-(OH)2 D3 in DU-145 and PC-3 cells. We also examined the effect of the inhibitors alone and in combination with 1,25-(OH)2 D3 on the regulation of VDR target genes, CYP24, p21waf1/cip1 and GADD45␣.
2. Materials and methods 2.1. Chemicals 1␣,25-Dihydroxyvitamin D3 (1,25-(OH)2 D3 ) was a kind gift from Dr. Milan R. Uskokovic (Hoffman-La Roche, Nutley, NJ). 25-Hydroxyvitamin D3 (25-(OH)D3 ) (Fluka chemicals, Dorset, UK) and 1,25-(OH)2 D3 compounds were stored at 1 mM in ethanol at −20 ◦ C in the dark. 25-Hydroxy-[26,27-methyl-3 H]-vitamin D3 (30 Ci/mmol) was purchased from Amersham Biosciences (Buckinghamshire, UK). 2-(4-Hydroxybenzyl)-6-methoxy3,4-dihydro-2H-naphthalen-1-one (4) was chemically synthesised in the laboratory as described previously [13] and ketoconazole (Sigma, Poole, UK) stored as a 1 mM stock solution in phosphate buffered saline (PBS) pH 7.5 at 4 ◦ C. (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) for cell-proliferation assay was purchased from Sigma (Poole, UK). All solvents used for HPLC were of
HPLC grade and were purchased from Fisher Scientific (Leicestershire, UK). 2.2. Cell culture The androgen-independent prostate cancer cell lines PC3 and DU-145 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained in RPMI 1640 medium (GibcoBRL, Paisley), supplemented with 10% fetal calf serum (Gibco-BRL), 100 units/mL penicillin and 100 g/mL streptomycin. The cells were passaged by trypsinising with 0.25% trypsin–EDTA (Gibco-BRL). The cells were grown at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. 2.3. Preparation of rat kidney mitochondria Mitochondria were isolated from male Wistar rats. Three Wistar rats (250 g each) were fed for 2 weeks with calcium and Vitamin D3 replete diet (calcium carbonate was added to the feed to achieve a 1% calcium level and Vitamin D3 was added to achieve 2200 i.u./kg in the feed). The isolated kidneys were washed with ice-cold phosphate buffer (50 mM, pH 7.4) containing 0.25 M sucrose, then resuspended in icecold Tris–acetate buffer (15 mM, pH 7.4) containing 0.25 M sucrose. The kidney was cut into smaller pieces using scissors and the tissues were homogenised using an Elvejhm-Potter homogeniser. The nuclei and unbroken cells were pelleted by centrifugation for 20 min at 600 × g at 4 ◦ C. The above supernatant was then centrifuged for 20 min at 12 000 × g at 4 ◦ C. The pellet containing the mitochondria was washed with the Tris–acetate buffer and resuspended in ice-cold 20% glycerol and 15 mM Tris–acetate pH 7.4, containing 0.6% sodium cholate. This 20% (w/w) homogenate was stirred on ice for 1 h and the homogenate was centrifuged at 12 000 × g for 1 h to disrupt the mitochondria pellet. The suspension was then distributed into 1.5 mL capped microcentrifuge tubes, frozen in liquid N2 and stored at −80 ◦ C until needed.
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2.4. High performance liquid chromatography (HPLC) analysis of 25-hydroxyvitamin D3 metabolism The assay performed was based on a modification of the general procedure previously described [12,15]. Tubes in duplicate, with a total volume of 500 L, containing 25-hydroxy-[26,27-methyl-3 H]-vitamin D3 (0.05 Ci as a tracer) with unlabelled 25-(OH)D3 (25 M) (10 L), inhibitor (10 L), NADPH (4 mM, 50 L), phosphate buffer (50 mM, pH 7.4, 400 L) and rat kidney mitochondria (30 L) were incubated in a shaking water bath for 30 min at 37 ◦ C. The reaction was terminated by the addition of acetic acid (1% (v/v), 100 L). The Vitamin D3 metabolites were obtained by extraction with ethyl acetate (2 mL) containing 0.02% butylated hydroxyl anisole. After evaporation of the organic solvent, the residue was analysed by HPLC system connected to a -RAM online scintillation detector, connected to a Compaq PC running Laura data acquisition and analysis software (LabLogic Ltd.). The separated [3 H]-vitamin D3 metabolites were separated on the 5 M ODS EXSIL® 4.6 mm × 200 mm column using an isocratic method: 750 mL acetonitrile/250 mL 1% (w/v) ammonium acetate/1 mL acetic acid at a flow rate of 1.5 mL/min. 25-(OH)D3 metabolites were identified by matching their elution rates to the retention times of the known standards, 1,25-(OH)2 D3 , 24,25-(OH)2 D3 and 1,24,25-(OH)3 D3 (generously supplied by Dr. Lise Binderup, Leo Pharma, Denmark) by using a photodiode array detector to monitor UV absorption at 265 nm. The separated [3 H]-vitamin D3 metabolites were quantitatively calculated from the areas under the curves. Using a control with solvent (DMSO) instead of inhibitor, these results were expressed as ‘percentage inhibition relative to control’ (%) = 100[metabolites (control) − metabolites (inhibitor)/metabolites (control)]. 2.5. Inhibition of CYP24 enzyme activity in DU-145 cells A confluent DU-145 cell culture from a 75 cm2 sterile flask was treated with 10 nM 1,25-(OH)2 D3 for 24 h. Cells were then rinsed with 10 mL PBS and incubated with culture medium at 37 ◦ C in a humidified atmosphere of 5% CO2 for approximately 30 min to remove 1,25-(OH)2 D3 . Cells were detached by trypsinization and 2 × 106 cells were assayed in duplicate. Cells were incubated in 200 L medium (RPMI 1640 with 1% FBS) for 30 min at 37 ◦ C in a shaking water bath with 25-hydroxy-[26,27-methyl-3 H]-vitamin D3 (0.05 Ci/tube as tracer), 1 M 25-(OH)D3 and with ethanol or various concentrations of inhibitor (1, 10, 20 and 50 M). The reaction was terminated by the addition of 620 L methanol–chloroform (2:1). The tubes were then centrifuged at 500 × g for 10 min. The supernatant was then transferred into respective glass tubes containing 200 L of chloroform and 100 L of distilled water. After vortexing, the tubes were centrifuged at 500 × g for 15 min. The
lower organic phase was transferred into another clean glass tube, whereas the water phase was re-extracted with 200 L chloroform. The combined organic phases in the tubes were placed in a rotating evaporator for 20–30 min. The production of [3 H]-24,25-(OH)2 D3 was quantitated by HPLC using the mobile phase and HPLC column described above (Section 2.4). 2.6. Cell proliferation assays Cells were plated in 96-well plates (Appleton Woods, Birmingham, UK). Both the DU-145 and PC-3 cells were seeded at 2 × 103 cells/well. Cells were allowed to attach (left at least 7 h), and treated with growth media containing varying concentrations of the inhibitor alone, varying concentrations of the inhibitor with 10 nM 1,25-(OH)2 D3 , or 10 nM 1,25-(OH)2 D3 alone, resulting in a final volume of 100 L/well. The plates were incubated for 96 h, with re-dosing after 48 h. After 93 h incubation, 20 L of MTT solution (5 mg/mL in distilled water and filtered) was added to each well and the cells were left for 3 h in the incubator (37 ◦ C and 5% CO2 ). The 120 L of MTT-containing cells and medium were removed without disturbing the blue precipitate attached to the well. One hundred microliters of DMSO (Sigma–Aldrich, Dorset, UK) were added into each well and incubated for 15 min at room temperature. Finally, the absorption was measured at 550 nm using the absorbance microplate reader (EMax® , Molecular Devices Corporation). The growth inhibition was expressed as a percentage of control. 2.7. Extraction of RNA and reverse transcription DU-145 and PC-3 prostate cancer cells were both seeded at 3.5 × 105 cells/well in a six-well plate. Cells were dosed as indicated and total RNA was extracted using Tri Reagent (Sigma–Aldrich) following the manufacturer’s instructions. Total RNA amounts were quantified by measuring absorbance at 260 nm. The A260 /A280 nm absorption ratio was greater than 1.7. For real-time RT-PCR, cDNA was prepared from 2 g of total RNA by reverse transcription with M-MLV reverse transcriptase (SuperscriptTM II Reverse Transcriptase, Invitrogen, UK) and performed in a thermocycler (Applied Biosystems Gene Amp PCR System 9700). 2.8. Real-time quantitative RT-PCR The expression of the specific mRNAs (i.e. CYP24, p21waf1/cip1 and GADD45␣) was quantitated using the ABI PRISM 7700 Sequence Detection System. DU-145 cells were treated with DMSO vehicle, 10 nM 1,25-(OH)2 D3 for 7 h, inhibitor (10 M tetralone derivative (4) or 7.5 M ketoconazole) for 7 h, or pre-treatment with the inhibitor overnight prior to the addition of 10 nM 1,25-(OH)2 D3 for 7 h. Total RNA was isolated as described above. Each sample was amplified in triplicate wells in 20 L volumes
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Table 1 The primers and probe sequences Primers and probe
Sequence
CYP24 forward primer CYP24 reverse primer CYP24 probe p21waf1/cip1 forward primer p21waf1/cip1 reverse primer p21waf1/cip1 probe GADD45␣ forward primer GADD45␣ reverse primer GADD45␣ probe
5 -CAAACCGTGGAAGGCCTATC-3 5 -AGTCTTCCCCTTCCAGGATCA-3 5 -ACTACCGCAAAGAAGGCTACGGGCTGT-3 5 -GCAGACCAGCATGACAGATTTC-3 5 -GGATTAGGGCTTCCTCTTGGA-3 5 -CCACTCCAAACGCCGGCTGATCTTT-3 5 -AAGACCGAAAGGATGGATAAGGT-3 5 -GTGATCGTGCGCTGACTCA-3 5 -TGCTGAGCACTTCCTCCAGGGCAT-3
containing 2× qPCRTM QuickGoldStar Master Mix (5 mM MgCl2 , dNTPs, QuickGoldStar DNA polymerase, UracilN-glycosylase), 2.5 pmol FAM-labelled TaqMan probe and 18 pmol primers. All-reactions were multiplexed with preoptimised control primers and VIC labelled probe for 18S ribosomal RNA (TaqMan® Ribosomal RNA control reagents, Applied Biosystems, Warrington, UK). Primer and probe sequences are given in Table 1. Reactions were cycled as follows: 50 ◦ C for 2 min, 95 ◦ C for 10 min (for activation of the QuickGoldStar DNA polymerase); then 44 cycles for 95 ◦ C for 15 s and 60 ◦ C for 1 min. Data were expressed as Ct values and used to determine ␦Ct values. ␦Ct = Ct of the target gene minus Ct of the housekeeping gene. The housekeeping gene is the 18S ribosomal RNA. The data was transformed through the equation 2−␦␦Ct to give fold changes in gene expression. ␦␦Ct = ␦Ct of the target gene from treated cells minus ␦Ct of the target gene from non-treated (control) cells. To exclude potential bias due to averaging of data all statistics were performed with ␦Ct values. Measurements were carried out at least two times each in triplicate wells for each condition. 2.9. Statistical analyses The data were obtained from at least two different experiments and are presented as mean ± S.E.M. All statistical analyses were performed using the Student’s t-test. P < 0.05 was considered statistically significant.
3. Results 3.1. Inhibition of Vitamin D3 metabolising enzymes in rat kidney mitochondria The HPLC analysis identified major polar metabolites of [3 H]-25-(OH)D3 , namely 1,25-(OH)2 D3 and 24,25(OH)2 D3 , and a minor polar metabolite, 1,24,25-(OH)3 D3 , in the organic solvent extracts of rat kidney mitochondria.
Fig. 2. DU-145 cells were treated with 10 nM 1,25-(OH)2 D3 for 24 h. Treated cells were detached by trypsinization and incubated with various concentrations of the inhibitor for 30 min, 37 ◦ C in water bath as described in Section 2.5. The separated [3 H]-25-(OH)D3 and [3 H]-24,25-(OH)2 D3 were quantitatively calculated from the areas under the curves by HPLC as described in Section 2.4. Using DMSO solvent as control instead of inhibitor, the results were expressed as percentage inhibition relative to control. This experiment was performed two times and each time in duplicate. Values represent mean ± S.E.M. (n = 4).
The rat kidney mitochondria express both CYP24 and CYP27B1 enzyme activities. Ketoconazole and tetralone derivative (4) have an inhibitory effect on both enzymes, and inhibit the metabolism of 25-(OH)D3 in a dose-dependent manner (between 1 and 100 M). The IC50 for the inhibition of vitamin D3 metabolising enzymes was 3.5 and 20 M, for tetralone derivative (4) and ketoconazole, respectively, as previously reported [13]. The IC50 values were the mean of two experiments and were determined from a dose–response curve. 3.2. Inhibition of CYP24 enzyme activity in DU-145 cells The CYP24 enzyme activity in DU-145 cells is enhanced by the presence of 10 nM 1,25-(OH)2 D3 for 12–24 h. Whereas, ketoconazole and tetralone derivative (4) alone, demonstrated no intrinsic ability to induce CYP24 enzyme activity. After DU-145 cells were treated with 10 nM 1,25(OH)2 D3 for 24 h to induce CYP24 enzyme activity, various concentrations of the inhibitor (1, 10, 20 and 50 M) were examined for their abilities to inhibit the CYP24 enzyme activity. As illustrated in Fig. 2, ketoconazole and tetralone derivative (4), were able to inhibit directly CYP24 enzyme activity in a dose-dependent manner by blocking the conversion of [3 H]-25-(OH)D3 to [3 H]-24,25-(OH)2 D3 . Ketoconazole and tetralone derivative (4), at 10 M, resulted in approximately 50% and 65% inhibition of CYP24 enzyme activity. 3.3. Effects of Vitamin D3 hydroxylases inhibitor and 1,25-(OH)2 D3 on prostate cancer cell growth DU-145 and PC-3 are relatively insensitive to the antiproliferative effect of 1,25-(OH)2 D3 with ED50 > 100 nM
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Fig. 3. The effect of Vitamin D3 metabolising enzymes inhibitor, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 on the growth of DU-145 (a and b) and PC-3 (c and d) cells. These effects were assessed by MTT assay as described in Section 2. Results are expressed as a mean percentage (±S.E.M.) of untreated control wells (n = 3). Each point represents a mean of three experiments with triplicate dishes. * P < 0.05, ** P < 0.01 when samples were compared to samples treated with the inhibitor alone.
compared to non-malignant prostate epithelial cells, as demonstrated previously by our group [17]. In the current study 10 nM 1,25-(OH)2 D3 resulted in approximately 5% growth inhibition after 96 h. DU-145 cells, treated with either ketoconazole or the tetralone derivative (4) alone showed significant anti-proliferation effect at >10 M (with >20% growth inhibition) (Fig. 3a and b). However, the combination treatment with either 5 M ketoconazole or 10 M of the tetralone derivative (4) in combination with 10 nM 1,25(OH)2 D3 caused more than 30% growth inhibition in DU-145 (Fig. 3a and b), suggesting a co-operative effect. High doses of ketoconazole and the tetralone derivative (4) alone showed significant anti-proliferation effects at, ≥10 and >25 M, respectively in PC-3. In contrast, PC-3 cells were not only recalcitrant to 1,25-(OH)2 D3 (at 10 nM) but also did not show significant anti-proliferation when the inhibitors and 1,25-(OH)2 D3 were used together (Fig. 3c and d).
have significant effects on the CYP24 mRNA level in DU-145 cells. Ketoconazole (100 M), when combined with 1,25(OH)2 D3 (10 nM) reduced the CYP24 mRNA to 362-fold change in DU-145 cells, relative to the cells treated with 10 nM 1,25-(OH)2 D3 alone (P < 0.05). By contrast, 7.5 M ketoconazole when combined with 10 nM 1,25-(OH)2 D3 increased the CYP24 mRNA transcription level strikingly
3.4. Effect of the Vitamin D3 metabolising enzymes inhibitor and 1,25-(OH)2 D3 on CYP24 mRNA expression The untreated DU-145 cells expressed very low level of CYP24 mRNA. However, when the cells were treated with 10 nM 1,25-(OH)2 D3 for 7 h, DU-145 cells showed approximately 2000-fold induction in CYP24 mRNA relative to the untreated cells (Fig. 4). Ketoconazole and the tetralone derivative (4) alone at high and low concentrations did not
Fig. 4. Quantitative real-time RT-PCR determination of CYP24 mRNA levels in DU-145 cells treated with, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 . * P < 0.05, ** P < 0.01 and *** P < 0.001 when samples were compared to cells treated with the 10 nM 1,25-(OH)2 D3 alone or with untreated cells. Values are shown as mean ± S.E.M. (n = 3).
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Fig. 5. Quantitative real-time RT-PCR determination of (a) p21waf1/cip1 and (b) GADD45␣ mRNA levels in DU-145 cells treated with, ketoconazole or tetralone derivative (4) alone or in combination with 10 nM 1,25-(OH)2 D3 . * P < 0.05. Values are shown as mean ± S.E.M. (n = 3).
(5340-fold change) in DU-145 compared to cells treated with 1,25-(OH)2 D3 alone (2000-fold change) (P < 0.01), as shown in Fig. 4. On the other hand, combination treatment with the tetralone derivative (4) (100 or 10 M) and 10 nM 1,25(OH)2 D3 in DU-145 significantly enhanced the CYP24 mRNA (>10 000-fold change) compared to the cells treated with 1,25-(OH)2 D3 alone (1999-fold change) (P < 0.001). 3.5. Effect of the Vitamin D3 metabolising enzymes inhibitor and 1,25-(OH)2 D3 on p21waf1/cip1 and GADD45α mRNA expression The results here showed that treatment with 10 nM 1,25(OH)2 D3 alone had no significant effect on p21waf1/cip1 and GADD45␣ mRNA expression in DU-145 cells compared to untreated cells (Fig. 5a and b). However, when the cells were treated with the inhibitor alone (at 100 M) or combination with 1,25-(OH)2 D3 (10 nM), the p21waf1/cip1 and GADD45␣ mRNA were enhanced significantly (P < 0.01) (data not shown here). This observation indicated that the inhibitor itself at 100 M has a significant effect on p21waf1/cip1 and GADD45␣ mRNA, and reflects the direct anti-proliferative effect of the (100 M) alone. Lower concentrations of the inhibitor alone (10 M of the tetralone derivative (4)), did not have an effect on p21waf1/cip1 and GADD45␣ mRNA level in DU-145 (Fig. 5a and b). It is interesting to observe the co-operatively enhanced p21waf1/cip1 and GADD45␣ mRNA level by the tetralone derivative (4) (10 M) and 1,25-(OH)2 D3 in DU-145 (Fig. 5a and b) (P < 0.05). However, this co-operative enhancement of p21waf1/cip1 and GADD45␣ mRNA level was not observed in combination treatment with ketoconazole (7.5 M) and 1,25-(OH)2 D3 , as ketoconazole (at 7.5 M) alone still has an enhanced effect on the p21waf1/cip1 and GADD45␣ mRNA compared to untreated cells (P < 0.05) (Fig. 5a and b).
4. Discussion The tetralone derivative (4) displayed potent inhibitory activity for 25-(OH)D3 metabolism, as revealed using the rat kidney mitochondria assay, compared with ketoconazole. This quick and robust in vitro assay is a more rapid and facile assay in identifying potential inhibitors compared with the inhibition studies carried out using DU-145 or other intact cell-lines and also allows screening of large libraries of potential inhibitors. It is known from various studies that ketoconazole inhibits the activity of Vitamin D3 metabolising enzymes, i.e. both CYP27B1 and CYP24 enzymes [18,19] and thus limits the conversion of active metabolite, 1,25-(OH)2 D3 , to inactive metabolites. The rat kidney mitochondria assay described here demonstrated that ketoconazole and the tetralone derivative (4) inhibit the activities of these Vitamin D3 metabolising enzymes. Likewise in the CYP24-induced DU-145 cell assay, ketoconazole and the tetralone derivative (4) both demonstrated CYP24 enzyme inhibition. As a result this probably resulted in the prolonged half-life of 1,25-(OH)2 D3 , which in turn potentiated the anti-proliferative action of 1,25-(OH)2 D3 (Fig. 3). However, this potentiation of the anti-proliferative effect of 1,25-(OH)2 D3 was not significant in PC-3 cells. This demonstrated that the mechanisms of resistance to 1,25(OH)2 D3 are multifactorial [17]. In the real-time quantitative RT-PCR studies in DU-145 cells, two different concentrations of the inhibitors were used, based on the cell proliferation (MTT) assay and the kidney mitochondrial assay. We decided to choose 100 M of the inhibitors, ketoconazole or tetralone derivative (4), as at such concentration, significant inhibition (>70%) of the Vitamin D3 metabolising enzymes were observed. At lower concentration of the inhibitor, 7.5 M ketoconazole or 10 M tetralone derivative (4), it was possible to observe more cooperative effects.
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From the real-time RT-PCR analysis, there was no inhibition of the basal CYP24 mRNA expression with the inhibitor alone. However, we saw an increase in 1,25-(OH)2 D3 induced CYP24 mRNA with either tetralone derivative (4) (10 and 100 M) or ketoconazole (7.5 M) co-treatment. This co-operatively enhanced CYP24 mRNA level observed from the combination treatment could be the result of the prolonged half-life of 1,25-(OH)2 D3 , which in turn enhanced the genomic actions of 1,25-(OH)2 D3 to induce VDR target genes such as CYP24. The multiple VDR response elements in CYP24 made this target gene a highly sensitive indicator of VDR effects. Similarly Cross and co-workers demonstrated that genistein at higher concentration (50 M) reduced the basal CYP24 mRNA and the 1,25-(OH)2 D3 -induced CYP24 mRNA but not when a lower concentration of genistein (5 and 25 M) was used [8,16]. Intracellular metabolic profiles are required to resolve if there is a disparity between the CYP24 mRNA levels and actual enzyme activity in the presence of these inhibitors. Consistent with the cell-proliferation assay, ketoconazole or the tetralone derivative (4) (at 100 M) either alone or co-treated with 1,25-(OH)2 D3 , caused significant antiproliferative effects, which correlated with enhanced levels of p21waf1/cip1 and GADD45␣ mRNA in DU-145 (Fig. 5a and b). The p21waf1/cip1 protein, a cell-cycle regulatory protein, inhibits the activity of cyclin-dependent kinases in the G0 /G1 cell-cycle phase, resulting in cell-cycle arrest. Whereas, induced expression of GADD45␣ inhibits cell proliferation and it is induced by various agents that damage DNA and arrest cell growth [20]. Induction of GADD45␣ contributes to the growth-inhibitory effects of 1,25-(OH)2 D3 in 1,25-(OH)2 D3 -sensitive cell lines [17]. The tetralone derivative (4) (at 10 M) did not alter the p21waf1/cip1 and GADD45␣ mRNA level, however, combination with 1,25-(OH)2 D3 (10 nM) co-operatively increased the p21waf1/cip1 and GADD45␣ mRNA levels in DU-145 (Fig. 5a and b). The increase in the p21waf1/cip1 and GADD45␣ mRNA seen with this combination treatment is reflective of the significant co-operative growth inhibition observed from the cell-proliferation assay (Fig. 3b). In contrast, the combination treatment of ketoconazole (7.5 M) with 1,25-(OH)2 D3 did not enhance the p21waf1/cip1 and GADD45␣ mRNA level in DU-145 despite the enhanced anti-proliferative effect observed with this combination treatment in the cellproliferation assay. This could be due to the less targeted effects of ketoconazole. In summary, the current study demonstrates that the sensitivity of DU-145 cells to the growth inhibitory actions of 1,25-(OH)2 D3 is increased by co-treatment with the inhibitor of Vitamin D3 metabolising enzymes, ketoconazole and the tetralone derivative (4). It is possible that the tetralone derivative (4) enhances the anti-proliferative action of 1,25-(OH)2 D3 in DU-145 by inhibiting the vitamin D3 metabolising enzymes and thus preventing the metabolism of the 1,25-(OH)2 D3 . By impeding the inactivation of 1,25(OH)2 D3 , this could lead to enhanced activation of VDR-
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