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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0779
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 1 69-78
Copyright © 2006 by The Endocrine Society

Dexamethasone Stimulation of Retinoic Acid-Induced Sodium Iodide Symporter Expression and Cytotoxicity of 131-I in Breast Cancer Cells

S. Unterholzner, M. J. Willhauck, N. Cengic, M. Schütz, B. Göke, J. C. Morris and C. Spitzweg

Department of Internal Medicine II (S.U., M.J.W., N.C., M.S., B.G., C.S.), 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}med.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: The sodium iodide symporter (NIS) mediates the active iodide uptake in the thyroid gland as well as lactating breast tissue. Recently induction of functional NIS expression was reported in the estrogen receptor-positive human breast cancer cell line MCF-7 by all-trans retinoic acid (atRA) treatment in vitro and in vivo, which might offer the potential to treat breast cancer with radioiodine.

Objective: In the current study, we examined the effect of dexamethasone (Dex) on atRA-induced NIS expression and therapeutic efficacy of 131-I in MCF-7 cells.

Design: For this purpose, NIS mRNA and protein expression levels in MCF-7 cells were examined by Northern and Western blot analysis after incubation with Dex (10–9 to 10–7 M) in the presence of atRA (10–6 M) as well as immunostaining using a mouse monoclonal human NIS-specific antibody. In addition, NIS functional activity was measured by iodide uptake and efflux assay, and in vitro cytotoxicity of 131-I was examined by in vitro clonogenic assay.

Results: After incubation with Dex in the presence of atRA, NIS mRNA levels in MCF-7 cells were stimulated up to 11-fold in a concentration-dependent manner, whereas NIS protein levels increased up to 16-fold and iodide accumulation was stimulated up to 3- to 4-fold. Furthermore, iodide efflux was modestly decreased after stimulation with Dex in the presence of atRA. Furthermore, in the in vitro clonogenic assay, selective cytotoxicity of 131-I was significantly increased from approximately 17% in MCF-7 cells treated with atRA alone to 80% in MCF-7 cells treated with Dex in the presence of atRA.

Conclusion: Treatment with Dex in the presence of atRA significantly increases functional NIS expression levels in addition to inhibiting iodide efflux, resulting in an enhanced selective killing effect of 131-I in MCF-7 breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRESENTLY NO CURATIVE therapy exists for recurrent and metastatic breast cancer, which represents the most common cancer type in women and the fifth most common cancer in the world according to the 2002 World Health Organization report. Although a broad spectrum of therapies is available for breast cancer, including mastectomy, lumpectomy, radiotherapy, systemic hormone-modulating therapy, and chemotherapy, the prognosis of patients with recurrent and metastatic mammary carcinoma has remained poor, and therefore new therapeutic approaches need to be investigated (1, 2).

In contrast to mammary cancer, differentiated thyroid cancer has been effectively treated by application of radioiodine (131-I) for more than 60 yr. Thyroidal iodide accumulation allows imaging as well as effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine, thereby improving the prognosis and treatment of thyroid cancer significantly (3). The ability to concentrate iodide is a fundamental property of thyroid tissue, which was first reported in 1915 (4) and was later discovered to be mediated by the sodium iodide symporter (NIS) localized at the basolateral membrane of thyroid follicular cells (5, 6, 7).

In addition to thyroid tissue, iodide is also concentrated in the lactating mammary gland, which has been known for at least 60 yr (8). In the lactating mammary gland, iodide is actively transported and secreted into milk, thereby supplying iodide to the infant for the biosynthesis of thyroid hormones that are essential for the development of the nervous system, skeletal muscle, and lung (5).

In 2000 Tazebay et al. (9) reported the identification of NIS protein in mammary gland and demonstrated that NIS catalyzes iodide accumulation in lactating mammary gland. In normal mammary tissue NIS is present exclusively during gestation and lactation, in contrast to the constitutive expression in thyroid gland, suggesting that hormones involved in active lactation stimulate NIS expression and/or its functional activity. Hormonal regulation studies in intact and ovariectomized mice showed rather complicated regulation mechanisms for NIS in mammary gland by estrogen, prolactin, and oxytocin. Moreover, immunohistochemical analysis showed NIS expression in more than 80% of human invasive breast cancer cases, indicating that NIS is up-regulated with a high frequency during malignant transformation in human breast (9). These observations suggest that NIS expression in mammary carcinoma may offer the possibility of radioiodine application in the diagnosis as well as treatment of breast cancer.

Recently it has been demonstrated that all-trans retinoic acid (atRA) induced both NIS gene expression as well as iodide accumulation in vitro in a well-differentiated estrogen receptor-positive human breast cancer cell line (MCF-7) (10), which has also been confirmed in mouse breast cancer models in vivo (11). Retinoids, synthetic and natural analogs of vitamin A, play a well-characterized role in cancer development, cell differentiation, and cell growth (12). atRA has been reported to inhibit cell cycle progression and induce apoptosis in many tumor cell lines (12). Retinoids have also been used in a number of clinical studies to investigate the therapeutic effect in a variety of tumors, including human breast cancer, and have been described to inhibit the growth of several human hormone-dependent breast cancer cell lines (12).

In addition, glucocorticoids have been used for many years in patients with advanced breast cancer, also in combination with other endocrine agents to potentiate the action of primary endocrine therapy (13, 14) and seem particularly helpful in palliating advanced breast cancer in elderly women (15).

In the current study, we therefore examined the effect of dexamethasone (Dex) alone and in combination with atRA on NIS expression and therapeutic response to 131-I in the estrogen receptor-positive human breast cancer cell line MCF-7.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments

MCF-7 cells were grown in MEM medium (Invitrogen Life Technologies, Inc., Karlsruhe, Germany) supplemented with 10% fetal bovine serum (PAA, Cölbe, Germany), L-glutamine, and penicillin/streptomycin at 37 C and 5% CO2. Twenty-four hours after plating, cells were incubated with atRA (10–6 M) (Sigma, Taufkirchen, Germany), and Dex (10–9 to 10–7 M) (Sigma) in the presence of 10% charcoal-stripped fetal bovine serum for 24 and 48 h.

Iodide uptake studies

Uptake of 125-I by untreated and treated MCF-7 cells was determined at steady-state conditions as described by Weiss et al. (16, 17). In brief, cells were plated on six-well plates (3 x105 cells/well), and after incubation with atRA (10–6 M) and Dex (10–9 to 10–7 M), respectively, iodide uptake studies were performed in Hanks’ balanced salt solution (HBSS) supplemented with 10 µM NaI, 0.1 µCi Na 125-I per milliliter, and 10 mM HEPES (pH 7.3). A 100 µM concentration of KClO4 was added to control wells. After incubation for 1 h, 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 viability assay (see below) and expressed as counts per minute per A490 nm.

Cell viability assay

Cell viability was measured using the commercially available 3-(4,5-dimethylthiazol)-2-yl-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega Corp., Mannheim, Germany) according to the manufacturer’s recommendations. Cells were incubated with freshly prepared MTS/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 efflux studies

Efflux of 125-I was determined as described by Weiss et al. (16, 17). In brief, cells were plated on six-well plates (3 x 105 cells/well), after incubation with atRA (10–6 M) and Dex (10–7 M), respectively, cells were incubated with HBSS supplemented with 10 µM NaI, 0.1 µCi Na 125-I per milliliter and 10 mM HEPES (pH 7.3) at 37 C for 1 h. A 100 µM concentration of KClO4 was added to control wells before application of 125-I. Medium was then replaced every 2–5 min with fresh HBSS. The content of 125-I in the collected supernatant was measured by a {gamma}-counter. After the last time point, trapped 125-I was removed from cells by a 20-min incubation in 1 N NaOH and measured by {gamma}-counting.

RNA preparation and Northern blot analysis

Total RNA was isolated from untreated and treated MCF-7 cells, respectively, by the modified acid guanidinium thiocyanate-phenolchloroform method according to Chomczynski and Sacchi (18) using the RNeasy minikit (QIAGEN, Hilden, Germany). Aliquots (20 µg) of RNA were electrophoresed on a 1% agarose gel containing 2 M formaldehyde and transferred overnight in 20x saline sodium citrate (SSC) to a positively charged nylon membrane (Amersham, Freiburg, Germany). The human NIS cDNA fragment (nucleotides 1184–1667), which was generated as described previously (19), was radiolabeled with [{alpha}-32P]dCTP by random priming (PerkinElmer, Köln, Germany) and used as a hybridization probe. Blots were prehybridized at 68 C in hybridization solution (Express Hyb solution, CLONTECH Laboratories, Inc., Heidelberg, Germany) for 30 min, followed by hybridization at 68 C for 1 h. Blots were then rinsed four times in 2x SSC/0.05% sodium dodecyl sulfate (SDS) at room temperature for 10 min and twice in 0.1 x SSC/0.1% SDS at 50 C for 20 min, respectively. Exposures were made at –80 C for 48 h using Biomax MS films (Sigma). To strip off the NIS cDNA probe, blots were treated in 0.5% SDS at 95 C for 10 min and reprobed with a human ß-actin cDNA probe to monitor RNA integrity and quantity. Computer-assisted densitometric analysis of band intensities was performed (ImageJ, National Institutes of Health), and NIS measurements were normalized for ß-actin signal intensity.

Membrane preparation

Cell membranes were prepared from treated and untreated MCF-7 cells by a modification of a previously described procedure (20). In brief, cells plated on 100-mm dishes were washed with 1x PBS, harvested, and resuspended in buffer A, consisting of 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 cell lysate was centrifuged twice at 500 x g for 15 min at 4 C. After centrifugations, 100 µl 1 M Na2CO3 per milliliter 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 consisting of 250 mM sucrose, 10 mM HEPES (pH 7.5), and 1 mM MgCl2. Protein concentrations were determined by a protein assay (DC protein assay, Bio-Rad Laboratories, Inc., Munich, Germany).

Western blot analysis

For Western blot analysis, the NuPAGE electrophoresis system (Invitrogen) was used. Equal amounts of membrane protein as determined by DC protein assay (20 µg; Bio-Rad) were reduced by incubation with 0.5 M dithiothreitol for 10 min at 70 C and loaded on 4–12% Bis-Tris-HCl-buffered polyacrylamide gels. After gel electrophoresis for 1 h, proteins were transferred to nitrocellulose membranes using electroblotting. After blotting, membranes were preincubated for 1 h in 5% low-fat dried milk in 20 mM Tris, 137 mM NaCl, and 0.1% Tween 20 to block nonspecific binding sites. Membranes were then incubated with a mouse monoclonal antibody directed against amino acid residues 468–643 of human NIS (dilution 1:3000) (21) for 2 h at room temperature. After washing with TBS-T, horseradish 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 1 min using BIOMAX MR films (Sigma). Prestained protein molecular-weight standards (Bio-Rad) were run in the same gels for comparison of molecular weight and estimation of transfer efficiency.

For quantitative analysis blots were reprobed with a monoclonal anti-ß-actin antibody (Sigma), and computer-assisted densitometric analysis of band intensities was performed (ImageJ, National Institutes of Health). NIS protein measurements were normalized for ß-actin signal intensity.

Immunocytochemical staining

Immunocytochemical staining was performed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). MCF-7 cells were plated in six-well plates and grown to 60% confluency. Monolayers were washed with 1x PBS, air dried at room temperature overnight followed by incubation at 40 C for 45 min. Air-dried slides were rehydrated in 1x PBS and preincubated for 45 min with blocking serum to inhibit nonspecific binding. Cell monolayers were then incubated with a mouse monoclonal antibody directed against amino acid residues 468–643 of human NIS (21) at a dilution of 1:3000 for 90 min at room temperature. Cell monolayers were washed and incubated with biotin-conjugated antimouse immunoglobulin for 60 min at room temperature, followed by incubation with preformed avidin and biotinylated horseradish peroxidase macromolecular complex. Diaminobenzidine was used as the chromogene and yielded a bluish black precipitate indicative of human NIS-specific immunoreactivity. Counterstaining was performed with malachite green (0,1%). Parallel monolayers with the primary and secondary antibodies replaced in turn by PBS and isotype-matched nonimmune IgGs were examined to assure specificity and exclude cross-reactivities between the antibodies and conjugates used.

In vitro clonogenic assay

The in vitro clonogenic assay was performed as described by Mandell et al. (22). In brief, untreated and treated MCF-7 cells were grown in 75-cm2 flasks, washed with HBSS, and incubated for 7 h with 10 ml of 80 µCi/ml Na 131-I in HBSS supplemented with 10 µM NaI and 10 mM HEPES (pH 7.3). After incubation with radioiodine, cells were washed with HBSS; trypsinized using 0.05% trypsin-EDTA; and plated at cell densities of 500, 1000, 2000, 3000, 5000, and 7000 cells/well (10 wells each) in 12-well plates. Two weeks later, after 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 treatment 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. For the clonogenic assay, 10 wells were evaluated for each condition and cell density. Results are presented as means ± SD of triplicates. 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

The effect of Dex (10–9 to 10–7 M) and atRA (10–6 M) on iodide accumulation was studied in MCF-7 cells after treatment with or without atRA and Dex, respectively, as well as in control cells without treatment after 24 h (Fig. 1AGo) and 48 h (Fig. 1BGo). Treatment with atRA alone induced perchlorate-sensitive iodide uptake of approximately 800 cpm per A490 nm, which was significantly stimulated by the additional treatment with Dex in a concentration-dependent manner (Fig. 1Go, A and B). Maximal stimulation of iodide accumulation up to 3- to 4-fold was seen at 10–8 and 10–7 M Dex in the presence of atRA, compared with treatment with atRA only after 24 h (Fig. 1AGo) (*, P < 0.001). No iodide accumulation above background level was observed in untreated MCF-7 cells or cells treated with Dex alone in the absence of atRA (Fig. 1Go, A and B). Iodide accumulation in MCF-7 cells treated with atRA (10–6 M) or Dex (10–7 M) in the presence of atRA (10–6 M) reached half-maximal levels within 10 min and a plateau at 40 min (Fig. 2Go).



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  		FIG. 1. Iodide accumulation studies: effect of Dex in the presence of atRA on iodide accumulation in MCF-7 cells. Treatment with atRA (10–6 M) alone induced perchlorate-sensitive iodide uptake of approximately 800 cpm per A490 nm, which was significantly stimulated by the additional treatment with Dex (10–9 to 10–7 M) in a concentration-dependent manner (A and B). Maximal stimulation of iodide accumulation up to 3- to 4-fold was seen at 10–8 and 10–7 M Dex in the presence of atRA (*, P < 0.001) after 24 h (A). No perchlorate-sensitive iodide uptake above background level was observed in untreated cells and cells treated with Dex only (A and B). Results have been normalized to living cells measured by MTS cell viability assay, which directly correlates with number of living cells. Results represent means ± SD of triplicate experiments, and are expressed as amount of iodide accumulation in counts per minute per A490 nm.

 


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  		FIG. 2. Time course of iodide accumulation: time course of iodide uptake in MCF-7 cells treated with atRA (10–6 M) alone or in combination with Dex (10–7 M). Iodide uptake reached half-maximal levels within 10 min and a plateau at 40 min. Results represent means ± SD of triplicate experiments and are expressed as amount of iodide uptake in counts per minute per A490 nm.

 
Iodide efflux studies

To determine the effect of Dex and atRA on iodide efflux in MCF-7 cells, cells were incubated with or without Dex (10–7 M) in the presence of atRA (10–6 M), and an iodide efflux assay was performed (Fig. 3Go, A–C). Iodide efflux in cells treated with atRA alone was rapid; approximately 80% of the accumulated 125-I was released into the medium during the initial 2 min. (Fig. 3AGo). In contrast, a modest decrease in efflux was observed by additional treatment with Dex; after 10 min approximately 20% of initially trapped iodide remained in the cells (*, P < 0.05) (Fig. 3AGo). As shown in Fig. 3BGo, the percentage of released iodide was modestly decreased in cells treated with Dex in the presence of atRA, compared with cells treated with atRA only, which was mainly due to a lower initial release rate in atRA/Dex-treated cells (*, P < 0.05), as shown in Fig. 3CGo.



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  		FIG. 3. Iodide efflux. Iodide efflux was studied in vitro in MCF-7 cells after stimulation with atRA (10–6 M) and Dex (10–7 M) in comparison with MCF-7 cells treated with atRA (10–6 M) alone (A–C). In contrast to atRA-treated MCF-7 cells, in which approximately 80% of the accumulated 125-I was released into the medium during the initial 2 min (A), in MCF-7 cells treated with Dex in the presence of atRA, efflux was modestly reduced; approximately 20% of the accumulated radioiodine remained in the cells after 10 min (*, P < 0.05) (A). The percentage of released iodide is modestly decreased in atRA/Dex-treated cells, compared with atRA-treated cells (B), which is mainly due to a lower initial release rate in atRA/Dex-treated cells (*, P < 0.05) (C). Results represent means ± SD of triplicate experiments.

 
Northern blot analysis

After incubation of MCF-7 cells for 24 h with Dex (10–9 to 10–7 M) in the presence or absence of atRA (10–6 M), NIS mRNA steady-state levels were examined by high-stringency Northern blot analysis using a 32-P-labeled human NIS-specific cDNA probe. NIS mRNA was detected as a single species of approximately 4 kb (Fig. 4AGo). When normalized for ß-actin mRNA signal intensities, treatment with Dex in the presence of atRA increased NIS mRNA levels up to 11-fold in a concentration-dependent manner, compared with treatment with atRA alone (Fig. 4Go, A and B). No NIS mRNA expression was detected in MCF-7 cells when incubated with Dex only (10–9 to 10–7 M) in the absence of atRA (10–6 M). After treatment with atRA alone (10–6 M), only weak NIS mRNA expression could be detected (Fig. 4Go, A and B).



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  		FIG. 4. Northern blot analysis: effect of Dex in the presence of atRA on NIS mRNA expression. Northern blot analysis of mRNA derived from untreated MCF-7 cells and MCF-7 cells treated for 24 h with atRA (10–6 M) with or without Dex (10–9 to 10–7 M) using a 32-P-labeled NIS-specific cDNA probe was performed. NIS mRNA was detected as a single species of approximately 4 kb (A). No NIS mRNA was detected in untreated MCF-7 cells and cells treated with Dex only. In contrast, NIS mRNA levels were increased up to 11-fold in a concentration-dependent manner after incubation with Dex in the presence of 10–6 M atRA, compared with treatment with atRA alone (10–6 M), in which only weak NIS mRNA expression could be detected (A and B). Hybridization with ß-actin probe served as a control. Computer-assisted densitometric analysis of band intensities was performed, and NIS measurements were normalized for ß-actin signal intensity; bars represent NIS to ß-actin mRNA ratios (B).

 
Western blot analysis

After incubation of MCF-7 cells for 24 h with Dex (10–9 to 10–7 M) in the presence or absence of atRA (10–6 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. 5AGo). When normalized for ß-actin protein signal intensities, treatment with Dex in the presence of atRA increased NIS protein levels up to approximately 16-fold in a concentration-dependent manner (Fig. 5Go, A and B). No NIS protein expression was detected in MCF-7 cells when incubated with Dex (10–9 to 10–7 M) in the absence of atRA (10–6 M). Furthermore, weak NIS protein expression was detected in MCF-7 cells incubated with atRA (10–6 M) alone (Fig. 5Go, A and B). The additional band, which migrates above the 75-kDa molecular mass marker and is present in untreated as well as treated cells, results from the human NIS-antibody cross-reacting with a nonspecific protein in MCF-7 cells (Fig. 5AGo).



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  		FIG. 5. Western blot analysis: effect of Dex in the presence of atRA on NIS protein expression. Western blot analysis of membrane proteins derived from untreated MCF-7 cells and MCF-7 cells incubated with atRA (10–6 M) with or without Dex (10–9 to 10–7 M) using a mouse monoclonal NIS-specific antibody was performed. NIS protein was detected as a band of approximately 90 kDa (A). After a 24-h incubation with Dex in the presence of 10–6 M atRA, NIS protein expression was increased up to 16-fold in a concentration-dependent manner, compared with weak NIS protein expression in MCF-7 cells incubated with atRA (10–6 M) alone (A and B). No NIS protein was detected in MCF-7 cells when incubated with Dex (10–9 to 10–7 M) in the absence of atRA (10–6 M) (A). Computer-assisted densitometric analysis of band intensities was performed, and NIS measurements were normalized for ß-actin signal intensity; bars represent NIS to ß-actin protein ratios (B).

 
Immunocytochemical staining

Using a highly sensitive immunostaining technique and a mouse monoclonal human NIS-specific antibody, heterogenous, primarily membrane-associated (arrows) NIS-specific immunoreactivity was detected in MCF-7 cells treated with Dex (10–7 M) in the presence of 10–6 M atRA (Fig. 6AGo). Treatment with 10–6 M atRA alone showed a weak but distinguishable immunoreactivity in a subset of treated MCF-7 cells (Fig. 6BGo). In contrast, untreated MCF-7 cells and cells treated with Dex in the absence of atRA (Fig. 6Go, C and D) did not show NIS-specific immunoreactivity. In addition, parallel control slides with the primary and secondary antibodies replaced in turn by PBS and isotype-matched nonimmune immunoglobulin were negative (not shown).



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  		FIG. 6. Immunocytochemistry. Immunocytochemical staining of MCF-7 cells was performed after treatment with Dex in the presence of atRA using a mouse monoclonal NIS-specific antibody. Heterogenous, primarily membrane-associated (arrows) NIS-specific immunoreactivity was detected in MCF-7 cells treated with Dex (10–7 M) in the presence of 10–6 M atRA (A). MCF-7 cells treated with 10–6 M atRA showed only a weak but distinguishable NIS-specific immunostaining (B). In contrast, untreated MCF-7 cells (C) and cells treated with Dex alone (D) did not show NIS-specific immunoreactivity.

 
In vitro clonogenic assay

To determine the effect of Dex and atRA treatment on cytotoxicity of 131-I in vitro in MCF-7 cells incubated with Dex (10–7 M) in the presence of atRA (10–6 M), a clonogenic assay was performed (Fig. 7Go). No cell killing was observed in untreated cells by exposure to 131-I, and no significant change of cytotoxicity was observed after treatment with Dex alone. In contrast, approximately 17% of MCF-7 cells incubated for 24 h with 10–6 M atRA alone were killed by 131-I in an in vitro clonogenic assay (*, P < 0.05). After additional treatment with Dex (10–7 M) for 24 h in the presence of atRA (10–6 M), this cytotoxic effect of 131-I was increased to approximately 80% (***, P < 0.001) (Fig. 7Go A). In the presence of perchlorate, approximately 95% of MCF-7 cells treated with Dex (10–7 M) in the presence of atRA (10–6 M) survived the treatment with 131-I. Furthermore, in the control group (no 131-I) of the in vitro clonogenic assay, we observed a slight, nonsignificant proliferative effect of Dex (10–7 M) and a significant proliferative effect of atRA (10–6 M) (approximately 20%) (**, P < 0.01), compared with untreated cells or cells treated with atRA plus Dex (10–7 M) (Fig. 7BGo).



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  		FIG. 7. Clonogenic assay. A clonogenic assay was performed to determine whether MCF-7 cells, stimulated with Dex in presence of atRA, can be selectively killed by 131-I treatment. No cell killing was observed in untreated MCF-7 cells by exposure to 131-I, and no significant change of cytotoxicity was observed after treatment with Dex alone. In contrast, approximately 17% of MCF-7 cells were killed after incubation for 24 h with 10–6 M atRA alone (*, P < 0.05) (A). In MCF-7 cells treated with Dex (10–7 M) in the presence of 10–6 M atRA, the killing effect of 131-I was significantly increased to approximately 80% (***, P < 0.001) (A). In the presence of perchlorate, approximately 95% of MCF-7 cells treated with Dex (10–7 M) in the presence of atRA (10–6 M) survived the treatment with 131-I (A). In the absence of 131-I, no effect of Dex in the presence of atRA was observed on proliferation of MCF-7 cells, whereas colony-forming capacity was slightly increased by Dex alone and significantly increased (~20%) in MCF-7 cells treated with atRA (10–6 M) alone (**, P < 0.01) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present study was to examine the regulation of the NIS expression in the estrogen receptor-positive human breast cancer cell line MCF-7 by the synthetic glucocorticoid Dex in combination with atRA. The combined treatment with Dex and atRA significantly increased NIS mRNA and protein levels as well as iodide-accumulating activity, whereas iodide efflux was reduced. These effects of combined treatment with atRA and Dex resulted in a highly increased selective killing effect of 131-I in MCF-7 cells.

NIS is an intrinsic membrane protein that mediates the active iodide uptake across the basolateral membrane of benign and malignant thyroidal cells (5, 23, 24, 25). Diagnostic imaging and effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine are based on thyroidal expression of functionally active NIS protein. The high success rate of radioiodine therapy is reflected in the low mortality of patients suffering from metastatic thyroid cancer who are treated with 131-I (3%) as compared with those who are not (12%). Even young patients with diffuse pulmonary metastases at initial presentation can be successfully treated by 131-I, achieving a 10-yr survival of more than 80% (3).

Since cloning of the NIS gene in 1996 (23), NIS has been applied as a novel therapeutic gene to achieve radioiodine accumulation in extrathyroidal tumors allowing the therapeutic application of radioiodine. In recent studies we were able to demonstrate tissue-specific iodide accumulation that was sufficient to elicit a therapeutic response of 131-I in vitro and in vivo after transcriptionally targeted NIS gene delivery in different tumor models including prostate, colon, and medullary thyroid cancer. 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 (26, 27, 28, 29, 30, 31, 32). In addition to induction of NIS expression by NIS gene transfer, endogenous NIS mRNA and protein expression has also been reported in a variety of extrathyroidal tissues, including salivary and lacrimal glands, gastric mucosa, kidney, placenta, and in particular mammary gland, suggesting that iodide transport in these tissues is mediated by the expression of functional NIS protein (7, 9, 19, 33, 34). In breast tissue, a role for iodine in the prevention of breast dysplasia and hyperplasia has been suggested (35, 36, 37). The expression and regulation of NIS in the mammary gland as well as its physiological and pathophysiological role have been described by Tazebay et al. (9) in 2000, reporting NIS expression in lactating mammary gland as well as more than 80% of human breast cancer samples analyzed by immunohistochemistry (9).

Kogai et al. (10, 38) reported increased functional NIS expression in the human breast cancer cell line MCF-7 in vitro after administration of various retinoid receptor ligands, including 9-cis-retinoic acid (RA); AGN190168, a RA receptor (RAR)-ß/{gamma}-agonist; and atRA, which was also shown to be stimulated by the additional treatment with Dex in a very recent paper (38). In addition, significant induction of NIS mRNA and protein expression and markedly increased iodide accumulation was demonstrated in vivo in xenograft tumors after atRA treatment (11). Furthermore, in a recent study, we showed that treatment with atRA increases NIS expression levels and selective killing effect of 131-I in prostate cancer cells stably expressing NIS under the control of the prostate-specific antigen promoter (29).

Retinoid receptors belong to the steroid receptor superfamily, and their action is mediated through two families of nuclear receptors: RARs, which are activated by both atRA and 9-cis-RA, and retinoid X receptors (RXRs), which are activated by 9-cis-RA only. Both receptor types are expressed in MCF-7 cells. The activated nuclear receptors bind to RA-responsive elements in the promoter regions of target genes and act as ligand-dependent transcription factors (39, 40).

Retinoids are known to play a crucial role in the control of normal epithelial cell proliferation and differentiation and inhibit neoplastic processes in many organs including mammary gland (41). The vitamin A metabolite RA inhibits cell proliferation (42, 43) and induces apoptosis in a variety of tumor types (44) including MCF-7 cells (45). Liu et al. (46) reported a correlation between RARß expression, growth-inhibitory effects and induction of apoptosis after atRA treatment in MCF-7 cells.

In 1975 Horwitz et al. (47) reported the expression of the glucocorticoid receptor in MCF-7 cells, as another member of the nuclear receptor superfamily. An antiproliferative activity of the synthetic glucocorticoid Dex in breast cancer was reported in 1976 by Lippman et al. (48). In addition Dex has been described to act synergistically with atRA in different cell lines, as in up-regulation of GHRH receptor mRNA in fetal rat pituitary gland (49), up-regulation of alkaline phosphatase expression in MCF-7 cells (50), and up-regulation of a mouse mammary tumor virus promoter expression in human embryonic kidney cells transfected with a mouse mammary tumor virus promoter-luciferase reporter construct (51).

In view of these experimental data, in the current study, we examined the combined effect of Dex and atRA on NIS expression and therapeutic efficacy of 131-I in MCF-7 cells. An 11-fold stimulation of NIS mRNA expression as well as a 16-fold stimulation of NIS protein expression was observed in MCF-7 cells after incubation with Dex in the presence of atRA, resulting in a 3- to 4-fold increase of iodide accumulation. The discrepancy between the degree of atRA/Dex-mediated stimulation of NIS expression on RNA, protein, and functional levels may result from posttranscriptional or posttranslational effects that are currently unknown. In addition to stimulation of NIS mRNA and protein expression, treatment with Dex in the presence of atRA might have inhibitory effects on NIS functional activity explaining the only 3- to 4-fold increase in functional activity in contrast to a more than 10-fold increase in NIS mRNA and protein expression. These data are mostly consistent with the data from Kogai et al. (38) in their very recent paper demonstrating significant induction of iodide uptake and NIS mRNA expression in MCF-7 cells treated with atRA and Dex, although the level of iodide uptake in cells treated with atRA (10–6 M) alone was higher than in our study and therefore the magnitude of the stimulatory effect of Dex in the presence of 10–6 M atRA was less pronounced. In addition, Kogai et al. (38) reported a slight induction of iodide uptake in MCF-7 cells treated with Dex alone, which we did not observe. Furthermore, in our study iodide efflux was modestly decreased by simultaneous treatment with atRA and Dex, which was not observed in the study from Kogai et al. (38). These differences between the results in our study and the recent study from Kogai et al. (38) might be due to different strains of MCF-7 cells that were used in these studies. Ultimately, in our study these effects resulted in a significantly stimulated selective killing effect of 131-I in MCF-7 cells of approximately 80% after treatment with Dex plus atRA. In the control experiments without 131-I, no cytotoxic effect was detected in MCF-7 cells treated with Dex in the presence or absence of atRA. These data suggest that the enhanced therapeutic effect of 131-I after treatment with Dex and atRA was due to stimulation of iodide accumulation based on increased NIS mRNA and protein expression and decreased iodide efflux.

Several groups reported Dex-induced stimulation of RXR{alpha} expression in hepatocytes (52, 53). RXRs have been described as a common accessory nuclear protein required for high-affinity DNA binding of a group of receptors for nonsteroid ligands such as RARs, thyroid receptor, peroxisomal proliferator-activated receptor-{gamma}, and vitamin D receptor and further to homodimerize to itself. The heterodimerization of RXR with RAR stabilizes the binding of the receptors to their cognate response elements (54, 55) and leads to a promoted ligand-dependent transcriptional regulation (56, 57). In a recent study, Tanosaki et al. (58) hypothesized that NIS activity could be augmented by activation of both RAR/RXR and RXR/RXR pathways. They reported NIS expression after treatment with various nuclear hormone receptor ligands in breast cancer cells. Combining RAR and RXR selective ligands, both NIS mRNA expression and iodide uptake in MCF-7 cells were enhanced (58). These data suggest that the increased iodide accumulation in MCF-7 cells after stimulation with atRA and Dex might be mediated by Dex-potentiated RAR/RXR heterodimerization and therefore increased ligand-dependent transcription of the NIS gene. In addition, Audouin-Chevallier et al. (59) reported enhanced translocation of the glucocorticoid receptor from the cytosolic compartment to the nuclear compartment and stimulation of the binding capacity of glucocorticoid receptor after RA administration. This mechanism might further explain the additive effect of Dex and atRA in MCF-7 cells. Further studies assessing the exact interactions of glucocorticoid- and retinoid-regulated processes in breast cancer cells are needed to fully understand the mechanisms by which simultaneous treatment with atRA and Dex enhances functional NIS expression.

In the thyroid gland, after transport of iodide across the apical membrane into the colloid by pendrin, iodide is oxidized and incorporated into tyrosyl residues along the thyroglobulin backbone (6). This complex reaction at the cell/colloid interface is catalyzed by thyroid peroxidase and termed iodide organification, referring to the incorporation of iodide into organic molecules. Iodide organification results in an increased retention time of trapped iodide in the thyroid gland. In addition, as with organification of iodide in the thyroid gland, about 20% of the trapped iodide has been shown to be organified in lactating mammary gland as a result of iodide oxidation by peroxidases expressed in mammary alveolar cells followed by binding to tyrosyl residues of caseins and other milk proteins (60, 61, 62, 63). In our study, treatment with atRA in the presence of Dex resulted in a modestly reduced iodide efflux, which might result from atRA- and Dex-mediated effects on iodide efflux mechanisms, which are not known in MCF-7 cells, or from partial organification of accumulated iodide, which has to be addressed in future studies. Reduced iodide efflux after combined treatment with Dex and atRA may represent an advantage for therapeutic application of 131-I because it is capable of increasing the achieved radiation dose due to enhanced retention time and biological half-life of 131-I in the target tissue.

In conclusion, simultaneous administration of atRA and Dex increases functional NIS expression levels, thereby significantly enhancing iodide accumulation and the selective killing effect of 131-I in MCF-7 cells. Therefore, based on the extensive experience with radioiodine in diagnosis and therapy of thyroid cancer, treatment with Dex and atRA may allow diagnostic and therapeutic application of radioiodine in breast cancer in the future.


   Acknowledgments
 
The authors are grateful to Dr. S. M. Jhiang (Ohio State University, Columbus, OH) for supplying the full-length human NIS cDNA.


   Footnotes
 
This study was supported by Grants Sp 581/3-1, 3-2, Sp 581/4-1, and 4-2 (to C.S.) from the German Research Council (Deutsche Forschungsgemeinschaft, Bonn, Germany) and Mayo Foundation Prostate Cancer SPORE Grant CA91956 (to J.C.M.).

This work represents a portion of the medical thesis of M.S. at Munich Medical School, Ludwig-Maximilians-University, Munich, Germany.

First Published Online October 18, 2005

Abbreviations: atRA, All-trans retinoic acid; Dex, dexamethasone; HBSS, Hanks’ balanced salt solution; MTS, 3-(4,5-dimethylthiazol)-2-yl-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NIS, sodium iodide symporter; RA retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate.

Received April 8, 2005.

Accepted October 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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