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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 3 1108-1116
Copyright © 2004 by The Endocrine Society

Dexamethasone Enhances the Cytotoxic Effect of Radioiodine Therapy in Prostate Cancer Cells Expressing the Sodium Iodide Symporter

I. V. Scholz, N. Cengic, B. Göke, J. C. Morris and C. Spitzweg

Department of Internal Medicine II (I.V.S., N.C., B.G., C.S.), Klinikum Grosshadern, Ludwig-Maximilians-University 81377 Munich, Germany; and Department of Endocrinology (J.C.M.), Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Christine Spitzweg, M.D., Klinikum Grosshadern, Medizinische Klinik II, Marchioninistrasse 15, 81377 Muenchen, Germany. E-mail: christine.spitzweg{at}med2.med.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we have reported the induction of prostate-specific radioiodine accumulation in prostate cancer cells (LNCaP) using a prostate-specific antigen (PSA)-promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine.

The aim of our current study was to examine the regulation of PSA-promoter-directed NIS expression in NIS-transfected LNCaP cells (NP-1) by dexamethasone (Dex). For this purpose, NIS mRNA and protein expression levels were examined in NP-1 cells by Northern and Western blot analysis, respectively, after incubation with Dex (10-8–10-6 M) in the presence of 10-9 M mibolerone. NIS functional activity was measured by iodide uptake assay. In addition, we examined regulation of in vitro cytotoxicity of 131-I by Dex in an in vitro clonogenic assay. After incubation with Dex, iodide accumulation in NP-1 cells increased up to 1.5-fold, whereas NIS mRNA and protein expression levels were increased up to 1.7-fold. This effect of Dex was blocked by the androgen receptor antagonist casodex (10-6 M). The killing effect of 131-I in NP-1 cells was increased from 55% when incubated with mibolerone alone to 95% when treated with Dex (10-7 M) plus mibolerone. Treatment of NP-1 cells with Dex resulted in an additional antiproliferative effect as measured by clonogenic assay and nonradioactive proliferation assay.

In conclusion, in addition to an antiproliferative effect, treatment with Dex increases androgen-dependent NIS mRNA and protein expression as well as iodide accumulation, resulting in an increased cytotoxic effect of 131-I in prostate cancer cells stably expressing NIS under the control of the PSA-promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CURRENTLY NO CURATIVE therapy exists for metastatic prostate cancer, which represents the second leading cause of mortality in men after lung cancer (1). Despite various treatment options including radical surgery, external radiotherapy, interstitial brachytherapy, androgen ablation, cryotherapy, and systemic cytotoxic chemotherapy, the prognosis of patients with locally relapsed and metastatic prostate cancer is poor (2). The development of novel treatment strategies, such as gene therapy, is therefore urgently needed. Gene therapeutic approaches for the treatment of prostate cancer are attractive because of the possibility of selective targeting of therapeutic genes to tumor cells, thereby avoiding extratumoral toxicities associated with treatments such as systemic cytotoxic chemotherapy, which has been shown to be of limited benefit in prostate cancer patients (3).

The sodium iodide symporter (NIS) localized at the basolateral membrane of thyroid cells is responsible for the capacity of the thyroid gland to concentrate iodide, thereby allowing effective radioiodine therapy of thyroid cancer, even in advanced metastatic disease (4). Cloning of the NIS gene and its characterization as novel therapeutic gene now offers the possibility of NIS gene transfer into nonthyroidal tumors followed by extrathyroidal radioiodine therapy (5, 6). NIS gene expression under the control of tissue-specific promoters provides a way of selectively targeting the NIS gene to tumor cells, thereby maximizing tissue-specific cytotoxicity with minimal toxic side effects in other organs (7).

In recent studies, we investigated the feasibility of radioiodine therapy in prostate cancer after prostate-specific antigen (PSA)-driven NIS gene transfer. We used a 6-kb PSA-promoter fragment to induce prostate-specific, androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP. The induced iodide accumulation was high enough to allow a therapeutic effect of 131-I in an in vitro clonogenic assay in NIS-transfected LNCaP cells as well as in vivo in xenografts in nude mice (8, 9). The enormous potential of NIS as a novel therapeutic gene allowing radioiodine therapy of nonthyroidal cancers, in particular prostate cancer, has therefore been clearly demonstrated in these studies.

Glucocorticoids have been widely used in the treatment of advanced prostate cancer due to their overall palliative effect (10). Despite multiple studies on glucocorticoid use in prostate cancer, their exact therapeutic role has remained unclear. Although glucocorticoids do not appear to induce apoptosis in prostate cancer cells, a growth-inhibitory effect is well documented (10). Glucocorticoid activity in prostate cancer cells has been shown to be heterogeneous and depends on the chemical agent used, dosage, type of prostate cancer cells, glucocorticoid receptor expression, and presence of androgen receptor mutations (10).

The aim of our current study was to examine the effects of dexamethasone (Dex), a synthetic glucocorticoid frequently used in the treatment of advanced prostate cancer, on growth, androgen-dependent NIS expression, and therapeutic efficacy of 131-I in LNCaP cells expressing the NIS gene under the control of the PSA-promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs and establishment of stably transfected LNCaP cell lines

The expression and control vectors have been generated as previously described (8). We used the pEGFP-1 vector from Clontech Laboratories, Inc. (Heidelberg, Germany) that had been precut with HindIII and NotI restriction enzymes, thereby removing the 800-bp enhanced green fluorescent protein (EGFP) fragment. The resulting expression plasmid construct contained full-length human NIS cDNA coupled to the 6-kb PSA-promoter fragment (NIS/PSA-pEGFP-1). Two control vectors were designed containing NIS cDNA without the PSA-promoter (NIS-pEGFP-1) and the PSA-promoter without NIS cDNA (PSA-pEGFP-1).

Stable transfection of LNCaP cells was performed as previously described (8). In brief, the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP was transfected with NIS/PSA-pEGFP-1 and the control vectors NIS-pEGFP-1 and PSA-pEGFP-1, respectively, using Lipofectamine Plus Reagent (Life Technologies, Inc., Karlsruhe, Germany). Selection was performed with geneticin (Life Technologies), and surviving clones were isolated and subjected to screening for androgen-dependent iodide uptake activity. NP-1, the clone with the highest androgen-dependent iodide uptake activity, was chosen for the studies, as well as the stably transfected (PSA-pEGFP-1) control cell line P-1.

Cell culture and treatments

LNCaP cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 C and 5% CO2 (Life Technologies). After incubation in serum-free RPMI 1640 medium for 24 h, cells were incubated with 10-9 M mibolerone (synthetic androgen, which is not metabolized by LNCaP cells and has similar affinity to the androgen receptor as dihydrotestosterone) (NEN Life Science Products, Köln, Germany), Dex (10-8–10-6 M) (Sigma, Deisenhofen, Germany), and the antiandrogen casodex (bicalutamide, 10-6 M, which was a generous gift from AstraZeneca, Macclesfield, UK) in the presence of 10% charcoal-stripped fetal bovine serum for 48 h.

Cell proliferation assay

Cell proliferation was measured using the commercially available MTS-assay according to the manufacturer’s recommendations (Promega, Mannheim, Germany). Cells were incubated with freshly prepared MTS [3-(4,5-dimethylthiazol)-2-yl-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]/phenazine methosulfate solution (ratio, 1:1 by volume) for 1.5 h at 37 C in a humidified 5% CO2 atmosphere. The absorbance of the formazan product was read at 490 nm, which is directly proportional to the number of living cells in culture.

Iodide uptake studies

Iodide uptake was determined as described by Weiss et al. (11). In brief, cells were plated on six-well plates at a density of 2 x 105 cells/well followed by incubation with mibolerone and Dex as described above. Iodide uptake studies were performed in HBSS supplemented with 10 µM NaI, 0.1 µCi Na125-I/ml, and 10 mM HEPES at pH 7.3. KClO4 (100 µM) was added to control wells. After incubation for 45 min, trapped iodide was removed from cells by a 20-min incubation in 1 N NaOH and measured by {gamma}-counting. Results were normalized to cell survival measured by cell proliferation assay (see above) and expressed as cpm/A490nm.

Northern blot analysis

Total RNA was isolated from untreated and treated NP-1 and P-1 cells using the RNeasy Mini Kit (Qiagen, Hildesheim, Germany). Ten micrograms of total RNA were electrophoresed on a 1% agarose gel containing 2 M formaldehyde and transferred to a positively charged nylon membrane (PALL, Gelman Laboratory, Dreieich, Germany). The human NIS-gene-specific cDNA-fragment (nucleotides 1184–1667) was radiolabeled with [{alpha}-32P] deoxyadenosine-5'-triphosphate by random priming (Amersham, Braunschweig, Germany) and used as hybridization probe. Blots were prehybridized for 30 min at 68 C in hybridization solution (Express Hyb solution, Clontech Laboratories), followed by hybridization at 68 C for 1 h. Blots were then rinsed four times in 2x saline sodium citrate/0.05% sodium dodecyl sulfate (SDS) at room temperature for 10 min and twice in 0.1x saline sodium citrate/0.1% SDS at 50 C for 20 min, respectively. Exposures were made at -80 C for 48 h using Kodak X-OMAT AR films (Sigma). To strip off the NIS cDNA probe, blots were treated in 0.5% SDS at 95 C for 10 min and were reprobed with a human ß-actin cDNA probe to monitor RNA integrity and quantity. Computer-assisted densitometric analysis of band intensities was performed, and NIS-measurements were normalized for ß-actin signal intensity.

Membrane preparation

Cell membranes were prepared from treated and untreated NP-1 cells by a modification of a previously described procedure (12). In brief, cells plated on 100-mm dishes were washed with PBS, harvested, and resuspended in buffer A (250 mM sucrose; 10 mM HEPES, pH 7.5; 1 mM EDTA; 10 µg/ml leupeptin; 10 µg/ml aprotinin; and 1 mM phenylmethanesulfonyl fluoride). The homogenate was centrifuged twice at 500 x g for 15 min at 4 C. After centrifugations, 100 µl 1 M Na2CO3/ml buffer A was added to the supernatant and incubated at 4 C for 45 min with continuous shaking. Then a further centrifugation at 100,000 x g was performed for 15 min, and the pellet was resuspended in an appropriate volume of buffer B (250 mM sucrose; 10 mM HEPES, pH 7.5; and 1 mM MgCl2). Protein concentrations were determined by a protein assay (Bio-Rad DC Protein Assay, Bio-Rad, Munich, Germany).

Western blot analysis

Equal amounts of membrane protein (10 µg), as determined by Bio-Rad DC Protein Assay, were reduced (0.5 M dithiothreitol) and subjected to electrophoresis on a 4–12% Bis-Tris-HCl buffered polyacrylamide gel. After transfer of proteins to nitrocellulose membranes by electroblotting, transfer of equal amounts of membrane protein was confirmed by colloidal gold staining (Bio-Rad). Membranes were then preincubated in 5% low-fat dried milk in TBS-T (20 mM Tris, 137 mM NaCl, and 0,1% Tween 20) to block nonspecific binding sites and then incubated for 2 h at room temperature with a mouse monoclonal antibody directed against amino acid residues 468–643 of human NIS (dilution, 1:3000) (13). After washing with TBS-T, peroxidase-labeled goat antimouse antibody was applied (dilution, 1:5000) for 1 h at room temperature before incubation with enhanced chemiluminescence Western blotting detection reagents (Amersham) for 1 min. Exposures were made at room temperature for approximately 5 min using Kodak BIOMAX MR films (Sigma).

PSA microparticle enzyme immunoassay (MEIA)

In brief, cells were plated on 12-well plates at a density of 1 x 105 cells/well, followed by incubation with mibolerone (10-9 M) and Dex (10-8–10-6 M) as described above. Five hundred microliters of supernatant from each well were used, and total PSA was determined using a PSA MEIA test kit (Abbott, Wiesbaden, Germany) according to the manufacturer’s protocol. Total secreted PSA was normalized to cell survival as determined by cell proliferation assay (see above).

In vitro clonogenic assay

Untreated and treated LNCaP cells were grown to 50% confluency and incubated for 7 h with 0.8 mCi Na131-I in HBSS supplemented with 10 µM NaI and 10 mM HEPES at pH 7.3. After incubation with radioiodine, cells were washed with HBSS, trypsinized, and plated at cell densities of 5000, 7000, and 9000 cells/well (six wells each) in 12-well plates. Four weeks later, after cell colony development, cells were fixed with methanol and stained with crystal violet, and colonies containing more than 50 cells were counted. Parallel experiments were performed for each cell line using HBSS without 131-I, and all values were adjusted for plating efficiency. The percentage of survival represents the percentage of cell colonies after 131-I treatment compared with mock treatment with HBSS.

Statistical methods

All experiments were carried out in triplicates or quadruplicates. For the clonogenic assay, six wells were evaluated for each condition and cell density. Results are presented as means ± SD. Statistical significance was tested using Student’s t test. Results shown are representative of three experiments performed under the same conditions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Iodide uptake studies in vitro

Iodide accumulation was studied in LNCaP cells stably expressing NIS under the control of the PSA promoter (NP-1) and the control cell line P-1 after incubation with or without mibolerone (10-9 M) and Dex (10-8–10-6 M), respectively (Fig. 1Go). Treatment with Dex increased androgen-dependent and perchlorate-sensitive iodide accumulation in NP-1 cells up to 1.5-fold in a concentration-dependent manner. Maximal stimulation of iodide accumulation was seen at 10-7 M Dex. No iodide accumulation above background level was observed in NP-1 cells treated with Dex and mibolerone in the presence of the antiandrogen casodex (10-6 M) and in androgen-deprived NP-1 cells when incubated in the presence or in the absence of Dex. Further, no perchlorate-sensitive iodide uptake was observed in P-1 cells incubated in the presence or in the absence of mibolerone or Dex, respectively (data not shown).



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  		FIG. 1. A 48-h incubation with Dex (10-8–10-6 M) in the presence of mibolerone (Mib, 10-9 M) significantly increased androgen-dependent and perchlorate-sensitive iodide accumulation in LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1) up to 1.5-fold in a concentration-dependent manner. *, P < 0.05; **, P < 0.01. No perchlorate-sensitive iodide uptake was observed in androgen-deprived cells and in cells treated with Dex and Mib in the presence of the antiandrogen casodex (10-6 M). Results have been normalized to cell proliferation measured by cell proliferation assay and are expressed as cpm/A490nm.

 
Northern blot analysis

After incubation of NP-1 and P-1 cells for 48 h with Dex (10-8–10-6 M) in the presence or absence of mibolerone (10-9 M), NIS mRNA steady-state levels were examined by high stringency Northern blot analysis using a 32P-labeled human NIS-specific cDNA probe. NIS mRNA was detected as a single species of approximately 4 kb (Fig. 2AGo). When normalized for ß-actin mRNA signal intensities (Fig. 2BGo), treatment with Dex increased NIS mRNA levels up to 1.7-fold in a concentration-dependent manner (Fig. 2CGo). No NIS mRNA expression was detected in androgen-deprived NP-1 cells when incubated in the presence (data not shown) or in the absence of Dex (10-8–10-6 M). Further, no NIS mRNA expression was detected in P-1 cells incubated in the presence or in the absence of mibolerone and Dex, respectively (data not shown).



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  		FIG. 2. Northern blot analysis of mRNA derived from untreated LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1), and NP-1 cells treated for 48 h with Mib (10-9 M) and Dex (10-8–10-6 M) using a 32P-labeled NIS-specific cDNA probe. NIS mRNA was detected as a single species of approximately 4 kb (A). No NIS mRNA was detected in untreated NP-1 cells. NIS mRNA levels were increased up to 1.7-fold in a concentration-dependent manner after incubation with Dex in the presence of 10-9 M Mib. Hybridization with ß-actin probe served as a control (B). Computer-assisted densitometric analysis of band intensities was performed, and NIS measurements were normalized for ß-actin signal intensity (C). *, P < 0.05.

 
Western blot analysis

After incubation of NP-1 and P-1 cells for 48 h with Dex (10-8–10-6 M) in the presence or absence of mibolerone (10-9 M), NIS protein expression levels were examined by Western blot analysis using a mouse monoclonal human NIS-specific antibody. NIS protein was detected as a band of approximately 90 kDa (Fig. 3AGo). Treatment with Dex increased NIS protein levels up to approximately 1.7-fold in a concentration-dependent manner (Fig. 3BGo). No NIS protein expression was detected in androgen-deprived NP-1 cells when incubated in the presence (data not shown) or in the absence of Dex (10-8–10-6 M). Further, no NIS protein expression was detected in P-1 cells incubated in the presence or in the absence of mibolerone and Dex, respectively (data not shown).



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  		FIG. 3. Western blot analysis of membrane protein derived from untreated LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1), and NP-1 cells incubated with Mib (10-9 M) and Dex (10-8–10-6 M) using a mouse monoclonal NIS-specific antibody. NIS protein was detected as a band of approximately 90 kDa. After a 48-h incubation with Dex in the presence of 10-9 M Mib, NIS protein expression was increased up to 1.7-fold in a concentration-dependent manner (A). Androgen-deprived NP-1 cells did not show NIS protein expression. Computer-assisted densitometric analysis of band intensities was performed (B). *, P < 0.05.

 
PSA MEIA

PSA secretion was measured in NP-1 cells incubated with Dex (10-8–10-6 M) in the presence or absence of mibolerone using a PSA MEIA test kit. PSA secretion increased in a dose-dependent manner by up to 25% in NP-1 cells treated with Dex (10-8–10-7 M) in the presence of 10-9 mibolerone in comparison with NP-1 cells treated with mibolerone alone (Fig. 4Go). In the absence of mibolerone, treatment with Dex was able to increase PSA secretion (approximately 2.8-fold compared with NP-1 cells without treatment) only at high concentrations of Dex (10-6 M). The same experiment was performed with P-1 cells, showing similar results (data not shown).



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  		FIG. 4. A 48-h incubation with Dex (10-8–10-6 M) in the presence of Mib (10-9 M) significantly increased PSA secretion in LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1) by 25% in a concentration-dependent manner. *, P < 0.05. In the absence of Mib, treatment with Dex increased PSA secretion (approximately 2.8-fold compared with NP-1 cells without treatment) only at high concentrations of Dex (10-6 M). Results have been normalized to cell proliferation measured by cell proliferation assay and are expressed as ng/ml/A490nm.

 
In vitro clonogenic assay

To determine the effect of Dex treatment on cytotoxicity of 131-I in vitro in NP-1 and P-1 cells incubated in the presence of androgen, a clonogenic assay was performed (Fig. 5AGo). Whereas only about 15% of P-1 cells were killed by exposure to 131-I, approximately 55% of NP-1 cells incubated for 48 h with 10-9 M mibolerone were killed by 131-I in an in vitro clonogenic assay. After treatment with 10-7 M Dex for 48 h in the presence of 10-9 M mibolerone, approximately 95% of NP-1 cells were selectively killed by 131-I (Fig. 5AGo). Further, in the control group (no 131-I treatment) of the in vitro clonogenic assay, we observed an antiproliferative effect of Dex (10-7 M) in NP-1 cells in the presence of mibolerone (10-9 M) of approximately 15% in comparison with NP-1 cells incubated with mibolerone alone (Fig. 5BGo).



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  		FIG. 5. A, Whereas only about 15% of LNCaP cells stably transfected with the control vector PSA/pEGFP-1 (P-1) were killed by exposure to 131-I in an in vitro clonogenic assay, approximately 55% of LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1) were killed after incubation for 48 h with 10-9 M Mib alone. In NP-1 cells treated with 10-7 M Dex in the presence of 10-9 M Mib, the killing effect of 131-I was significantly increased to approximately 95%. **, P < 0.01. B, In the absence of 131-I, cell proliferation was decreased by approximately 15% in LNCaP cells stably transfected with the expression vector NP-1 after treatment with Dex (10-7 M) in the presence of 10-9 M Mib in comparison with NP-1 cells treated with 10-9 M Mib alone, which was set as 100% ± SD. *, P < 0.05.

 
Proliferation assay

NP-1 and P-1 cells were stimulated with Dex (10-8–10-6 M) with and without mibolerone for 48 h in 96-well plates, and cell survival was measured by the nonradioactive MTS-cell proliferation assay. Whereas treatment with Dex in the absence of mibolerone had no significant effect on NP-1 cell proliferation (Fig. 6AGo), we showed a significant antiproliferative effect in NP-1 cells treated with Dex in the presence of 10-9 M mibolerone of approximately 27% in comparison with NP-1 cells treated with 10-9 M mibolerone alone (Fig. 6BGo). Similar experiments were performed for P-1 cells, showing comparable results (data not shown).



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  		FIG. 6. Whereas a 48-h incubation with Dex (10-8–10-6 M) in the absence of Mib had no significant effect on NP-1 cell proliferation (A), treatment with Dex in the presence of 10-9 M Mib resulted in a significant antiproliferative effect of up to 27% in LNCaP cells stably transfected with the expression vector NIS/PSA-pEGFP-1 (NP-1) in comparison with NP-1 cells treated with 10-9 M Mib alone (*, P < 0.05; ***, P < 0.001) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present study was to examine the regulation of PSA-promoter-directed androgen-dependent NIS expression in the human prostate cancer cell line LNCaP by the synthetic glucocorticoid Dex. NIS mRNA and protein levels and iodide-accumulating activity were significantly increased after treatment with Dex in the presence of androgen in LNCaP cells stably expressing the human NIS gene under the control of the PSA-promoter (NP-1). This and an additional antiproliferative effect of Dex resulted in a significant increase of the efficacy of 131-I treatment in prostate cancer cells after PSA-promoter-directed NIS gene transfer.

Since cloning and characterization of the NIS gene (5), several investigators explored the potential of a novel cytoreductive gene therapy strategy based on NIS gene transfer into nonthyroidal tumor cells followed by radioiodine therapy. NIS gene transfer has been shown to be capable of inducing radioiodine accumulation in vitro and in vivo in several nonthyroidal cancer cell lines, including dedifferentiated thyroid cancer, glioma, neuroblastoma, melanoma, and cervix, breast, lung, liver, colon, and ovarian carcinoma cells (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

Recently, we were able to demonstrate prostate-specific iodide accumulation that was sufficient to elicit a therapeutic response of 13I-I in vitro and in vivo after PSA-promoter-mediated NIS gene delivery in prostate cancer cells (8, 9). These studies clearly showed, for the first time, that tissue-specific NIS gene delivery into nonthyroidal tumors is capable of inducing the accumulation of therapeutically effective radioiodine doses in vitro and in vivo. This novel gene therapy approach might therefore represent an effective and potentially curative therapy for extrathyroidal tumors, in particular prostate cancer.

In search for therapeutic strategies for prostate cancer, glucocorticoids have been found to provide a significant palliative effect in patients with advanced prostate cancer (25, 26, 27, 28). Miller and Hinman (29) were the first to report on the clinical utility of glucocorticoids in advanced prostate cancer. Eight of 10 patients with progressing metastatic prostate cancer despite prior orchiectomy and estrogen therapy, who had been treated with 50–200 mg/d cortisone, reported subjective symptomatic improvements (29). Since this initial publication, multiple clinical studies have evaluated the effects of glucocorticoids in the treatment of advanced prostate cancer and were able to confirm an overall palliative effect, such as improvement in pain, appetite and weight loss, and reduction of ureteric obstruction (25, 28, 30, 31). Although well known for its palliative activity, and therefore widely used in the treatment of advanced prostate cancer, preclinical data regarding the exact mechanisms of the glucocorticoid action in prostate cancer cells are limited. Whereas the antiinflammatory activity and cytotoxic effect of glucocorticoids in hematologic cells is well defined (32, 33), the role of glucocorticoids as antineoplastic agents in epithelial tumors, such as prostate carcinoma, is less well characterized. Glucocorticoids do not appear to induce apoptosis in prostate cancer cells; several studies in different prostate cancer cell lines (DU145, PAIII, PC-3), however, showed a growth inhibitory effect of Dex (34, 35, 36, 37). Dondi et al. examined the effect of Dex on the cell proliferation of DU145 cells (androgen-independent human prostatic adenocarcinoma cell line). Treatment with Dex (10-11–10-5 M) for 4 d produced a significant and dose-dependent decrease in the number of DU145 cells, with maximal growth inhibition at 1–10 µM Dex (34). Similar results were obtained by Koutsilieris et al. in PAIII cells (androgen-independent rat prostatic adenocarcinoma cell line) and by Reyes-Moreno et al. (35, 38) in PC3 cells (androgen-independent human prostatic adenocarcinoma cell line). In LNCaP cells (androgen-dependent human prostatic adenocarcinoma cell line), which carry the T877A mutation within the androgen receptor, Chang et al. (39) showed that Dex inhibits cell proliferation in the presence of 0.1 nM dihydrotestosterone, whereas there was no significant effect on cell proliferation in the absence of androgen. In support of these data, using an in vitro clonogenic assay as well as a nonradioactive proliferation assay, in the present study we demonstrated a significant and dose-dependent antiproliferative effect of Dex in LNCaP cells in the presence of mibolerone, a synthetic androgen. In contrast to these results, glucocorticoids have been shown to exhibit a growth stimulatory effect in MDA PCa cells (androgen-independent human prostatic adenocarcinoma cell line), which express an androgen-receptor with the two mutations L701H and T887A (40). Collectively, the effect of Dex on prostate cancer cell growth appears to be mainly inhibitory but depends on dosage, prostate cancer cell type, presence of androgen receptor-mutations, and presence or absence of androgens. Although there is evidence that modulation of cellular growth factors, such as lipocortin, tumor growth factor ß-1, urokinase-type plasminogen activator, and IL-6, plays a role in the inhibition of prostate cancer cell proliferation by glucocorticoids, the exact mechanisms are still unknown and need to be addressed in further studies (10).

In view of these clinical and experimental data, in the current study we further examined the effect of Dex on androgen-dependent NIS expression and the therapeutic response to 131-I in LNCaP cells after PSA promoter-directed NIS gene transfer (NP-1). When normalized for ß-actin mRNA signal intensities, NIS mRNA levels in NP-1 cells were stimulated in a concentration-dependent manner after incubation with Dex plus androgen. Both NIS protein levels and iodide accumulation also increased in a concentration-dependent manner after treatment with Dex in the presence of androgen. Further, the selective killing effect of 131-I in NP-1 cells was increased significantly, when cells were incubated with androgen and Dex (10-7 M). In the control experiments, without 131-I, an additional antiproliferative effect of Dex was detected in the presence of androgen, suggesting that the increased therapeutic effect in the clonogenic assay was caused by a combination of enhanced 131-I cytotoxicity due to increased functional NIS expression and an additional antiproliferative effect of Dex.

Whereas LNCaP cells do not express a glucocorticoid receptor (41), they are characterized by the expression of an androgen receptor that bears the T877A mutation in the ligand-binding domain (threonine-to-alanine substitution at amino acid 877). This androgen receptor mutant, which is frequently detected in specimens of hormone refractory metastatic prostate cancer, has been reported to reveal an altered ligand specificity recognizing a number of nonandrogenic compounds, such as progesterins, estrogens, and even antiandrogens as androgens (42, 43, 44, 45, 46, 47, 48). In a recent study, Chang et al. (39) have further characterized this clinically important androgen receptor mutant and found that it can be also activated by a number of endogenous glucocorticoids as well as Dex, a synthetic glucocorticoid frequently used in various contexts for prostate cancer therapy. They have further shown that incubation with Dex stimulates mRNA expression as well as secretion of PSA, an androgen receptor-regulated glycoprotein produced mainly in the prostate (39). In our study, PSA secretion in LNCaP cells stably expressing NIS under the control of the PSA-promoter was increased in a dose-dependent manner after incubation with Dex in the absence or presence of androgen. Further, the stimulatory effect of Dex on androgen-induced iodide accumulation in NIS-transfected LNCaP cells was completely blocked by the androgen receptor antagonist casodex, indicating that the effect is androgen receptor-mediated. Thus, the increase of PSA-promoter-driven NIS expression in LNCaP cells after treatment with Dex might be caused by stimulation of PSA-promoter activity through activation of the mutant androgen receptor. The fact, however, that Dex had no effect on PSA-promoter-driven NIS expression in the absence of androgen, suggests the involvement of further, so far unknown mechanisms, which have to be investigated in further studies. One explanation might be the relatively low levels of secreted PSA after treatment with Dex alone, suggesting weak stimulation of PSA promoter activity, which might not be sufficient to allow detectable levels of functional NIS expression.

The requirement of sufficient androgen stimulation raises the question of potential adverse effects of androgen application in patients with prostate cancer. Although long-term application of androgen in these patients is certainly not recommended, exposure to androgen for only a short time period (24–48 h), which would be sufficient for PSA promoter-directed NIS gene transfer, seems to be acceptable, in particular as it serves as a prerequisite for the application of 131-I, a potentially curative therapeutic approach as our previously reported data have clearly shown (8, 9, 49). In addition, in our current study, we show that treatment with Dex increases androgen-induced functional NIS expression, which not only enhances the killing effect of 131-I in LNCaP cells stably expressing NIS under the control of the PSA promoter but also allows reduction of the applied androgen dose.

In conclusion, treatment with Dex increases androgen-induced functional NIS expression levels and the selective killing effect of 131-I in LNCaP cells stably expressing NIS under the control of the PSA-promoter. Furthermore, Dex potentiates the therapeutic effectiveness of 131-I by an additional antiproliferative effect in prostate cancer cells. Treatment with Dex may therefore be considered as a potent adjunct to 131-I therapy after tissue-specific NIS gene transfer in prostate cancer, providing a new therapeutic approach, in particular for advanced prostate cancer.


   Acknowledgments
 
The authors are grateful to S. M. Jhiang, Ph.D., Department of Physiology, Ohio State University, Columbus, OH, for supplying the full-length human NIS cDNA. Further, the authors thank D. J. Tindall, Ph.D. and C. Y. F. Young, Ph.D., Department of Urology, Mayo Clinic, Rochester, MN, for providing the PSA promoter and the LNCaP prostate cancer cell line.


   Footnotes
 
This work was supported, in part, by Grant Sp 581/3-1 (to C.S.) from the German Research Council (Deutsche Forschungsgemeinschaft, Bonn, Germany) and by the Mayo Foundation Prostate Cancer Specialized Program of Research Excellence Grant CA91956 (to J.C.M.).

Abbreviations: Dex, Dexamethasone; EGFP, enhanced green fluorescent protein; MEIA, microparticle enzyme immunoassay; NIS, sodium iodide symporter; PSA, prostate-specific antigen; SDS, sodium dodecyl sulfate.

Received May 28, 2003.

Accepted November 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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