Eur J Nucl Med Mol Imaging (2009) 36:1434–1442 DOI 10.1007/s00259-009-1117-x

ORIGINAL ARTICLE

Characterization of choline uptake in prostate cancer cells following bicalutamide and docetaxel treatment Sebastian A. Müller & Korbinian Holzapfel & Christof Seidl & Uwe Treiber & Bernd J. Krause & Reingard Senekowitsch-Schmidtke

Received: 23 October 2008 / Accepted: 2 March 2009 / Published online: 8 April 2009 # Springer-Verlag 2009

Abstract Purpose Choline derivatives labelled with positron emitters are successfully used for PET imaging of prostate cancer patients. Since little is known about uptake mechanisms, the aim of this study was to characterize choline uptake in prostate cancer cells, also following anti-androgen treatment or chemotherapy. Methods Choline uptake in prostate cancer cells (LNCaP, PC-3) and Michaelis-Menten kinetics were analysed using different concentrations of 3H-choline via liquid scintillation counting. Inhibition of 3H-choline uptake was assayed in the presence of hemicholinium-3 (HC-3), unlabelled choline, guanidine and tetraethylammonium (TEA), an inhibitor of the organic cation transporter (OCT). Changes in choline uptake triggered by bicalutamide and docetaxel were evaluated and choline transporters were detected via Western blotting. Results Michaelis-Menten kinetics yielded a saturable transport with Km values of 6.9 and 7.0 µmol/l choline for LNCaP and PC-3 cells, respectively. Treatment of cells with bicalutamide and docetaxel caused an increase in total choline uptake but had no significant effect on Km values. Uptake of 3H-choline was NaCl dependent and 4.5-fold higher in LNCaP cells than in PC-3 cells. 3H-Choline uptake was reduced by 92–96% using HC-3 and unlabelled S. A. Müller : K. Holzapfel : C. Seidl : B. J. Krause : R. Senekowitsch-Schmidtke (*) Department of Nuclear Medicine, Technische Universität München, Ismaninger Strasse 22, Munich 81675, Germany e-mail: [email protected] U. Treiber Department of Urology, Technische Universität München, Munich, Germany

choline, by 63–69% using guanidine and by 20% using TEA. The high-affinity choline transporter was detected via Western blotting. Conclusion Choline uptake in prostate cancer cells is accomplished both by a transporter-mediated and a diffusion-like component. Results of inhibition experiments suggest that uptake is mediated by a selective choline transporter rather than by the OCT. Bicalutamide- and docetaxel-induced changes in total choline uptake could affect PET tumour imaging. Keywords [methyl-3H]Choline . Prostate cancer cell lines LNCaP and PC-3 . Anti-androgen bicalutamide . Chemotherapeutic agent docetaxel . 11C-Choline PET

Introduction Prostate cancer is the most common cancer in men and the second most common cancer type leading to death following lung cancer. In 2007, 218,890 new cases and 27,050 deaths from prostate cancer were estimated in the USA [1]. The clinical appearance of prostate cancer is widely spread from low-grade indolent to aggressively growing and metastasizing cancers [2]. Most prostate cancers show androgen dependence early in the course of disease. In many patients there is tumour progression from a slow-growing, androgen-dependent carcinoma toward a more and more aggressive, androgen-independent tumour [3, 4]. Therefore, approximately half of the patients relapse and there is a great variability concerning the period of response to an anti-androgen therapy. To achieve a better therapy response rate for patients with less androgen-dependent cancer or with advanced stages of disease, combination therapies with chemotherapy

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are a therapeutic option. Chemotherapy has not been shown to significantly improve survival rates in advanced prostate cancer in a number of studies [5–7]. However, in a recent study Berthold et al. [8] could demonstrate a significant survival benefit following therapy with docetaxel plus prednisone. Recent clinical trials have shown that chemotherapy can play an important role also in palliative therapy of prostate cancer, and thus docetaxel has become a standard chemotherapeutic agent for treatment of metastatic hormone-refractory prostate cancer [9–11]. In order to optimize the therapy regimen, it is important to determine the extent of disease. Unfortunately the value of clinical examination, ultrasound and current imaging techniques for staging and re-staging of advanced tumour stages is limited [12]. PET and PET/CT studies using 11Cor 18F-labelled choline derivatives recently have shown promising results for re-staging prostate cancer in patients with biochemical recurrence [13–17] and in patients with advanced prostate cancer [18]. The rationale for the use of choline for prostate cancer imaging is based on an increased content of phosphorylcholine and an increased phosphatidylcholine turnover in prostate cancer cells [19, 20]. Accordingly, prostate cancer tissue shows a characteristic increase in choline compared to normal prostate tissue, which can be detected by magnetic resonance spectroscopy (MRS) [21, 22]. Key enzymes of choline metabolism are upregulated [23, 24], and prostate cancer cells have shown an increased expression of choline transporters and an upregulated transport rate [25–28]. The quaternary amine choline as a charged hydrophilic cation needs specific transporters to pass the cell membrane. Mainly there are two groups of choline transporters: the high-affinity (Km <10 µM), NaCl-dependent choline transporter (CHT1) that provides choline for acetylcholine synthesis in the presynaptic cholinergic nerve terminal and the low-affinity (Km 30–100 µM), NaCl-independent choline transporter, which is ubiquitously located and provides choline for phospholipid synthesis [29]. Also other choline transport systems have been described [30, 31]. Choline transport has been investigated in different tumour cells. Amongst these, little has been published on the choline transport in prostate cancer cells [28, 32]. Transport kinetics have been evaluated in the androgenindependent cell line PC-3, derived from human prostate cancer cells [28]. In the cell lines PC-3 and LNCaP, [3H] choline uptake kinetics have been analysed with respect to irradiation [32]. The results of these studies indicate that choline is transported through a system that in its kinetic properties is similar to the human choline transporter-like protein (hCTL1). Therapeutic approaches for the treatment of advanced prostate cancer include anti-androgen therapy, chemotherapy and targeted therapies. Anticancer therapies in prostate

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cancer trigger inhibition of angiogenesis, proliferation and tumour growth involving regulation of the Ras signalling pathway activation. The Ras signalling pathway is also regulated through other modulators like those of androgen receptor signalling. Many of the therapeutic approaches in prostate cancer have in common that they modulate signalling pathways and therefore potentially modulate choline uptake and turnover in prostate cancer cells [28]. The question to be addressed in this study was if choline can be used to characterize and quantify the modulation of choline uptake and transport caused by pharmacotherapy. The aims of these in vitro studies were: (1) to characterize the choline uptake and transport in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines with respect to uptake kinetics, competitive inhibition and sodium dependence, (2) to assess the influence of anti-androgen (bicalutamide) and cytostatic (docetaxel) therapy on choline uptake and transport and (3) to identify the CHT1 protein in prostate cancer cell lines by Western blotting using a polyclonal anti-CHT1 antibody.

Materials and methods Cell lines and cell culture The human prostate cancer cell lines LNCaP and PC-3 used for the uptake studies were provided by the Department of Urology of the Technische Universität München. For Western blot analysis of the CHT1 the following cell lines were additionally assayed for comparison: DU145 (human prostate cancer, provided by the Department of Urology, Technische Universität München), SW707 (human colon cancer, provided by the German Cancer Research Centre Heidelberg, Germany), PC-12 (rat pheochromocytoma, provided by the Department of Pathology, Technische Universität München, Germany), Kelly and Sy5y (human neuroblastoma, provided by the Department of Pediatrics, Universität Tübingen, Germany). Human erythrocytes, freshly prepared from volunteers, were provided by the blood bank from the Technische Universität München, Germany. All tumour cells were grown in RPMI medium (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum and 1% non-essential amino acids in the cases of DU145, LNCaP and PC-3, 10% fetal bovine serum in the case of SW707, 5% fetal bovine serum in the case of Kelly and Sy5y and 10% horse serum in the case of PC-12. Supplements were obtained from Biochrom (Berlin, Germany). Cells were kept in an incubator at 37ºC in a humidified 5% CO2 atmosphere and subcultured every 7 days. For culture of PC-12 cells collagen-covered flasks were used (Greiner, Solingen, Germany).

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Radiotracer uptake studies [methyl-3H]Choline chloride (specific activity 3.03 TBq/ mmol) was purchased from GE Healthcare (Freiburg, Germany). For uptake studies 1×105 subconfluent tumour cells in 100 μl uptake buffer (25 mM Tris/HCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 140 mM NaCl, pH 7.4) were transferred to Eppendorf vials and preincubated for 10 min at 37ºC. To investigate NaCl dependence of the uptake, NaCl was replaced by 140 mM of N-methyl-D-glucamine (NMDG) [30]. Uptake was initiated by addition of 100 μl [methyl- 3 H]choline (1.85 kBq, 0.61 pmol) diluted in uptake buffer together with unlabelled choline (10 μM). Uptake was allowed to proceed for 10 min at 37ºC in standard uptake experiments and between 1 and 240 min in experiments for determination of uptake kinetics. Uptake was stopped by addition of 1 ml of ice-cold phosphate-buffered saline (PBS) (Biochrom, Berlin, Germany). Subsequently, cells were washed three times with ice-cold PBS (1 ml each). Prior to determination of [methyl-3H]choline uptake, cells were lysed in 0.5 ml of 2% sodium dodecyl sulphate (SDS) overnight. 3H-Activity was then quantified by liquid scintillation counting using a beta-counter (Wallac, Turku, Finland). Cellular protein content was determined according to the method of Bradford (Bio-Rad, Munich, Germany). Uptake was expressed as pmol/mg cell protein. Inhibition of [methyl-3H]choline uptake Inhibitory effects on uptake of [methyl-3H]choline in LNCaP and PC-3 cells were assayed using increasing concentrations (0.01 μM–10 mM) of unlabelled choline and hemicholinium3 (HC-3), as well as guanidine and tetraethylammonium (TEA) (1 mM each). HC-3 is known as a specific competitive inhibitor of the CHT1 [29] and TEA has been described to inhibit all enzymes from the organic cation transporter (OCT) family [33]. After preincubation of cells (1×105) with inhibitors for 15 min at 37ºC, [methyl- 3 H]choline (1.85 kBq, 0.61 pmol) was added and uptake was allowed to proceed for 10 min. Termination of uptake was achieved by addition of 1 ml ice-cold PBS. Uptake was quantified via liquid scintillation counting as described above. Calculation of Michaelis-Menten kinetics A total of 1×105 cells (LNCaP or PC-3) in 100 μl uptake buffer per vial were preincubated for 15 min at 37ºC. Uptake kinetics were started by addition of 100 μl uptake buffer containing [methyl-3H]choline (1.85 kBq, 0.61 pmol) and different concentrations of unlabelled choline yielding final concentrations between 1 μM and 200 μM. Uptake was terminated after 10 min at 37ºC by 1 ml of ice-cold

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PBS. Liquid scintillation counting of 3H-activity was performed as described above. For determination of nonsaturable, unspecific uptake, experiments were carried out in the presence of an excess amount of unlabelled choline (10 mM). Specific, transporter-mediated choline uptake was calculated via subtraction of nonsaturable, unspecific uptake from total uptake. From the resulting graph the kinetic parameters Km (Michaelis-Menten constant) and vmax (maximum velocity) were calculated by non-linear regression using Prism 4 software (GraphPad Software, San Diego, CA, USA). Treatment of cells with bicalutamide and docetaxel To investigate the effects of the anti-androgen bicalutamide (Casodex®, AstraZeneca, Luton, UK) and the chemotherapeutic agent docetaxel (Taxotere®, Sanofi-Aventis, Frankfurt, Germany) on choline uptake, LNCaP and PC-3 cells were incubated with IC50 concentrations of the compounds for 5 days prior to analysis of uptake. For that purpose the following concentrations were applied as determined via clonogenic assays: bicalutamide, 243 μM (LNCaP) and 227 μM (PC-3); docetaxel, 0.75 ng/ml (LNCaP) and 1.2 ng/ml (PC-3). IC50 was chosen because the therapeutic window of both docetaxel and bicalutamide is relatively narrow. This means that the pharmacological efficacy of both compounds is low close below IC50 and high close above IC50. Uptake studies using [methyl-3H]choline (1.85 kBq, 0.61 pmol) and unlabelled choline in final concentrations varying from 1 μM to 10 mM per 1×105 cells and liquid scintillation counting of 3H-activity were carried out as described above. Detection of choline transporters via Western blotting Subconfluent tumour cells of the cell lines DU145, LNCaP, PC-3, SW707, PC-12, Kelly and Sy5y were harvested using 1 mM ethylenediaminetetraacetate (EDTA) in PBS. Total protein of the tumour cells and of human erythrocytes was extracted using a proteome extraction kit (ProteoExtract Complete, Calbiochem, Darmstadt, Germany) according to the instructions of the manufacturer. Extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (8% acrylamide) and blotted onto a polyvinylidene difluoride (PVDF) membrane. Unspecific binding was blocked with 5% non-fat powdered milk and the membrane was probed with primary (polyclonal anti-CHT1 antibody from rabbit, Chemicon, Temecula, CA, USA) and secondary antibodies (goat anti-rabbit IgG coupled with alkaline phosphatase, Sigma, Taufkirchen, Germany) using standard techniques. Protein bands representing choline transporters were detected via colour reaction using nitroblue tetrazolium (NBT)/5-bromo-4chloro-3-indolyl phosphate (BCIP) as substrates.

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LNCaP cells as well as 17.3 μM (HC-3) and 7.3 μM (unlabelled choline) for PC-3 cells. At concentrations of 1 mM each, HC-3 and unlabelled choline reduced uptake of [methyl-3H]choline by 92% in LNCaP and by 96% in PC-3, respectively. At the same concentration (1 mM) the inhibitors guanidine and TEA were less efficient. While guanidine decreased uptake by 63% in LNCaP and 69% in PC-3, TEA only caused a reduction by approximately 20% in both LNCaP and PC-3 (Fig. 3).

Statistics Each value represents the mean ± standard error of the mean (SEM) of at least three measurements in quadruplicate. The significance level was determined by means of Student’s t test (p<0.05).

Results [methyl-3H]Choline uptake kinetics and NaCl dependence in prostate cancer cells

Determination of Michaelis-Menten kinetics As determined via [methyl-3H]choline, uptake of choline increased with increasing choline concentrations in the uptake buffer in both LNCaP and PC-3 prostate cancer cells. Choline uptake drastically increased from 1 μM to 10 μM, almost reached a plateau between 10 μM and 50 μM and moderately increased between 50 μM and 200 μM of choline (Fig. 4, total). Even at a choline concentration of 1 mM, saturation was not attained (not shown). Subtraction of the unspecific uptake—represented by the line through origin expressing choline uptake at 10 mM choline (Fig. 4, unspecific)—from the total, unsaturable uptake yielded the specific, transportermediated uptake (Fig. 4, specific). K m values for transporter-mediated uptake derived from the graphs in Fig. 4 using Prism 4 software were in the high-affinity range (< 10 μM choline) [29] for both LNCaP (6.9 μM) and PC-3 (7.0 μM) prostate cancer cells.

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Uptake of [methyl- H]choline constantly increased up to 240 min in the prostate cancer cell lines LNCaP and PC-3 (Fig. 1). Within the first hour a linear increase in uptake was observed in both cell lines. Uptake of [methyl-3H] choline after 240 min was significantly higher in LNCaP cells (9.5 pmol/mg cell protein) compared to PC-3 cells (2.1 pmol/mg cell protein). In the presence of NaCl, [methyl-3H]choline uptake in LNCaP was up to 1.9 times higher and in PC-3 up to 2.7 times higher than uptake in NaCl-free, NMDG-substituted buffer (Fig. 1). Thus, choline uptake in both prostate cancer cell lines at least in part is driven by a NaCl-dependent component. Competitive inhibition of [methyl-3H]choline uptake For inhibition studies, [methyl-3H]choline uptake was allowed to proceed for 10 min and was subsequently stopped with ice-cold PBS. Competitive inhibition using HC-3 and unlabelled choline ranging from 0.01 μM to 10 mM resulted in sigmoid inhibition graphs (Fig. 2). As calculated using GraphPad Prism, IC50 values were 24.5 μM (HC-3) and 6.7 μM (unlabelled choline) for

Treatment of prostate cancer cells with IC50 concentrations of the anti-androgen bicalutamide and the chemotherapeutic

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Fig. 1 Uptake of [3H]choline in LNCaP and PC-3 cells in the presence and absence of NaCl (140 mM) at different time points after addition of the tracer (1.85 MBq) to 1×105 cells. NaCl was substituted by 140 mM NMDG

Changes in choline uptake of prostate cancer cells after treatment with bicalutamide and docetaxel

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docetaxel increased Km in LNCaP cells to 8.6 μM and to 8.2 μM, respectively, but slightly decreased Km in PC-3 cells to approximately 6 μM with both compounds. Following docetaxel treatment of LNCaP, vmax increased almost threefold, whereas bicalutamide induced only a 100 80 60 40 20 0

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Fig. 2 Inhibition of [3H]choline uptake in LNCaP (a) and PC-3 (b) by increasing concentrations of HC-3 and unlabelled choline; 1×105 cells were incubated with [3H]choline (1.85 MBq) and HC-3 or unlabelled choline at concentrations of 0.01 µM to 10 mM

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Fig. 3 Reduction of [3H]choline uptake (% of control) as induced by unlabelled choline, HC-3, TEA and guanidine (1 mM each)

Fig. 4 Michaelis-Menten kinetics for choline uptake in LNCaP (a) and PC-3 (b) prostate cancer cells. Specific, high-affinity uptake was calculated by subtracting unspecific uptake from total choline uptake

slight increase. In PC-3 cells both compounds caused negligible changes of vmax (Table 1). These results suggest that specific, transporter-mediated choline uptake is only faintly affected by bicalutamide and docetaxel. However, total choline uptake increased depending on choline concentration. In LNCaP cells choline uptake following bicalutamide treatment did not significantly differ from untreated controls at physiological choline concentrations of 5μM (p=0.581), 10 μM (p=0.066) and 20 μM (p=0.786). In contrast, bicalutamide significantly increased choline uptake at concentrations from 50 μM choline upwards, peaking at 355% of control at 10 mM choline (p<0.001 in each case). Docetaxel treatment of LNCaP cells significantly increased choline uptake at choline concentrations from 1 μM to 10 mM ranging from 146% (at 1 μM) to 210% (at 200 μM) of controls (p<0.001 in each case except for p= 0.008 at 500 μM and p=0.007 at 10 mM) (Fig. 5). In PC-3 cells bicalutamide significantly increased choline uptake at choline concentrations from 50 μM (159% of control) to 10 mM (296% of control) (p<0.001 in each case). Docetaxel treatment of PC-3 induced an average increase in choline

Eur J Nucl Med Mol Imaging (2009) 36:1434–1442 Table 1 Km and vmax values (± SD) for LNCaP and PC-3 cells following treatment with bicalutamide or docetaxel compared to untreated controls

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Treatment Control (untreated) Bicalutamide Docetaxel Control (untreated) Bicalutamide Docetaxel

uptake of 127% of control at all choline concentrations assayed (Fig. 5). Increases ranging from 112% (at 20 μM choline) to 151% (at 1 mM choline) were statistically significant (p<0.001 to p=0,031) except for 20 μM choline (p=0.051) and 500 μM choline (p=0.062). Detection of CHT1s via Western blotting For detection of choline transporters, protein extracts of different tumour cell lines and erythrocytes were assayed

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with a polyclonal antibody against the CHT1. In all tumour cell lines analysed, DU145, LNCaP and PC-3 prostate cancer cells, SW707 colon adenocarcinoma cells, PC-12 pheochromocytoma cells (rat), as well as Kelly and Sy5y neuroblastoma cells, however not in erythrocytes, a double band at 94 and 98 kDa could be detected, representing the CHT1 in these cells (Fig. 6). Additional CHT1-positive bands were observed in PC-12 cells (44 kDa) and Kelly cells (72 kDa) (not shown). In erythrocytes a double band at 186/200 kDa and a single band at 62 kDa could be demonstrated (not shown).

Discussion

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In the disease management of patients with prostate cancer, exact tumour staging at any time during therapy is of major importance because it implies important clinical consequences with respect to the therapy regimen. For PET imaging of prostate cancer 11C- or 18F-labelled choline derivatives have turned out to be superior to [18F]FDG [12, 34–40]. Anti-androgen therapy and chemotherapy potentially influence the uptake of [11C]choline and it has been reported that the choline uptake decreases after initiation of anti-hormonal therapy [41]. Characterization of choline uptake

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Choline uptake in prostate cancer cells was analysed in a buffer containing 140 mM NaCl. Using NaCl-free uptake buffer, choline uptake decreased to approximately 50% in LNCaP and PC-3 (Fig. 1). If the choline transporter in both prostate cancer cell lines is truly NaCl dependent, there

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Fig. 5 Choline uptake (% of control) in LNCaP (a) and PC-3 (b) after treatment with bicalutamide and docetaxel; 1 × 105 cells were incubated with [3H]choline (1.85 MBq) and unlabelled choline at concentrations of 1 µM to 10 mM in the presence of IC50 concentrations of bicalutamide or docetaxel

Fig. 6 Detection of the CHT1 via Western blotting. Protein extracts of different tumour cell lines and erythrocytes were separated via SDS-PAGE (8% polyacrylamide) and probed with a polyclonal antibody against the CHT1. A 94/98 kDa double band could be detected in all samples except for erythrocytes

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should be only negligible choline uptake in NaCl-free uptake buffer. On the other hand, a choline transporter that functions independently of NaCl will not be inhibited by omission of NaCl. Thus, the results obtained suggest a combined transport system composed of two components: one being NaCl dependent and the other being NaCl independent [28]. In Ehrlich-Lettré ascites tumour cells also two components of choline transport, one of which is NaCl dependent, have been described [42]. Studying competitive inhibition of choline transport in both LNCaP and PC-3 prostate cancer cell lines revealed potent inhibition by a specific inhibitor of the choline transporter, HC-3, and by unlabelled choline and a somewhat less potent inhibition by guanidine, also a selective inhibitor of the choline transporter (at 1 mM each, Fig. 3). Hara et al. [28] also found that HC-3 inhibited the transporter-facilitated choline uptake for the PC-3 cell line. Inhibition of choline transport for the LNCaP prostate cancer cell line has not yet been described. We could show a potent inhibition of choline transport by HC-3, unlabelled choline and guanidine also for LNCaP cells. These results argue for similar mechanisms of choline transport in both prostate cancer cell lines analysed. IC50 values of 24.5 and 17.5 µM of HC-3 for LNCaP and PC-3, respectively (Fig. 2), are in between the respective values described for the CHT1 (0.001–0.1 µM) and low-affinity choline transporter CHT2 (~100 µM), again supporting a combined transport system [29]. TEA only caused weak inhibition of choline uptake in the prostate cancer cell lines analysed (Fig. 3). Since TEA specifically inhibits OCT-mediated transport, an OCT-mediated choline uptake in prostate cancer cells can be excluded [31]. Michaelis-Menten kinetics yielded an unsaturable total choline uptake with a drastic increase from 1 to 10 µM choline, an almost plateau between 10 and 50 µM and a moderate increase between 50 and 200 µM choline (Fig. 4). The two increases in choline uptake can be ascribed to the two components of choline transport in prostate cancer cells: the first increase is thought to be mediated by the high-affinity, transporter-mediated component with a Km value of 7.0 µM choline in the PC-3 cell line and the second increase by the low-affinity, unsaturable component. The Km value of 7.0 µM is in the same order of magnitude as the respective Km values already published by Holzapfel et al. [32] (7.5 µM) and Hara et al. [28] (9.7 µM). For the LNCaP prostate cancer cell line a Km value of 6.9 µM choline was calculated for the first increase which corresponds to the respective Km value recently published [32]. Choline transport systems CHT1s have been characterized in different tissues including the cholinergic nervous system [29], fibroblasts [43], rat brain

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microvessel endothelial cells [30] and human keratinocytes [31]. Moreover, malignant transformation of cells often is accompanied by elevation of choline uptake [44, 45]. Hara et al. [28] evaluated the transport kinetics of choline in the androgen-independent human prostate cancer cell line PC-3. The transport rate of PC-3 cells was found to be in between the one known for the human CHT1 (hCHT1) and the organic cationic transporter (hOCT1). Thus, it was in the range of the lower affinity human choline transporter-like protein (hCTL1) which is in line with our findings. Our Western blot results confirm the assumption that at least part of the choline uptake is mediated by the CHT1. Using the anti-CHT1 antibody we could detect specific bands of 94 and 98 kDa in the prostate cancer cells LNCaP and PC-3 as well as in other cancer cell lines known to express the choline transporter (Fig. 6). According to Apparsundaram et al. [46] who cloned the human, hemicholinium-3-sensitive choline transporter, the molecular weight of this transmembrane protein varies between 30 and 90 kDa. These findings correspond to our results. Treatment with cytostatic drugs and anti-androgens It is known that treatment of cells with cytostatic drugs induces changes in tracer uptake, for example of FDG, methionine or thymidine [47–49]. However, no data are available on the effects of chemotherapy and anti-androgen treatment on choline transport in prostate cancer cells. The chemotherapeutic docetaxel (Taxotere®) has been described to inhibit cell proliferation of PC-3 cells both in vitro and in vivo [50–52]. For evaluation of the effect of docetaxel on choline uptake, the prostate cancer cell lines PC-3 and LNCaP were treated with 227 µM and 243 µM docetaxel, respectively, resulting in approximately 50% survival as assayed via the clonogenic assay. In this study treatment with docetaxel induced an increase in total choline uptake but had no significant effect on Km values. Androgens are the primary regulators of prostate cancer cell growth and proliferation. Bicalutamide is a pure antiandrogen that inhibits gene expression and cell growth stimulated by androgens via blockage of the androgen receptor [53]. The effect of bicalutamide on choline uptake in the androgen-dependent LNCaP cell line was evaluated following treatment with 0.75 ng/ml bicalutamide. This resulted in 50% survival. Though the androgen receptor in LNCaP cells reveals a point mutation in the steroid-binding domain, bicalutamide inhibited growth in LNCaP cells [54] as has been confirmed in our study. The effect of bicalutamide on choline uptake in the androgen-independent cell line PC-3 was assessed following treatment with 1.2 ng/ml bicalutamide. This resulted in reduction of survival by approximately 50%. This indicates that bicalutamide also significantly affects growth of

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androgen-independent prostate cancer cells. The findings by Floyd et al. [55] according to which bicalutamide induces apoptosis in PC-3 cells are in accordance with our results. Interestingly, docetaxel treatment of LNCaP and PC-3 cells induced a significant increase in choline uptake both at physiological and supraphysiological choline concentrations but had no significant effect on K m values. The bicalutamide-triggered increase of choline uptake was not significant at physiological but significant at supraphysiological concentrations in both cell lines. Thus, this increase should be due to an increase of unspecific choline transport. The increase in uptake of total choline might be due to the following reasons: (1) Drug treatment induces cellular stress which in turn results in an increase of cellular metabolism and thus raises the need for choline as used for synthesis of cell membranes. (2) Alterations in the cell membrane due to induction of apoptosis could finally result in an increased permeability for choline. This is corroborated by an increase of apoptotic nuclei and 3H-choline uptake following 2methoxyestradiol treatment of PC-3 cells [56]. (3) Timing of choline uptake assessment might influence the choline uptake results. In our experiments the effects of docetaxel and bicalutamide on choline uptake were assessed at 10 min and thus might differ at other time points.

Conclusions Our results suggest that choline uptake in LNCaP and PC-3 prostate cancer cells is mediated by both a NaCl-dependent, saturable, specific, high-affinity transporter (CHT1) and a NaCl-independent, unsaturable, low-affinity component. Choline uptake is not mediated by the OCT -family of transporters. CHT1 could be identified via Western blotting both in LNCaP and PC-3 cells. Growth inhibition triggered by docetaxel and bicalutamide treatment had no significant effect on transport affinity in LNCaP and PC-3. However, total choline uptake was significantly elevated in both cell lines by docetaxel at both physiological and supraphysiological choline concentrations and by bicalutamide at supraphysiological choline concentrations. Acknowledgements We would like to thank A. Lehmer from the Department of Urology, Technische Universität München, for providing the human prostate cancer cell lines LNCaP, PC-3 and DU145. Conflicts of interest None.

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