Differential Regulation of Sodium/Iodide Symporter Gene Expression by Nuclear Receptor Ligands in MCF-7 Breast Cancer Cells
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内分泌学杂志 2005年第7期
Molecular Endocrinology Laboratory (T.K., Y.K., A.I.L., L.H.C., E.O., K.T., G.A.B.), Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, California 90073; Vitae Pharmaceuticals (R.A.C.), Irvine, California 92618; and Third Department of Medicine (K.T., T.S.), Yamanashi University, Yamanashi 409-3898, Japan
Address all correspondence and requests for reprints to: Dr. Gregory A. Brent, Molecular Endocrinology Laboratory, Building 114, Room 230, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent@ucla.edu.
Abstract
The sodium/iodide symporter (NIS) mediates iodide uptake in lactating breast tissue and is expressed in some breast cancers. We have previously demonstrated that all-trans retinoic acid (tRA) stimulates NIS gene expression and the selective cytotoxic effect of ?-emitting radioiodide-131 (131I) in both in vitro and in vivo MCF-7 breast cancer cell systems. We studied the ability of natural and synthetic retinoids, in combination with other nuclear receptor ligands, to achieve greater and more sustained induction of NIS in MCF-7 cells and enhance 131I-mediated cytotoxicity. Selective stimulation of retinoic acid receptor (RAR) ?/ produced marked NIS induction; and selective stimulation of RAR, RAR, or retinoid X receptor produced more modest induction. Maximal NIS induction was seen with 9-cis retinoic acid and AGN190168, a RAR ?/-agonist. Dexamethasone (Dex), but not the other nuclear receptor ligands, in combination with tRA synergistically induced iodide uptake and NIS mRNA expression, predominantly by prolonging NIS mRNA half-life. The addition of Dex reduced the EC50 of tRA for NIS stimulation to approximately 7%, such that 10 –7 M tRA with addition of Dex enhanced iodide uptake and selective cytotoxicity of 131I greater than 10–6 M tRA alone. AGN190168 combined with Dex synergistically increased iodide uptake and significantly prolonged induction (5 d) of iodide uptake compared with that induced by the combination of tRA/Dex or 9-cis retinoic acid/Dex. The addition of Dex reduced the effective dose of retinoid and prolonged the induction of NIS, especially with AGN190168, suggesting higher efficacy of 131I after combination treatment.
Introduction
THE SODIUM/IODIDE SYMPORTER (NIS) is expressed on the basolateral membrane of alveolar cells in lactating breast tissue and functions to concentrate iodide in breast milk (1, 2, 3). Iodide is required by the developing neonate for thyroid hormone synthesis. Oxytocin is a major stimulus to NIS expression in the lactating mammary gland (1, 2), and both prolactin and estrogen enhance NIS induction by oxytocin (1). More than 60% of breast cancer tissue also expresses NIS (1, 4), and correlation between 99mTc-pertechnetate uptake and NIS expression in breast tumor tissues has recently been confirmed (5, 6).
The ?-emitting isotope, radioiodide-131 (131I), is commonly used in the treatment of differentiated thyroid cancer after thyroidectomy. NIS is predominantly expressed in the follicular cells within the thyroid gland, and TSH up-regulates NIS expression and iodide uptake (7, 8). Thyroid cancer is associated with reduced NIS activity, and a high level of TSH stimulation is required to maximize 131I uptake. This is accomplished by cessation of thyroid hormone supplementation after thyroidectomy to raise endogenous serum TSH or by exogenous administration of recombinant TSH.
The retinoic acid receptor (RAR) ligand, all-trans retinoic acid (tRA), stimulates NIS gene expression and iodide uptake in the estrogen receptor-positive MCF-7 breast cancer cell line (9). A recent study has suggested that activity of a cardiac homeobox transcription factor, Nkx-2.5, is critical for induction of NIS by tRA in MCF7 cells (10). Administration of 131I in MCF-7 cells after augmentation of NIS gene expression by tRA results in selective cytotoxicity (9). The retinoid X receptor (RXR) ligand 9-cis retinoic acid (9-cis RA) and some synthetic retinoids also induce iodide uptake and NIS mRNA in MCF-7 cells (11). RAR has three isoforms (, ?, and ) with a specific pattern of developmental and tissue distribution. tRA and 9-cis RA stimulate formation of heterodimers of RAR and RXR. Other nuclear hormone receptors, such as vitamin D3 receptor and thyroid hormone receptor, also heterodimerize with RXR. The RXR-nuclear hormone receptor heterodimer binds to cis-elements, recruits coactivators, and enhances gene expression (12, 13).
Systemic tRA treatment of immunodeficient mice with MCF-7 cell xenografts markedly stimulated NIS expression and iodide uptake (15-fold) (14). The tRA dose for the maximum NIS induction in that model, however, is more than 10-fold above the maximum tolerable tRA dose, on a milligram-per-square-meter basis, used in humans for treatment of solid tumors (14). In addition, the duration of the NIS induction is relatively short, followed by a significant reduction (more than 60%) within 2 d of the maximum induction (14). To reduce tRA toxicity and increase the efficacy of radioiodide, additional agents to enhance NIS expression in breast cancer were investigated.
Retinoid isomers and RAR isoform-selective retinoid agonists have been shown to enhance the response of some RA-stimulated genes. Systemic treatment of rodents with 13-cis RA, an isomer of tRA, has been shown to have a significantly lower toxicity than tRA (15). The elimination half-life of 13-cis RA in plasma is about 10 times longer than tRA (16). Systemic administration of isoform-selective retinoids may be associated with greater efficacy and reduced adverse effects, compared with nonselective retinoids. We therefore studied some isoform-selective synthetic retinoids, to gain insight into the retinoid pathway of NIS induction and evaluate efficacy of NIS induction in MCF-7 cells. We found that an RAR ?/-agonist and 9-cis RA induced NIS mRNA and iodide uptake as well as tRA.
Dexamethasone (Dex), T3, and estradiol (E2) have been shown to influence NIS expression in FRTL-5 rat thyroid cells (17, 18). We tested the influence of these ligands, as well as other nuclear receptor ligands, on NIS expression in MCF-7 cells. It has been reported in some cancer cells that there is a synergistic effect of retinoids with other nuclear receptor ligands in gene regulation (19, 20) and cell proliferation (21). We therefore investigated the influence of tRA, in combination with other nuclear receptor ligands, on NIS expression, and we found that Dex significantly enhanced retinoid-induced NIS expression and cell-selective cytotoxicity with I131 in MCF-7 cells.
Materials and Methods
Chemicals and cells
MCF-7 breast cancer cells (lot no. F15100 and 205623) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained as described (22). An RAR-selective ligand AGN195183, an RAR ?/-selective ligand AGN190168 (tazarotene) (23), an RAR-selective ligand AGN194433, and an RXR-selective ligand AGN194204 (24) were provided by Allergan, Inc. (Irvine, CA). These retinoids were dissolved in dimethylsulfoxide (DMSO) to a concentration of 10–2 M and stored at –20 C. tRA, 9-cis RA, Dex, and other cell culture reagents were purchased from Sigma (St. Louis, MO). Cells were maintained with 0.1% DMSO vehicle, when treated with nuclear receptor ligands, and fed with fresh media with ligands every 24 h. In some experiments, fetal bovine serum (ATCC) in the medium was replaced with charcoal-stripped serum or Knockout Serum Replacement (Invitrogen, Carlsbad, CA) 24 h before the treatment with ligands, as noted.
Iodide uptake
The iodide uptake assay was performed as described (9, 25), with minor modifications. Briefly, cells were grown in 12-well dishes, washed with Hanks’ Balanced Salt Solution (HBSS), and incubated for 1 h at 37 C with 500 μl HBSS containing approximately 0.1 μCi carrier-free Na125I (Amersham Biosciences, Piscataway, NJ) and 10 μM NaI. The specific activity under these conditions was 20 mCi/mmol. After incubation, the cells were washed twice with ice-cold HBSS, scraped from each well, and radioactivity measured in a -counter. Cell number was determined by counting in a hemocytometer. The radioactivity was normalized to the cell number at the time of the assay. For kinetic studies, cells were incubated with 1–600 μM NaI and 20 mCi/mmol Na125I at 37 C for 5 min.
Iodide efflux
The iodide efflux assay was described previously (9, 26). Briefly, cells in 12-well dishes were incubated with HBSS containing 10 μM NaI and 20 mCi/mmol Na125I at 37 C for 1 h, and the medium was replaced every 5 min with fresh HBSS without NaI. The content of 125I in the collected supernatant was measured by -counter. After the last time point (60 min), the cells were extracted with 400 μl ethanol to count residual radioactivity.
cDNA preparation for RT-PCR
Total RNA was isolated by RNeasy mini kit (QIAGEN, Valencia, CA) from cells grown in 100-mm-diameter culture dishes. On-column DNase I digestion was performed as recommended by QIAGEN. Three micrograms of purified total RNA were reverse-transcribed by using 50 U Superscript III reverse transcriptase (Invitrogen) in 20 μl reaction with oligo(deoxythymidine)12–18 primer (1 μg).
Quantitative real-time PCR analysis
Primers for PCR were designed with Primer3 software (http://frodo.wi.mit.edu), and those sequences were checked by nucleotide BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) to avoid cross-reactivity with other known sequences. The sequences were the following; NIS forward, 5'-GTTCTACACTGACTGCGACCCTC-3'; NIS reverse, 5'-GCAGCCGAGGTTTGATGAG-3'; glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) forward, 5'-GAGTCAACGGATT-TGGTCGTA-3'; GAPDH reverse, 5'-CATGGGTGGAATCATATTGGA-3'. PCR mixture contained 10 μl of 2x QuantiTect SYBR Green PCR Master Mix (QIAGEN), 1 μl of the reverse-transcription reaction, and 6 pmol of forward and reverse primers. PCR was carried out using the DNA Engene Opticon System (MJ Research, Inc., Waltham, MA) with the following cycle parameters: polymerase activation for 15 min at 95 C and amplification for 35 cycles of 20 sec at 96 C, 45 sec at 55 C, and 45 sec at 72 C. Melting curve analysis was performed after the amplification as recommended. Standard curves representing six-point serial dilution of mixed cDNA of the corresponding control group, MCF-7 cells treated with 10–6 M tRA for 12 h, unless otherwise noted, were analyzed in each assay and used for calculation of relative expression values. The sample quantifications were normalized by the internal control GAPDH mRNA.
131I-Cytotoxic assay
The procedure was carried out as previously described (9), with minor modifications. Briefly, cells grown in 25-cm2 flasks were treated with or without tRA and/or Dex for 48 h. The cells were then incubated for 6 h at 37 C with 5 ml HBSS containing 0 or 60 μCi/ml Na131I (PerkinElmer, Boston, MA) and 0 or 6 μM NaI, respectively. The reaction was terminated by removing the radioiodide-containing medium and washing cells twice with HBSS. The cells were then trypsinized, counted, and plated at densities of 250 and 1000 cells/well with growth medium in 6-well plates. Uptake of 131I– was confirmed by a Geiger Mueller counter before the plating. Cells were grown for 12 d, fixed with 3:1 methanol:acetic acid, and stained with crystal violet, and the number of macroscopic colonies were counted. The survival rate was calculated as the percentage of surviving cell colonies in plates treated with 131I– compared with those treated with only HBSS.
Statistical analysis
Statistical comparison was performed using STAT VIEW 5.0 software (SAS Institute, Cary, NC) with significance at a P value < 0.05. Comparison between groups was determined by conducting a paired Student’s t test. The synergistic effect of two stimulatory factors was determined by two-factor factorial ANOVA test.
Results
Iodide uptake in MCF-7 cells treated with retinoid receptor ligands
All-trans RA (tRA) and 4-[E2–5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl] benzoic acid, a selective RAR ligand, increase iodide uptake about 9-fold above baseline in the well-differentiated breast cancer cell line, MCF-7 (9). tRA also markedly induces iodide uptake in MCF-7 cell xenografts in immunodeficient mice, up to 15-fold above baseline (14). The dose of systemic tRA required for NIS induction, however, is relatively high in the xenograft model. To reduce tRA toxicity, synthetic retinoids with more selective actions and longer half-life, such as 13-cis RA and tazarotene (AGN190168), are commonly used in clinical applications, especially topical skin treatment. We tested the ability of these, as well as other retinoid receptor ligands, to induce iodide uptake in MCF-7 cells.
13-cis RA, an isomer of tRA that is converted to tRA in some cells (27, 28), significantly induced iodide uptake in a dose-dependent manner in MCF-7 cells, approximately 7-fold above baseline, at a concentration of 10–6 M (Fig. 1A). 9-cis RA, a ligand for both RAR and RXR, also significantly induced the uptake in a dose-dependent manner, approximately 11-fold above baseline, at a concentration of 10–6 M (Fig. 1A). The magnitude of induction by tRA (9-fold) was higher than with 13-cis RA and lower than 9-cis RA; however, these differences were not statistically significant (Fig. 1A). We also tested the ability of synthetic retinoids to induce iodide uptake in MCF-7 cells. AGN190168, an RAR ligand that predominantly stimulates the ?- and -isoforms, significantly induced radioiodide uptake in a dose-dependent manner, approximately 8-fold above baseline at a concentration of 10–6 M (Fig. 1B). The dose response and maximal induction was similar to that of tRA (Fig. 1B), with similar estimated median effective concentration (EC50), approximately 9.1 x 10–8 M for AGN190168 and approximately 9.3 x 10–8 M for tRA. AGN194204, an RXR-specific ligand, significantly induced iodide uptake at 10–7 M, but the induction was relatively modest and was reduced at higher ligand concentrations (Fig. 1B). The -isoform-specific ligand AGN195183 and a -isoform-specific ligand AGN194433 also significantly induced iodide uptake. The magnitude of induction with these ligands, however, was lower than for other retinoids, consistent with a previous study using RAR-ligands (11). These data suggest that signal transduction through RAR, especially RAR?, is important for the induction of iodide uptake in MCF-7 cells, and selective RXR stimulation results in only a modest induction of uptake. The induced uptake stimulated by these retinoids was almost completely blocked by KClO4, a specific inhibitor of NIS-mediated iodide transport (29) (data not shown).
FIG. 1. Effect of retinoids on iodide uptake in MCF-7 breast cancer cells. A, Effects of isomers of tRA. Cells were incubated with the indicated concentration of retinoids for 48 h, and iodide uptake assay was performed with 500 μl HBSS containing approximately 0.1 μCi Na125I and 10 μM NaI. The reactions with or without 30 μM KClO4 were terminated at 1 h, and the content of iodide in the cells was determined with a -counter. Replacement of fetal bovine serum to 10% Knockout SR (Invitrogen) did not significantly change the iodide uptake induced by these retinoids (data not shown). B, Effects of synthetic retinoid receptor ligands in MCF-7 cells. Cells were incubated with the indicated concentration of retinoids for 48 h, and iodide uptake assay was performed. Replacement of fetal bovine serum to 10% Knockout SR (Invitrogen) did not significantly change the iodide uptake induced by these retinoids (data not shown). C, Combination study of RAR-specific ligands and an RXR-specific ligand on iodide uptake in MCF-7 cells. Cells were treated for 48 h with an RAR ligand, tRA, AGN190168, or AGN194433, in combination with AGN194204, an RXR ligand, as indicated, and an iodide uptake assay was performed with () or without () 30 μM KClO4. Values are expressed as mean ± SD (n = 3). Values of iodide uptake with KClO4 were between 0.82 and 0.95 in each conditions and not shown in A and B. *, P < 0.02; **, P < 0.001 compared with untreated cells.
RAR and RXR bind ligand, and the resulting heterodimer binds to specific cis-elements in the regulatory regions of RA-responsive genes. Some RA-responsive genes can be synergistically induced by combinations of RAR and RXR ligands (30, 31, 32). We therefore tested some RAR-specific ligands in combination with the RXR-specific ligand AGN194204 to induce iodide uptake in MCF-7 cells. The RAR ligands, tRA, AGN190168 (RAR ?/), and AGN194433 (RAR), however, did not synergistically induce iodide uptake in combination with AGN194204 treatment (Fig. 1C).
NIS mRNA expression in MCF-7 cells treated with retinoid receptor ligands
tRA up-regulates NIS gene expression predominantly at the transcriptional level (9). To evaluate whether the NIS mRNA expression is correlated with iodide uptake in MCF-7 cells, we performed quantitative RT-PCR of NIS with MCF-7 cells treated with several retinoids at the dose for the highest induction of iodide uptake shown in Fig. 1. NIS mRNA levels, as determined by RT-PCR, were significantly increased by retinoids that induced iodide uptake in MCF-7 cells (Fig. 2), and the magnitude of induction of mRNA was correlated with that of iodide uptake (Fig. 1, A and B). tRA, 9-cis RA, and the RAR ?/-ligand AGN190168 stimulated the greatest magnitude of NIS mRNA (more than 70-fold induction), consistent with the maximal effect on iodide uptake. The inductions by 13-cis RA and an RXR ligand AGN194204 were significant, but relatively modest (49-fold and 26-fold, respectively), consistent with a previous report with other RXR ligands (11).
FIG. 2. Effect of retinoids on NIS mRNA expression in MCF-7 cells. Cells were treated with indicated ligands (10–6 M, except for 10–7 M of AGN194204) for 12 h, and RT-PCR for NIS and GAPDH was performed with a real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C for NIS and 79 C for GAPDH (data not shown). The average ratio of NIS/GAPDH in untreated cells was set as 1. Values are expressed as means ± SD (n = 3). *, P < 0.015 compared with untreated cells.
The magnitude of NIS mRNA induction by these retinoids was markedly greater than that of iodide uptake (8- to 10-fold in Fig. 1, A and B). The finding of greater NIS mRNA induction than functional iodide uptake is consistent with previous observations in breast models (9, 11, 14). This may be due, in part, to a predominance of NIS protein remaining in the cytoplasm, as shown in our previous immunocytochemical studies in MCF7 cells (14). NIS protein may also be in the cell membrane but not functional for promoting iodide uptake.
Iodide uptake in MCF-7 cells treated with nuclear hormone receptor ligands
tRA up-regulates NIS expression in MCF-7 cells (9) but significantly reduces NIS mRNA expression and iodide uptake in FRTL-5 rat thyroid cells (33). The nuclear receptor ligands, Dex, T3 (17), and E2 (18), modestly reduce TSH-induced NIS expression in FRTL-5 cells. To determine whether ligands for other nuclear receptors could stimulate NIS expression and iodide uptake in MCF7 breast cancer cells, we tested various nuclear receptor ligands and combinations: Dex, E2, pioglitazone (PGZ), a synthetic peroxisome proliferator-activated receptor--ligand, progesterone (Prog), T3, and 1,25 dihydroxyvitamin D3 (VD3) compared with tRA. Treatment with 10–6 M Dex alone significantly increased iodide uptake in MCF-7 cells, approximately 1.8-fold above baseline. The Dex-induced iodide uptake, however, was only approximately 30% of that induced by 10–6 M tRA. Other nuclear receptor ligands did not significantly influence uptake, with concentrations in the range of 10–6 M to 10–8 M (data not shown).
Recent studies have suggested so-called cross-talk of RAR signaling with that of other nuclear receptor ligand pathways. Peroxisome proliferator-activated receptor--ligand with retinoid, for example, synergistically inhibits growth and induces apoptosis in MCF7 cells (21). Signaling by cAMP pathways stimulates NIS in samples from transgenic breast cancer models (34). We studied the ability of Dex, E2, PGZ, Prog, T3, and VD3 to enhance tRA-induced iodide uptake in MCF-7 cells. Dex significantly enhanced the tRA (10–6 M)-induced iodide uptake, approximately 1.5-fold of that without tRA. VD3 and E2 at concentrations of 10–7 M decreased the tRA-induced iodide up to 22 and 27%, respectively, although the differences were not significant (data not shown). Nuclear receptor ligands, PGZ, Prog, VD3, E2, T3, and tamoxifen, were tested at concentrations of 10–6 to 10–8 M, and no significant effect on tRA-induced uptake was seen (data not shown).
Effects of Dex on iodide uptake in MCF-7 cells
Dex increased iodide uptake induced by tRA in MCF-7 cells in a dose-dependent manner (Fig. 3A). Dex (10–7 M) increased the uptake about 3-fold above 10–7 M RA alone and significantly higher than that with only 10–6 M tRA (Fig. 3A). A two-factor factorial ANOVA test indicated that the addition of 10–7 M Dex and 10–7 M tRA synergistically increased iodide uptake in MCF-7 cells (P < 0.0001, Fig. 3A). The maximal synergistic effect of Dex on RA stimulation was seen at a dose of 10–7 M. According to the dose-response curve, the estimated EC50 of tRA for iodide uptake in MCF-7 cells was approximately 9.6 x 10–8 M, whereas the estimated EC50 of tRA with Dex was approximately 6.8 x 10–9 M.
FIG. 3. Effect of Dex on iodide uptake in MCF-7 cells. A, Various dose combinations of Dex and tRA were tested to induce iodide uptake in MCF-7 cells. Cells were treated with indicated agents for 48 h, and the iodide uptake assay was performed with () or without () 30 μM KClO4. *, P < 0.02; **, P < 0.001 for comparison as shown; ***, significant difference in two-factor factorial ANOVA test among the four groups treated with 0 or 10–7 M tRA and/or 0 or 10–7 M Dex (P < 0.0001). B, Time course of up-regulation of iodide uptake induced by Dex and tRA. Cells were treated with 10–7 M Dex and 10–7 M tRA (), 10–7 M tRA (), or 10–6 M tRA () for 48 h. **, P < 0.01 when compared with untreated cells. C, Effect of Dex on tRA-treated MCF-7 cells. Cells were treated with 10–7 M tRA for 36 h, and then 0 M () or 10–7 M () Dex was added to the culture medium. ****, P < 0.01 when compared with values at 36 h. D, Iodide efflux from MCF-7 cells treated with 10–7 M Dex and 10–7 M tRA () or 10–6 M tRA (). Cells were treated with tRA and/or Dex for 48 h before the assay. Data are means ± SD (n = 3).
Our time course study showed that the iodide uptake was significantly increased by the addition of both 10–7 M Dex and 10–7 M tRA in 12 h, and it reached a maximum at 36 h (Fig. 3B). When 10–7 M Dex was added to the MCF-7 cells treated with 10–7 M tRA for 36 h, the uptake was significantly increased in 12 h and reached a maximum in 24 h but quickly fell in 36 h (Fig. 3C).
Variation in the net iodide uptake is predominantly the result of changes in iodide influx, although the influence of iodide efflux needs to be considered. We performed iodide efflux assays in MCF-7 treated with both 10–7 M Dex and 10–7 M tRA for 48 h, compared with cells treated with tRA (10–6 M) alone. The time point at which 50% of iodide remained in MCF-7 cells after RA treatment was approximately 21 min, consistent with previous data (9), and Dex did not influence this time point (Fig. 3D).
We analyzed the kinetics of iodide uptake in MCF-7 cells treated with 10–6 M tRA and 10–7 M Dex, compared with that with only 10–6 M tRA treatment. When iodide uptake was initiated with approximately 20 mCi/mmol Na125I in MCF-7 cells treated with both Dex and tRA, the uptake reached a half-maximal level of activity in 10 min and saturation at about 30 min (data not shown), consistent with the time course in tRA-treated MCF-7 cells reported previously (9). The initial velocity of iodide uptake was determined by incubation for 5 min with 1–600 μM NaI (Fig. 4A). Excess external iodide (>100 μM) saturated the iodide transport, and Lineweaver-Burk double-reciprocal plots yielded the Michaelis-Menten constant (Km) and maximum velocity values (Fig. 4B). The apparent Km for iodide was 18.6 ± 1.51 μM (the mean ± SD) with tRA and Dex, which was not significantly different from that with only tRA (17.9 ± 1.1 μM). These results are consistent with the range of values reported in MCF-7 cells (9) and FRTL-5 thyroid cells (25, 35). In contrast, the maximum velocity of iodide uptake with tRA and Dex (4.99 ± 0.60 pmol/min/104 cells) was significantly (P = 0.001) higher than that with only tRA (3.01 ± 0.30 pmol/min/104 cells), indicating increased functional NIS after Dex treatment.
FIG. 4. Kinetics study of iodide uptake by MCF-7 cells treated with Dex and tRA. Cells were treated with 10–7 M Dex and 10–7 M tRA (), or 10–6 M tRA () for 48 h before assay. A, Dependency of initial velocity of iodide uptake (5 min) on the extracellular iodide concentration in MCF-7 cells. After treatment with Dex and/or tRA, cells were incubated at 37 C for 5 min with 500 μl HBSS containing 20 mCi/mmol 125I– and indicated concentration of NaI. Trapped 125I– was measured by -counter and normalized to the cell number. Nonspecific binding of 125I– was determined in duplicate assays in the presence of 30 μM KClO4, and this value was normalized to the cell number and subtracted from the values measured. The subtracted data (net uptake velocity) are expressed as means ± SD (n = 3). B, The data points of 1–600 μM NaI from A are graphed as a Lineweaver-Burk plot. All points from triplicate wells are plotted. Equation for the "best fit" line and correlation coefficient (R2) are shown.
Effects of Dex on NIS mRNA expression in MCF-7 cells
Treatment with Dex alone (10–7 M) increased NIS mRNA expression, as assessed by RT-PCR, approximately 1.9-fold, but the difference was not statistically significant (P = 0.07; Fig. 5A). NIS mRNA levels were significantly increased, by 10–7 M and 10–6 M tRA, approximately 39-fold and approximately 72-fold, respectively. Addition of 10–7 M Dex significantly enhanced tRA-stimulated mRNA expression, approximately 2.8-fold with 10–7 M tRA and approximately 2.0-fold with 10–6 M tRA (Fig. 5A), compared with tRA alone. A two-factor factorial ANOVA test indicated that the effect of 10–7 M Dex and 10–7 M tRA on mRNA expression was synergistic (P < 0.008). Induction of NIS mRNA by both Dex and tRA was observed rapidly, in 3 h, and reached a maximum at 12 h (Fig. 5B).
FIG. 5. Induction of NIS mRNA by Dex in MCF-7 cells and tRA. A, Cells were treated with indicated agents for 12 h, and RT-PCR of NIS and GAPDH was performed with a real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C in NIS and 79 C in GAPDH (data not shown). Values are expressed as means ± SD (n = 3). *, P < 0.03; **, P < 0.05 for comparison as shown; ***, significant difference in two-factor factorial ANOVA test among the four groups treated with 0 or 10–7 M tRA and/or 0 or 10–7 M Dex (P < 0.01). B, Time course of NIS mRNA up-regulation by Dex and/or tRA. Cells were treated with 10–7 M Dex and 10–6 M tRA (), or 10–6 M tRA () for the indicated times. The average ratio of NIS/GAPDH in untreated cells was set as 1. Values are expressed as means ± SD (n = 3). *, P < 0.05 compared with the group treated with only tRA for 12 h. C, Effect of Dex on the stability of NIS mRNA in MCF-7 cells. Cells were treated with 10–7 M Dex and 10–6 M tRA (), or 10–6 M tRA () for 6 h and then treated with 40 μM 5,6-dichloro-1-?-D-ribofuranosylbenzimidazole for the indicated times. Total RNA was extracted from triplicate dishes, and RT-PCR was performed with a real-time PCR system. The ratio of remaining NIS mRNA was calculated by comparing the activity at each time point to that of the time point zero. The best-fit line for data points was used to calculate the half-life of the NIS mRNA in each experiment. Three triplicate experiments were repeated, and the average of the data was used to determine the half-life. A representative of three experiments is shown. Values are means ± SD (n = 3).
Our previous data, using a nuclear run-on assay, indicated that tRA increases the NIS mRNA expression in MCF-7 cells predominantly at the transcriptional level. Glucocorticoids have been shown to enhance retinoic acid action at the transcriptional level (19). Glucocorticoids also regulate some genes at the posttranscriptional level by stabilizing mRNA (36, 37, 38, 39, 40). To investigate whether Dex affects the stability of the NIS mRNA induced by tRA, we assessed the half-life of the mRNA in the MCF-7 cells treated with or without Dex in addition to tRA. We used the mRNA synthesis inhibitor 5,6-dichloro-1-?-D-ribofuranosylbenzimidazole, which inhibits transcription and allows for determination of the decay of preexisting mRNA (41). The half-life of NIS mRNA in tRA-stimulated MCF-7 cells without Dex treatment was 6.2 ± 1.1 h, similar to previous data (7.2 h) in FRTL-5 rat thyroid cells (7), whereas treatment with 10–7 M Dex stabilized NIS mRNA, significantly (P = 0.002) extending the half-life to 14.2 ± 1.6 h (Fig. 5C). Dex treatment did not significantly change the half-life of GAPDH in MCF-7 cells (data not shown).
Effects of Dex in combination with various retinoids on iodide uptake
Isomers of tRA and synthetic retinoids induce NIS mRNA and iodide uptake in MCF-7 cells, similar to that seen with tRA (Fig. 1). We assessed whether Dex differentially regulates the iodide uptake induced by these retinoids in MCF-7 cells. Dex (10–7 M) significantly increased iodide uptake induced by 10–6 M 9-cis RA and 10–6 M AGN190168 (RAR ?/-ligand), approximately 1.6-fold and approximately 1.9-fold greater than induction with retinoid alone (Fig. 6). In the presence of Dex treatment, the estimated EC50 of 9-cis RA for iodide uptake was approximately 7.2 x 10–8 M, whereas the EC50 of AGN190168 was 7.6 x 10–9 M. These results indicate that Dex enhances the effect of AGN190168 to a greater extent than the effects of 9-cis RA, especially at lower concentrations of these retinoids. In contrast, the effects of Dex on the uptake induced by 13-cis RA were relatively modest (Fig. 6). Dex slightly increased the uptake induced by AGN194433 (RAR-ligand, 10–8 to 10–6 M) and AGN194204 (RXR ligand, 10–8 to 10–7 M) but not significantly (data not shown). The potency of Dex enhancement of iodide uptake varies among the retinoids.
FIG. 6. Effects of various retinoid receptor ligands in combination with Dex on iodide uptake in MCF-7 cells. Cells were treated with the indicated agents for 48 h, and iodide uptake assay was performed with () or without () 30 μM KClO4. Values are expressed as means ± SD (n = 3). *, P < 0.01; **, P < 0.02, comparisons as shown.
Long-term treatment with retinoids and Dex for the induction of iodide uptake
Our in vivo study with MCF-7 xenografts demonstrated that systemic tRA markedly induced iodide uptake and NIS mRNA expression in the xenograft, but the activity declined in the 2 d after maximal concentration (14). Greater and more sustained induction of NIS in MCF-7 cells would increase the efficiency of radioiodide therapy for breast cancer. We, therefore, evaluated the effects of Dex on three retinoids, tRA, 9-cis RA, and AGN190168, which were identified as producing the greatest induction, on NIS mRNA induction in MCF-7 cells (Fig. 1). tRA and 9-cis RA maximally increased iodide uptake at d 2, and NIS mRNA at 12 h, consistent with a previous report of tRA (9), and the addition of Dex significantly enhanced the uptake and NIS mRNA at these time points (Fig. 7, A and B, left and middle). The induced iodide uptake and NIS mRNA, however, were significantly reduced in 4–5 d and 48–72 h, respectively, with tRA or 9-cis RA, even with Dex (Fig. 7, A and B, left and middle). The time course of the uptake induction by AGN190168 was similar to that of 9-cis RA, maximum at d 2 and slowly reduced by 5 d. Interestingly, the mRNA induction by AGN190168 was relatively slow, reaching the maximum at 24 h. tRA and 9-cis RA induced NIS mRNA more than 60-fold (more than 70% of the maximum induction) in 6 h, whereas the induction by AGN190168 at 6 h was about 25-fold, only approximately 28% of the maximum induction (Fig. 7B). The addition of Dex prolonged the induction of iodide uptake and mRNA expression by AGN190168; the maximum uptake and mRNA were at d 3 and 48 h, respectively, and significant reduction after the maximum points was not observed in both iodide uptake (by 5 d) and NIS mRNA (by 72 h). The uptake at d 5 and NIS mRNA at 72 h induced by both Dex and AGN190168 were still significantly higher than by only AGN190168 at the same time points. These results indicate that the combination treatment of Dex and AGN190168 offers longer duration of NIS expression and iodide uptake in MCF-7 cells.
FIG. 7. Effects of long-term treatment with Dex and retinoid receptor ligands on iodide uptake (A) and NIS mRNA expression (B) in MCF-7 cells. Cells were treated with 10–6 M retinoid (, tRA; , 9-cis RA; or , AGN190168) or combinations of 10–7 M Dex and 10–6 M retinoids (, tRA; , 9-cis RA, or , AGN190168) for the indicated times. The cells were fed every day with fresh agents tested during the treatment. A, After the treatment shown, iodide uptake assay was performed with or without 30 μM KClO4. Values are expressed as means ± SD (n = 3). Values of iodide uptake with KClO4 (between 0.74 and 0.98) are not shown. B, After the treatment shown, RT-PCR of NIS and GAPDH was performed with real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C in NIS and 79 C in GAPDH (data not shown). Values are expressed as means ± SD (n = 4). *, P < 0.05; **, P < 0.01, comparisons with the group without Dex at the same time point. ***, P < 0.05; ****, P < 0.01, comparison with the group of maximum value (at 2 d in iodide uptake, and 12 or 24 h in NIS mRNA) in the same treatment.
Cytotoxic clonogenic assay in 131I-treated MCF-7 cells stimulated by tRA and Dex
We previously demonstrated, by clonogenic assay, selective cytotoxicity of 131I in MCF-7 cells after treatment with 10–6 M tRA (9). We performed a clonogenic assay in MCF-7 cells treated with Dex and tRA compared with tRA treatment alone (Fig. 8). The clonogenic assay indicated that the addition of Dex (10–7 M) markedly increased 131I cytotoxicity compared with that after tRA (10–7 M) treatment alone. The combination of 10–7 M Dex and 10–7 M tRA, followed by 131I, resulted in a reduction in colony formation to 16.0 ± 4.1% (the mean ± SD) remaining, whereas survival after tRA treatment (10–7 M) was 58.1 ± 4.6%. The survival rate of cells stimulated with 10–6 M tRA and treated with 131I was 20.8 ± 3.4%, similar to that after treatment with the Dex-tRA (10–7 M) combination. These results demonstrate that Dex treatment with reduced tRA (10–7 M) concentrations increases the cytotoxicity after 131I treatment in MCF-7 cells to a level seen in cells treated with a 10-times-higher concentration of tRA.
FIG. 8. Cytotoxicity of 131I after the combination treatment of Dex and tRA in MCF-7 cells. Cells were treated with or without tRA and/or Dex at the indicated concentration for 48 h and then incubated with HBSS containing 0, or 60 μCi/ml Na131I and 0, or 6 μM NaI, respectively, for 6 h. Cells were harvested and used for clonogenic assay. A, Uptake of 131I– by MCF-7 cells. After incubation with 131I, ?-ray activity of the trapped 131I was counted by a Geiger-Mueller counter. The count was normalized to the cell number. B, The survival rate of MCF-7 cells after the 131I treatment. The rate was calculated as a percentage of cell colonies treated with 131I– compared with those treated with only HBSS. Values are means ± SD (n = 4). *, P < 0.0001 when compared with the group of unstimulated MCF-7 cells.
Discussion
NIS expression in breast cancer has been identified as an important target for therapeutic 131I (42, 43). Extensive experience with 131I treatment of thyroid cancer has demonstrated the importance of maximizing the magnitude of iodide uptake and prolonging the period of tumor residence. In the case of thyroid cancer, NIS expression in most tumors is responsive to TSH stimulation (44), and the radiation is retained for days in the tumors, likely due to some organification of the radioiodide. In contrast, no iodide organification has been observed in breast cancer cells in vitro (9). The iodide concentration in breast cancer specimens is significantly lower than that in normal thyroid tissue (45). The primary agent that has been shown to stimulate NIS expression in breast cancer is retinoic acid (9, 11, 14). In limited in vitro and in vivo studies, the RA concentration required for this action is quite high (14).
Enhanced effectiveness of RA stimulation could potentially be achieved by more selective retinoid stimulation and with agents that work synergistically with RA. Our studies have demonstrated that an RAR ?/-specific ligand AGN190168, as well as tRA and 9-cis RA, effectively induced NIS expression and iodide uptake in MCF-7 breast cancer cells, and Dex significantly enhanced and prolonged these inductions, especially with the RAR ?/-ligand. The combination of tRA and Dex synergistically increased the NIS expression and cell-selective cytotoxicity by 131I. In contrast, inductions of iodide uptake by an RAR-selective ligand AGN195183, an RAR-selective ligand AGN194433, and an RXR-selective ligand AGN194204 were relatively modest, and Dex did not significantly enhance the iodide uptake induced by AGN194433 and AGN194204.
tRA is a so-called pan-retinoid agonist, which binds to a broad spectrum of RAR isoforms, , ?, and , but does not bind RXRs (46, 47, 48). 9-cis RA binds to a broader spectrum of retinoid receptors, RARs and RXRs (24, 46). The dissociation constant values of tRA to RARs and 9-cis RA to both RARs and RXRs are similar, with some variation in previous reports (24, 46, 47, 48). Although 9-cis RA can be converted to tRA in vitro and in vivo, the isomerization activity in epithelial cells is much lower than liver cells (49). Our study indicated that tRA, 9-cis RA, and AGN190168, which has approximately 100-times higher affinity for RAR ? and compared with (50), induced iodide uptake and NIS mRNA most effectively in MCF-7 cells, whereas stimulation by isoform-selective retinoid receptor ligands AGN195183 (RAR) and AGN194433 (RAR) were relatively low. These data suggest that RAR?-signaling is important for the NIS induction in MCF-7 cells. An RXR-selective agonist AGN194204, which has 10-times higher affinity for all RXR isoforms compared with 9-cis RA (24), modestly induced iodide uptake and NIS mRNA in MCF-7 cells. Because the RXR agonist has no binding affinity and transactivation activity for RARs (24), NIS is likely to be modestly induced by RXR signaling in MCF-7 cells. A previous report has shown additive or synergistic effects of RAR-ligands with RXR ligands on NIS mRNA and iodide uptake induction in MCF7 cells (11). In contrast, we observed that AGN194204 (pan-RXR ligand) did not significantly enhance the iodide uptake induced by tRA, AGN190168 (RAR ?/-ligand), and AGN194433 (RAR-ligand). Further studies are needed to clarify the mechanism of the differential regulation between RAR and ?/.
Although 13-cis RA has a low affinity for RARs (about 10-times lower than tRA and 9-cis RA) and RXRs (about 100-times lower than 9-cis RA) (47), it stimulates transactivation by RAR, likely after isomerization to tRA and 9-cis RA (51). Glutathione S-transferases effectively catalyze isomerization of 13-cis RA to tRA (52). Expression of one of the glutathione S-transferases, which is associated with resistance to doxorubicin, is reduced in wild MCF-7 cells, compared with less-differentiated breast cancer cells (53). The lower induction of iodide uptake in 13-cis RA-treated MCF-7, compared with tRA, might involve the reduced expression of glutathione S-transferase.
Although our study with isoform-specific retinoid receptor ligands suggests an important role of RAR? in the NIS expression in MCF-7 cells, previous reports demonstrated the abundant expression of RAR and and reduced RAR? in MCF-7 cells (14, 54). tRA treatment significantly increases RAR? mRNA in MCF-7 xenograft tumors (14). Our time course study indicated relatively slow induction of NIS mRNA by AGN190168 (RAR ?/-ligand). An RAR-selective ligand AGN195183 and an RXR-selective ligand AGN194204 modestly induced iodide uptake mediated by NIS. During the early phase of the NIS induction by tRA and 9-cis RA before the induction of RAR?, NIS could be modestly induced through RAR signaling, which would not be activated by AGN190168 (RAR ?/-ligand).
Dex and tRA have been shown to synergistically increase the expression of various genes (19, 20, 55). The mouse mammary tumor virus promoter has a retinoic acid response element and a glucocorticoid response element, both of which are located separately but can be synergistically activated by tRA and Dex without direct receptor interaction (19). In other genes, glucocorticoids regulate various genes at the posttranscriptional level by stabilizing mRNA (36, 37, 38, 39, 40). In this study, we showed that Dex stabilized NIS mRNA and synergistically induced iodide uptake and NIS mRNA with tRA in MCF-7 cells. tRA induces NIS mRNA partially at the transcriptional level (9), and the effect of Dex on tRA-induced NIS mRNA is rapidly observed within 3 h after the beginning of the treatment. Further studies with Dex on the NIS transcription may provide additional insight into the role of Dex on the NIS up-regulation.
Although the iodide uptake and NIS mRNA expression in TSH-stimulated FRTL-5 rat thyroid cells is reduced by tRA (33) and Dex (17, 56), our study indicates that these nuclear receptor ligands synergistically increase the NIS expression in MCF-7 breast cancer cells. In addition, T3 (17) and E2 (18) down-regulate the TSH-induced NIS expression in thyroid cells, whereas these hormones, as well as the estrogen receptor antagonist tamoxifen, do not significantly influence tRA-induced iodide uptake in MCF-7 cells. These observations suggest the differential regulatory mechanism of NIS in thyroid tissue and breast cancer.
The absence of an influence of T3 and E2 on RA-induced NIS expression in breast cancer is important for therapeutic applications. To use systemic radioiodide treatment targeted to breast cancer, iodide uptake in the thyroid gland must be blocked to protect the thyroid from the radioiodide and to maximally concentrate radioiodide into breast cancer. Our in vivo study with MCF-7 xenograft model mice demonstrated that both tRA and T4 significantly reduced iodide uptake in the thyroid gland (Ref. 14 and unpublished data), consistent with the previous in vitro studies (17, 33). Dex will likely also reduce iodide uptake into the thyroid. Tamoxifen is a commonly used agent in breast cancer, and it did not significantly affect the tRA-induced iodide uptake in MCF-7 cells. tRA treatment before radioiodide therapy, therefore, could be used with tamoxifen treatment in estrogen receptor-positive breast cancer.
We and other groups have observed tRA stimulation of NIS expression in MCF-7 cells in vitro (9, 11, 57). We have also reported on significant induction of NIS in vivo by systemic tRA treatment in a transgenic mouse model of breast cancer, murine mammary tumor virus-polyoma virus middle T antigen, as well as a MCF-7 xenograft model (14). Only two of eight breast cancer cell lines tested, however, show RA induction of NIS mRNA (11). Approximately 50% of metastatic thyroid cancer concentrates radioiodine with recombinant TSH after total thyroidectomy (44), yet relatively few normal thyroid cell lines (25, 58, 59), and no thyroid cancer cell lines (60, 61, 62), have NIS activity. The disparity between in vitro and in vivo breast cancer models, therefore, is similar to that which is seen in thyroid cancer.
Dex decreased the EC50 of tRA and RAR ?/-ligand AGN190168 for iodide uptake more than 10 times in MCF-7 cells. Indeed, our in vitro clonogenic assay indicated cell-selective cytotoxicity in MCF-7 cells treated with Dex and reduced concentration of tRA. The addition of Dex has a potential to achieve the NIS induction with a lower dose of tRA in vivo. The combination of Dex and the RAR?/-selective agonist AGN190168 provided a means for longer duration and higher induction of iodide uptake in vitro, suggesting higher efficacy of 131I after the combination treatment.
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Address all correspondence and requests for reprints to: Dr. Gregory A. Brent, Molecular Endocrinology Laboratory, Building 114, Room 230, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent@ucla.edu.
Abstract
The sodium/iodide symporter (NIS) mediates iodide uptake in lactating breast tissue and is expressed in some breast cancers. We have previously demonstrated that all-trans retinoic acid (tRA) stimulates NIS gene expression and the selective cytotoxic effect of ?-emitting radioiodide-131 (131I) in both in vitro and in vivo MCF-7 breast cancer cell systems. We studied the ability of natural and synthetic retinoids, in combination with other nuclear receptor ligands, to achieve greater and more sustained induction of NIS in MCF-7 cells and enhance 131I-mediated cytotoxicity. Selective stimulation of retinoic acid receptor (RAR) ?/ produced marked NIS induction; and selective stimulation of RAR, RAR, or retinoid X receptor produced more modest induction. Maximal NIS induction was seen with 9-cis retinoic acid and AGN190168, a RAR ?/-agonist. Dexamethasone (Dex), but not the other nuclear receptor ligands, in combination with tRA synergistically induced iodide uptake and NIS mRNA expression, predominantly by prolonging NIS mRNA half-life. The addition of Dex reduced the EC50 of tRA for NIS stimulation to approximately 7%, such that 10 –7 M tRA with addition of Dex enhanced iodide uptake and selective cytotoxicity of 131I greater than 10–6 M tRA alone. AGN190168 combined with Dex synergistically increased iodide uptake and significantly prolonged induction (5 d) of iodide uptake compared with that induced by the combination of tRA/Dex or 9-cis retinoic acid/Dex. The addition of Dex reduced the effective dose of retinoid and prolonged the induction of NIS, especially with AGN190168, suggesting higher efficacy of 131I after combination treatment.
Introduction
THE SODIUM/IODIDE SYMPORTER (NIS) is expressed on the basolateral membrane of alveolar cells in lactating breast tissue and functions to concentrate iodide in breast milk (1, 2, 3). Iodide is required by the developing neonate for thyroid hormone synthesis. Oxytocin is a major stimulus to NIS expression in the lactating mammary gland (1, 2), and both prolactin and estrogen enhance NIS induction by oxytocin (1). More than 60% of breast cancer tissue also expresses NIS (1, 4), and correlation between 99mTc-pertechnetate uptake and NIS expression in breast tumor tissues has recently been confirmed (5, 6).
The ?-emitting isotope, radioiodide-131 (131I), is commonly used in the treatment of differentiated thyroid cancer after thyroidectomy. NIS is predominantly expressed in the follicular cells within the thyroid gland, and TSH up-regulates NIS expression and iodide uptake (7, 8). Thyroid cancer is associated with reduced NIS activity, and a high level of TSH stimulation is required to maximize 131I uptake. This is accomplished by cessation of thyroid hormone supplementation after thyroidectomy to raise endogenous serum TSH or by exogenous administration of recombinant TSH.
The retinoic acid receptor (RAR) ligand, all-trans retinoic acid (tRA), stimulates NIS gene expression and iodide uptake in the estrogen receptor-positive MCF-7 breast cancer cell line (9). A recent study has suggested that activity of a cardiac homeobox transcription factor, Nkx-2.5, is critical for induction of NIS by tRA in MCF7 cells (10). Administration of 131I in MCF-7 cells after augmentation of NIS gene expression by tRA results in selective cytotoxicity (9). The retinoid X receptor (RXR) ligand 9-cis retinoic acid (9-cis RA) and some synthetic retinoids also induce iodide uptake and NIS mRNA in MCF-7 cells (11). RAR has three isoforms (, ?, and ) with a specific pattern of developmental and tissue distribution. tRA and 9-cis RA stimulate formation of heterodimers of RAR and RXR. Other nuclear hormone receptors, such as vitamin D3 receptor and thyroid hormone receptor, also heterodimerize with RXR. The RXR-nuclear hormone receptor heterodimer binds to cis-elements, recruits coactivators, and enhances gene expression (12, 13).
Systemic tRA treatment of immunodeficient mice with MCF-7 cell xenografts markedly stimulated NIS expression and iodide uptake (15-fold) (14). The tRA dose for the maximum NIS induction in that model, however, is more than 10-fold above the maximum tolerable tRA dose, on a milligram-per-square-meter basis, used in humans for treatment of solid tumors (14). In addition, the duration of the NIS induction is relatively short, followed by a significant reduction (more than 60%) within 2 d of the maximum induction (14). To reduce tRA toxicity and increase the efficacy of radioiodide, additional agents to enhance NIS expression in breast cancer were investigated.
Retinoid isomers and RAR isoform-selective retinoid agonists have been shown to enhance the response of some RA-stimulated genes. Systemic treatment of rodents with 13-cis RA, an isomer of tRA, has been shown to have a significantly lower toxicity than tRA (15). The elimination half-life of 13-cis RA in plasma is about 10 times longer than tRA (16). Systemic administration of isoform-selective retinoids may be associated with greater efficacy and reduced adverse effects, compared with nonselective retinoids. We therefore studied some isoform-selective synthetic retinoids, to gain insight into the retinoid pathway of NIS induction and evaluate efficacy of NIS induction in MCF-7 cells. We found that an RAR ?/-agonist and 9-cis RA induced NIS mRNA and iodide uptake as well as tRA.
Dexamethasone (Dex), T3, and estradiol (E2) have been shown to influence NIS expression in FRTL-5 rat thyroid cells (17, 18). We tested the influence of these ligands, as well as other nuclear receptor ligands, on NIS expression in MCF-7 cells. It has been reported in some cancer cells that there is a synergistic effect of retinoids with other nuclear receptor ligands in gene regulation (19, 20) and cell proliferation (21). We therefore investigated the influence of tRA, in combination with other nuclear receptor ligands, on NIS expression, and we found that Dex significantly enhanced retinoid-induced NIS expression and cell-selective cytotoxicity with I131 in MCF-7 cells.
Materials and Methods
Chemicals and cells
MCF-7 breast cancer cells (lot no. F15100 and 205623) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained as described (22). An RAR-selective ligand AGN195183, an RAR ?/-selective ligand AGN190168 (tazarotene) (23), an RAR-selective ligand AGN194433, and an RXR-selective ligand AGN194204 (24) were provided by Allergan, Inc. (Irvine, CA). These retinoids were dissolved in dimethylsulfoxide (DMSO) to a concentration of 10–2 M and stored at –20 C. tRA, 9-cis RA, Dex, and other cell culture reagents were purchased from Sigma (St. Louis, MO). Cells were maintained with 0.1% DMSO vehicle, when treated with nuclear receptor ligands, and fed with fresh media with ligands every 24 h. In some experiments, fetal bovine serum (ATCC) in the medium was replaced with charcoal-stripped serum or Knockout Serum Replacement (Invitrogen, Carlsbad, CA) 24 h before the treatment with ligands, as noted.
Iodide uptake
The iodide uptake assay was performed as described (9, 25), with minor modifications. Briefly, cells were grown in 12-well dishes, washed with Hanks’ Balanced Salt Solution (HBSS), and incubated for 1 h at 37 C with 500 μl HBSS containing approximately 0.1 μCi carrier-free Na125I (Amersham Biosciences, Piscataway, NJ) and 10 μM NaI. The specific activity under these conditions was 20 mCi/mmol. After incubation, the cells were washed twice with ice-cold HBSS, scraped from each well, and radioactivity measured in a -counter. Cell number was determined by counting in a hemocytometer. The radioactivity was normalized to the cell number at the time of the assay. For kinetic studies, cells were incubated with 1–600 μM NaI and 20 mCi/mmol Na125I at 37 C for 5 min.
Iodide efflux
The iodide efflux assay was described previously (9, 26). Briefly, cells in 12-well dishes were incubated with HBSS containing 10 μM NaI and 20 mCi/mmol Na125I at 37 C for 1 h, and the medium was replaced every 5 min with fresh HBSS without NaI. The content of 125I in the collected supernatant was measured by -counter. After the last time point (60 min), the cells were extracted with 400 μl ethanol to count residual radioactivity.
cDNA preparation for RT-PCR
Total RNA was isolated by RNeasy mini kit (QIAGEN, Valencia, CA) from cells grown in 100-mm-diameter culture dishes. On-column DNase I digestion was performed as recommended by QIAGEN. Three micrograms of purified total RNA were reverse-transcribed by using 50 U Superscript III reverse transcriptase (Invitrogen) in 20 μl reaction with oligo(deoxythymidine)12–18 primer (1 μg).
Quantitative real-time PCR analysis
Primers for PCR were designed with Primer3 software (http://frodo.wi.mit.edu), and those sequences were checked by nucleotide BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) to avoid cross-reactivity with other known sequences. The sequences were the following; NIS forward, 5'-GTTCTACACTGACTGCGACCCTC-3'; NIS reverse, 5'-GCAGCCGAGGTTTGATGAG-3'; glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) forward, 5'-GAGTCAACGGATT-TGGTCGTA-3'; GAPDH reverse, 5'-CATGGGTGGAATCATATTGGA-3'. PCR mixture contained 10 μl of 2x QuantiTect SYBR Green PCR Master Mix (QIAGEN), 1 μl of the reverse-transcription reaction, and 6 pmol of forward and reverse primers. PCR was carried out using the DNA Engene Opticon System (MJ Research, Inc., Waltham, MA) with the following cycle parameters: polymerase activation for 15 min at 95 C and amplification for 35 cycles of 20 sec at 96 C, 45 sec at 55 C, and 45 sec at 72 C. Melting curve analysis was performed after the amplification as recommended. Standard curves representing six-point serial dilution of mixed cDNA of the corresponding control group, MCF-7 cells treated with 10–6 M tRA for 12 h, unless otherwise noted, were analyzed in each assay and used for calculation of relative expression values. The sample quantifications were normalized by the internal control GAPDH mRNA.
131I-Cytotoxic assay
The procedure was carried out as previously described (9), with minor modifications. Briefly, cells grown in 25-cm2 flasks were treated with or without tRA and/or Dex for 48 h. The cells were then incubated for 6 h at 37 C with 5 ml HBSS containing 0 or 60 μCi/ml Na131I (PerkinElmer, Boston, MA) and 0 or 6 μM NaI, respectively. The reaction was terminated by removing the radioiodide-containing medium and washing cells twice with HBSS. The cells were then trypsinized, counted, and plated at densities of 250 and 1000 cells/well with growth medium in 6-well plates. Uptake of 131I– was confirmed by a Geiger Mueller counter before the plating. Cells were grown for 12 d, fixed with 3:1 methanol:acetic acid, and stained with crystal violet, and the number of macroscopic colonies were counted. The survival rate was calculated as the percentage of surviving cell colonies in plates treated with 131I– compared with those treated with only HBSS.
Statistical analysis
Statistical comparison was performed using STAT VIEW 5.0 software (SAS Institute, Cary, NC) with significance at a P value < 0.05. Comparison between groups was determined by conducting a paired Student’s t test. The synergistic effect of two stimulatory factors was determined by two-factor factorial ANOVA test.
Results
Iodide uptake in MCF-7 cells treated with retinoid receptor ligands
All-trans RA (tRA) and 4-[E2–5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl] benzoic acid, a selective RAR ligand, increase iodide uptake about 9-fold above baseline in the well-differentiated breast cancer cell line, MCF-7 (9). tRA also markedly induces iodide uptake in MCF-7 cell xenografts in immunodeficient mice, up to 15-fold above baseline (14). The dose of systemic tRA required for NIS induction, however, is relatively high in the xenograft model. To reduce tRA toxicity, synthetic retinoids with more selective actions and longer half-life, such as 13-cis RA and tazarotene (AGN190168), are commonly used in clinical applications, especially topical skin treatment. We tested the ability of these, as well as other retinoid receptor ligands, to induce iodide uptake in MCF-7 cells.
13-cis RA, an isomer of tRA that is converted to tRA in some cells (27, 28), significantly induced iodide uptake in a dose-dependent manner in MCF-7 cells, approximately 7-fold above baseline, at a concentration of 10–6 M (Fig. 1A). 9-cis RA, a ligand for both RAR and RXR, also significantly induced the uptake in a dose-dependent manner, approximately 11-fold above baseline, at a concentration of 10–6 M (Fig. 1A). The magnitude of induction by tRA (9-fold) was higher than with 13-cis RA and lower than 9-cis RA; however, these differences were not statistically significant (Fig. 1A). We also tested the ability of synthetic retinoids to induce iodide uptake in MCF-7 cells. AGN190168, an RAR ligand that predominantly stimulates the ?- and -isoforms, significantly induced radioiodide uptake in a dose-dependent manner, approximately 8-fold above baseline at a concentration of 10–6 M (Fig. 1B). The dose response and maximal induction was similar to that of tRA (Fig. 1B), with similar estimated median effective concentration (EC50), approximately 9.1 x 10–8 M for AGN190168 and approximately 9.3 x 10–8 M for tRA. AGN194204, an RXR-specific ligand, significantly induced iodide uptake at 10–7 M, but the induction was relatively modest and was reduced at higher ligand concentrations (Fig. 1B). The -isoform-specific ligand AGN195183 and a -isoform-specific ligand AGN194433 also significantly induced iodide uptake. The magnitude of induction with these ligands, however, was lower than for other retinoids, consistent with a previous study using RAR-ligands (11). These data suggest that signal transduction through RAR, especially RAR?, is important for the induction of iodide uptake in MCF-7 cells, and selective RXR stimulation results in only a modest induction of uptake. The induced uptake stimulated by these retinoids was almost completely blocked by KClO4, a specific inhibitor of NIS-mediated iodide transport (29) (data not shown).
FIG. 1. Effect of retinoids on iodide uptake in MCF-7 breast cancer cells. A, Effects of isomers of tRA. Cells were incubated with the indicated concentration of retinoids for 48 h, and iodide uptake assay was performed with 500 μl HBSS containing approximately 0.1 μCi Na125I and 10 μM NaI. The reactions with or without 30 μM KClO4 were terminated at 1 h, and the content of iodide in the cells was determined with a -counter. Replacement of fetal bovine serum to 10% Knockout SR (Invitrogen) did not significantly change the iodide uptake induced by these retinoids (data not shown). B, Effects of synthetic retinoid receptor ligands in MCF-7 cells. Cells were incubated with the indicated concentration of retinoids for 48 h, and iodide uptake assay was performed. Replacement of fetal bovine serum to 10% Knockout SR (Invitrogen) did not significantly change the iodide uptake induced by these retinoids (data not shown). C, Combination study of RAR-specific ligands and an RXR-specific ligand on iodide uptake in MCF-7 cells. Cells were treated for 48 h with an RAR ligand, tRA, AGN190168, or AGN194433, in combination with AGN194204, an RXR ligand, as indicated, and an iodide uptake assay was performed with () or without () 30 μM KClO4. Values are expressed as mean ± SD (n = 3). Values of iodide uptake with KClO4 were between 0.82 and 0.95 in each conditions and not shown in A and B. *, P < 0.02; **, P < 0.001 compared with untreated cells.
RAR and RXR bind ligand, and the resulting heterodimer binds to specific cis-elements in the regulatory regions of RA-responsive genes. Some RA-responsive genes can be synergistically induced by combinations of RAR and RXR ligands (30, 31, 32). We therefore tested some RAR-specific ligands in combination with the RXR-specific ligand AGN194204 to induce iodide uptake in MCF-7 cells. The RAR ligands, tRA, AGN190168 (RAR ?/), and AGN194433 (RAR), however, did not synergistically induce iodide uptake in combination with AGN194204 treatment (Fig. 1C).
NIS mRNA expression in MCF-7 cells treated with retinoid receptor ligands
tRA up-regulates NIS gene expression predominantly at the transcriptional level (9). To evaluate whether the NIS mRNA expression is correlated with iodide uptake in MCF-7 cells, we performed quantitative RT-PCR of NIS with MCF-7 cells treated with several retinoids at the dose for the highest induction of iodide uptake shown in Fig. 1. NIS mRNA levels, as determined by RT-PCR, were significantly increased by retinoids that induced iodide uptake in MCF-7 cells (Fig. 2), and the magnitude of induction of mRNA was correlated with that of iodide uptake (Fig. 1, A and B). tRA, 9-cis RA, and the RAR ?/-ligand AGN190168 stimulated the greatest magnitude of NIS mRNA (more than 70-fold induction), consistent with the maximal effect on iodide uptake. The inductions by 13-cis RA and an RXR ligand AGN194204 were significant, but relatively modest (49-fold and 26-fold, respectively), consistent with a previous report with other RXR ligands (11).
FIG. 2. Effect of retinoids on NIS mRNA expression in MCF-7 cells. Cells were treated with indicated ligands (10–6 M, except for 10–7 M of AGN194204) for 12 h, and RT-PCR for NIS and GAPDH was performed with a real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C for NIS and 79 C for GAPDH (data not shown). The average ratio of NIS/GAPDH in untreated cells was set as 1. Values are expressed as means ± SD (n = 3). *, P < 0.015 compared with untreated cells.
The magnitude of NIS mRNA induction by these retinoids was markedly greater than that of iodide uptake (8- to 10-fold in Fig. 1, A and B). The finding of greater NIS mRNA induction than functional iodide uptake is consistent with previous observations in breast models (9, 11, 14). This may be due, in part, to a predominance of NIS protein remaining in the cytoplasm, as shown in our previous immunocytochemical studies in MCF7 cells (14). NIS protein may also be in the cell membrane but not functional for promoting iodide uptake.
Iodide uptake in MCF-7 cells treated with nuclear hormone receptor ligands
tRA up-regulates NIS expression in MCF-7 cells (9) but significantly reduces NIS mRNA expression and iodide uptake in FRTL-5 rat thyroid cells (33). The nuclear receptor ligands, Dex, T3 (17), and E2 (18), modestly reduce TSH-induced NIS expression in FRTL-5 cells. To determine whether ligands for other nuclear receptors could stimulate NIS expression and iodide uptake in MCF7 breast cancer cells, we tested various nuclear receptor ligands and combinations: Dex, E2, pioglitazone (PGZ), a synthetic peroxisome proliferator-activated receptor--ligand, progesterone (Prog), T3, and 1,25 dihydroxyvitamin D3 (VD3) compared with tRA. Treatment with 10–6 M Dex alone significantly increased iodide uptake in MCF-7 cells, approximately 1.8-fold above baseline. The Dex-induced iodide uptake, however, was only approximately 30% of that induced by 10–6 M tRA. Other nuclear receptor ligands did not significantly influence uptake, with concentrations in the range of 10–6 M to 10–8 M (data not shown).
Recent studies have suggested so-called cross-talk of RAR signaling with that of other nuclear receptor ligand pathways. Peroxisome proliferator-activated receptor--ligand with retinoid, for example, synergistically inhibits growth and induces apoptosis in MCF7 cells (21). Signaling by cAMP pathways stimulates NIS in samples from transgenic breast cancer models (34). We studied the ability of Dex, E2, PGZ, Prog, T3, and VD3 to enhance tRA-induced iodide uptake in MCF-7 cells. Dex significantly enhanced the tRA (10–6 M)-induced iodide uptake, approximately 1.5-fold of that without tRA. VD3 and E2 at concentrations of 10–7 M decreased the tRA-induced iodide up to 22 and 27%, respectively, although the differences were not significant (data not shown). Nuclear receptor ligands, PGZ, Prog, VD3, E2, T3, and tamoxifen, were tested at concentrations of 10–6 to 10–8 M, and no significant effect on tRA-induced uptake was seen (data not shown).
Effects of Dex on iodide uptake in MCF-7 cells
Dex increased iodide uptake induced by tRA in MCF-7 cells in a dose-dependent manner (Fig. 3A). Dex (10–7 M) increased the uptake about 3-fold above 10–7 M RA alone and significantly higher than that with only 10–6 M tRA (Fig. 3A). A two-factor factorial ANOVA test indicated that the addition of 10–7 M Dex and 10–7 M tRA synergistically increased iodide uptake in MCF-7 cells (P < 0.0001, Fig. 3A). The maximal synergistic effect of Dex on RA stimulation was seen at a dose of 10–7 M. According to the dose-response curve, the estimated EC50 of tRA for iodide uptake in MCF-7 cells was approximately 9.6 x 10–8 M, whereas the estimated EC50 of tRA with Dex was approximately 6.8 x 10–9 M.
FIG. 3. Effect of Dex on iodide uptake in MCF-7 cells. A, Various dose combinations of Dex and tRA were tested to induce iodide uptake in MCF-7 cells. Cells were treated with indicated agents for 48 h, and the iodide uptake assay was performed with () or without () 30 μM KClO4. *, P < 0.02; **, P < 0.001 for comparison as shown; ***, significant difference in two-factor factorial ANOVA test among the four groups treated with 0 or 10–7 M tRA and/or 0 or 10–7 M Dex (P < 0.0001). B, Time course of up-regulation of iodide uptake induced by Dex and tRA. Cells were treated with 10–7 M Dex and 10–7 M tRA (), 10–7 M tRA (), or 10–6 M tRA () for 48 h. **, P < 0.01 when compared with untreated cells. C, Effect of Dex on tRA-treated MCF-7 cells. Cells were treated with 10–7 M tRA for 36 h, and then 0 M () or 10–7 M () Dex was added to the culture medium. ****, P < 0.01 when compared with values at 36 h. D, Iodide efflux from MCF-7 cells treated with 10–7 M Dex and 10–7 M tRA () or 10–6 M tRA (). Cells were treated with tRA and/or Dex for 48 h before the assay. Data are means ± SD (n = 3).
Our time course study showed that the iodide uptake was significantly increased by the addition of both 10–7 M Dex and 10–7 M tRA in 12 h, and it reached a maximum at 36 h (Fig. 3B). When 10–7 M Dex was added to the MCF-7 cells treated with 10–7 M tRA for 36 h, the uptake was significantly increased in 12 h and reached a maximum in 24 h but quickly fell in 36 h (Fig. 3C).
Variation in the net iodide uptake is predominantly the result of changes in iodide influx, although the influence of iodide efflux needs to be considered. We performed iodide efflux assays in MCF-7 treated with both 10–7 M Dex and 10–7 M tRA for 48 h, compared with cells treated with tRA (10–6 M) alone. The time point at which 50% of iodide remained in MCF-7 cells after RA treatment was approximately 21 min, consistent with previous data (9), and Dex did not influence this time point (Fig. 3D).
We analyzed the kinetics of iodide uptake in MCF-7 cells treated with 10–6 M tRA and 10–7 M Dex, compared with that with only 10–6 M tRA treatment. When iodide uptake was initiated with approximately 20 mCi/mmol Na125I in MCF-7 cells treated with both Dex and tRA, the uptake reached a half-maximal level of activity in 10 min and saturation at about 30 min (data not shown), consistent with the time course in tRA-treated MCF-7 cells reported previously (9). The initial velocity of iodide uptake was determined by incubation for 5 min with 1–600 μM NaI (Fig. 4A). Excess external iodide (>100 μM) saturated the iodide transport, and Lineweaver-Burk double-reciprocal plots yielded the Michaelis-Menten constant (Km) and maximum velocity values (Fig. 4B). The apparent Km for iodide was 18.6 ± 1.51 μM (the mean ± SD) with tRA and Dex, which was not significantly different from that with only tRA (17.9 ± 1.1 μM). These results are consistent with the range of values reported in MCF-7 cells (9) and FRTL-5 thyroid cells (25, 35). In contrast, the maximum velocity of iodide uptake with tRA and Dex (4.99 ± 0.60 pmol/min/104 cells) was significantly (P = 0.001) higher than that with only tRA (3.01 ± 0.30 pmol/min/104 cells), indicating increased functional NIS after Dex treatment.
FIG. 4. Kinetics study of iodide uptake by MCF-7 cells treated with Dex and tRA. Cells were treated with 10–7 M Dex and 10–7 M tRA (), or 10–6 M tRA () for 48 h before assay. A, Dependency of initial velocity of iodide uptake (5 min) on the extracellular iodide concentration in MCF-7 cells. After treatment with Dex and/or tRA, cells were incubated at 37 C for 5 min with 500 μl HBSS containing 20 mCi/mmol 125I– and indicated concentration of NaI. Trapped 125I– was measured by -counter and normalized to the cell number. Nonspecific binding of 125I– was determined in duplicate assays in the presence of 30 μM KClO4, and this value was normalized to the cell number and subtracted from the values measured. The subtracted data (net uptake velocity) are expressed as means ± SD (n = 3). B, The data points of 1–600 μM NaI from A are graphed as a Lineweaver-Burk plot. All points from triplicate wells are plotted. Equation for the "best fit" line and correlation coefficient (R2) are shown.
Effects of Dex on NIS mRNA expression in MCF-7 cells
Treatment with Dex alone (10–7 M) increased NIS mRNA expression, as assessed by RT-PCR, approximately 1.9-fold, but the difference was not statistically significant (P = 0.07; Fig. 5A). NIS mRNA levels were significantly increased, by 10–7 M and 10–6 M tRA, approximately 39-fold and approximately 72-fold, respectively. Addition of 10–7 M Dex significantly enhanced tRA-stimulated mRNA expression, approximately 2.8-fold with 10–7 M tRA and approximately 2.0-fold with 10–6 M tRA (Fig. 5A), compared with tRA alone. A two-factor factorial ANOVA test indicated that the effect of 10–7 M Dex and 10–7 M tRA on mRNA expression was synergistic (P < 0.008). Induction of NIS mRNA by both Dex and tRA was observed rapidly, in 3 h, and reached a maximum at 12 h (Fig. 5B).
FIG. 5. Induction of NIS mRNA by Dex in MCF-7 cells and tRA. A, Cells were treated with indicated agents for 12 h, and RT-PCR of NIS and GAPDH was performed with a real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C in NIS and 79 C in GAPDH (data not shown). Values are expressed as means ± SD (n = 3). *, P < 0.03; **, P < 0.05 for comparison as shown; ***, significant difference in two-factor factorial ANOVA test among the four groups treated with 0 or 10–7 M tRA and/or 0 or 10–7 M Dex (P < 0.01). B, Time course of NIS mRNA up-regulation by Dex and/or tRA. Cells were treated with 10–7 M Dex and 10–6 M tRA (), or 10–6 M tRA () for the indicated times. The average ratio of NIS/GAPDH in untreated cells was set as 1. Values are expressed as means ± SD (n = 3). *, P < 0.05 compared with the group treated with only tRA for 12 h. C, Effect of Dex on the stability of NIS mRNA in MCF-7 cells. Cells were treated with 10–7 M Dex and 10–6 M tRA (), or 10–6 M tRA () for 6 h and then treated with 40 μM 5,6-dichloro-1-?-D-ribofuranosylbenzimidazole for the indicated times. Total RNA was extracted from triplicate dishes, and RT-PCR was performed with a real-time PCR system. The ratio of remaining NIS mRNA was calculated by comparing the activity at each time point to that of the time point zero. The best-fit line for data points was used to calculate the half-life of the NIS mRNA in each experiment. Three triplicate experiments were repeated, and the average of the data was used to determine the half-life. A representative of three experiments is shown. Values are means ± SD (n = 3).
Our previous data, using a nuclear run-on assay, indicated that tRA increases the NIS mRNA expression in MCF-7 cells predominantly at the transcriptional level. Glucocorticoids have been shown to enhance retinoic acid action at the transcriptional level (19). Glucocorticoids also regulate some genes at the posttranscriptional level by stabilizing mRNA (36, 37, 38, 39, 40). To investigate whether Dex affects the stability of the NIS mRNA induced by tRA, we assessed the half-life of the mRNA in the MCF-7 cells treated with or without Dex in addition to tRA. We used the mRNA synthesis inhibitor 5,6-dichloro-1-?-D-ribofuranosylbenzimidazole, which inhibits transcription and allows for determination of the decay of preexisting mRNA (41). The half-life of NIS mRNA in tRA-stimulated MCF-7 cells without Dex treatment was 6.2 ± 1.1 h, similar to previous data (7.2 h) in FRTL-5 rat thyroid cells (7), whereas treatment with 10–7 M Dex stabilized NIS mRNA, significantly (P = 0.002) extending the half-life to 14.2 ± 1.6 h (Fig. 5C). Dex treatment did not significantly change the half-life of GAPDH in MCF-7 cells (data not shown).
Effects of Dex in combination with various retinoids on iodide uptake
Isomers of tRA and synthetic retinoids induce NIS mRNA and iodide uptake in MCF-7 cells, similar to that seen with tRA (Fig. 1). We assessed whether Dex differentially regulates the iodide uptake induced by these retinoids in MCF-7 cells. Dex (10–7 M) significantly increased iodide uptake induced by 10–6 M 9-cis RA and 10–6 M AGN190168 (RAR ?/-ligand), approximately 1.6-fold and approximately 1.9-fold greater than induction with retinoid alone (Fig. 6). In the presence of Dex treatment, the estimated EC50 of 9-cis RA for iodide uptake was approximately 7.2 x 10–8 M, whereas the EC50 of AGN190168 was 7.6 x 10–9 M. These results indicate that Dex enhances the effect of AGN190168 to a greater extent than the effects of 9-cis RA, especially at lower concentrations of these retinoids. In contrast, the effects of Dex on the uptake induced by 13-cis RA were relatively modest (Fig. 6). Dex slightly increased the uptake induced by AGN194433 (RAR-ligand, 10–8 to 10–6 M) and AGN194204 (RXR ligand, 10–8 to 10–7 M) but not significantly (data not shown). The potency of Dex enhancement of iodide uptake varies among the retinoids.
FIG. 6. Effects of various retinoid receptor ligands in combination with Dex on iodide uptake in MCF-7 cells. Cells were treated with the indicated agents for 48 h, and iodide uptake assay was performed with () or without () 30 μM KClO4. Values are expressed as means ± SD (n = 3). *, P < 0.01; **, P < 0.02, comparisons as shown.
Long-term treatment with retinoids and Dex for the induction of iodide uptake
Our in vivo study with MCF-7 xenografts demonstrated that systemic tRA markedly induced iodide uptake and NIS mRNA expression in the xenograft, but the activity declined in the 2 d after maximal concentration (14). Greater and more sustained induction of NIS in MCF-7 cells would increase the efficiency of radioiodide therapy for breast cancer. We, therefore, evaluated the effects of Dex on three retinoids, tRA, 9-cis RA, and AGN190168, which were identified as producing the greatest induction, on NIS mRNA induction in MCF-7 cells (Fig. 1). tRA and 9-cis RA maximally increased iodide uptake at d 2, and NIS mRNA at 12 h, consistent with a previous report of tRA (9), and the addition of Dex significantly enhanced the uptake and NIS mRNA at these time points (Fig. 7, A and B, left and middle). The induced iodide uptake and NIS mRNA, however, were significantly reduced in 4–5 d and 48–72 h, respectively, with tRA or 9-cis RA, even with Dex (Fig. 7, A and B, left and middle). The time course of the uptake induction by AGN190168 was similar to that of 9-cis RA, maximum at d 2 and slowly reduced by 5 d. Interestingly, the mRNA induction by AGN190168 was relatively slow, reaching the maximum at 24 h. tRA and 9-cis RA induced NIS mRNA more than 60-fold (more than 70% of the maximum induction) in 6 h, whereas the induction by AGN190168 at 6 h was about 25-fold, only approximately 28% of the maximum induction (Fig. 7B). The addition of Dex prolonged the induction of iodide uptake and mRNA expression by AGN190168; the maximum uptake and mRNA were at d 3 and 48 h, respectively, and significant reduction after the maximum points was not observed in both iodide uptake (by 5 d) and NIS mRNA (by 72 h). The uptake at d 5 and NIS mRNA at 72 h induced by both Dex and AGN190168 were still significantly higher than by only AGN190168 at the same time points. These results indicate that the combination treatment of Dex and AGN190168 offers longer duration of NIS expression and iodide uptake in MCF-7 cells.
FIG. 7. Effects of long-term treatment with Dex and retinoid receptor ligands on iodide uptake (A) and NIS mRNA expression (B) in MCF-7 cells. Cells were treated with 10–6 M retinoid (, tRA; , 9-cis RA; or , AGN190168) or combinations of 10–7 M Dex and 10–6 M retinoids (, tRA; , 9-cis RA, or , AGN190168) for the indicated times. The cells were fed every day with fresh agents tested during the treatment. A, After the treatment shown, iodide uptake assay was performed with or without 30 μM KClO4. Values are expressed as means ± SD (n = 3). Values of iodide uptake with KClO4 (between 0.74 and 0.98) are not shown. B, After the treatment shown, RT-PCR of NIS and GAPDH was performed with real-time PCR system. Melting curve analysis showed only one significant peak in each assay, with apparent melting temperature (Tm value) of 84 C in NIS and 79 C in GAPDH (data not shown). Values are expressed as means ± SD (n = 4). *, P < 0.05; **, P < 0.01, comparisons with the group without Dex at the same time point. ***, P < 0.05; ****, P < 0.01, comparison with the group of maximum value (at 2 d in iodide uptake, and 12 or 24 h in NIS mRNA) in the same treatment.
Cytotoxic clonogenic assay in 131I-treated MCF-7 cells stimulated by tRA and Dex
We previously demonstrated, by clonogenic assay, selective cytotoxicity of 131I in MCF-7 cells after treatment with 10–6 M tRA (9). We performed a clonogenic assay in MCF-7 cells treated with Dex and tRA compared with tRA treatment alone (Fig. 8). The clonogenic assay indicated that the addition of Dex (10–7 M) markedly increased 131I cytotoxicity compared with that after tRA (10–7 M) treatment alone. The combination of 10–7 M Dex and 10–7 M tRA, followed by 131I, resulted in a reduction in colony formation to 16.0 ± 4.1% (the mean ± SD) remaining, whereas survival after tRA treatment (10–7 M) was 58.1 ± 4.6%. The survival rate of cells stimulated with 10–6 M tRA and treated with 131I was 20.8 ± 3.4%, similar to that after treatment with the Dex-tRA (10–7 M) combination. These results demonstrate that Dex treatment with reduced tRA (10–7 M) concentrations increases the cytotoxicity after 131I treatment in MCF-7 cells to a level seen in cells treated with a 10-times-higher concentration of tRA.
FIG. 8. Cytotoxicity of 131I after the combination treatment of Dex and tRA in MCF-7 cells. Cells were treated with or without tRA and/or Dex at the indicated concentration for 48 h and then incubated with HBSS containing 0, or 60 μCi/ml Na131I and 0, or 6 μM NaI, respectively, for 6 h. Cells were harvested and used for clonogenic assay. A, Uptake of 131I– by MCF-7 cells. After incubation with 131I, ?-ray activity of the trapped 131I was counted by a Geiger-Mueller counter. The count was normalized to the cell number. B, The survival rate of MCF-7 cells after the 131I treatment. The rate was calculated as a percentage of cell colonies treated with 131I– compared with those treated with only HBSS. Values are means ± SD (n = 4). *, P < 0.0001 when compared with the group of unstimulated MCF-7 cells.
Discussion
NIS expression in breast cancer has been identified as an important target for therapeutic 131I (42, 43). Extensive experience with 131I treatment of thyroid cancer has demonstrated the importance of maximizing the magnitude of iodide uptake and prolonging the period of tumor residence. In the case of thyroid cancer, NIS expression in most tumors is responsive to TSH stimulation (44), and the radiation is retained for days in the tumors, likely due to some organification of the radioiodide. In contrast, no iodide organification has been observed in breast cancer cells in vitro (9). The iodide concentration in breast cancer specimens is significantly lower than that in normal thyroid tissue (45). The primary agent that has been shown to stimulate NIS expression in breast cancer is retinoic acid (9, 11, 14). In limited in vitro and in vivo studies, the RA concentration required for this action is quite high (14).
Enhanced effectiveness of RA stimulation could potentially be achieved by more selective retinoid stimulation and with agents that work synergistically with RA. Our studies have demonstrated that an RAR ?/-specific ligand AGN190168, as well as tRA and 9-cis RA, effectively induced NIS expression and iodide uptake in MCF-7 breast cancer cells, and Dex significantly enhanced and prolonged these inductions, especially with the RAR ?/-ligand. The combination of tRA and Dex synergistically increased the NIS expression and cell-selective cytotoxicity by 131I. In contrast, inductions of iodide uptake by an RAR-selective ligand AGN195183, an RAR-selective ligand AGN194433, and an RXR-selective ligand AGN194204 were relatively modest, and Dex did not significantly enhance the iodide uptake induced by AGN194433 and AGN194204.
tRA is a so-called pan-retinoid agonist, which binds to a broad spectrum of RAR isoforms, , ?, and , but does not bind RXRs (46, 47, 48). 9-cis RA binds to a broader spectrum of retinoid receptors, RARs and RXRs (24, 46). The dissociation constant values of tRA to RARs and 9-cis RA to both RARs and RXRs are similar, with some variation in previous reports (24, 46, 47, 48). Although 9-cis RA can be converted to tRA in vitro and in vivo, the isomerization activity in epithelial cells is much lower than liver cells (49). Our study indicated that tRA, 9-cis RA, and AGN190168, which has approximately 100-times higher affinity for RAR ? and compared with (50), induced iodide uptake and NIS mRNA most effectively in MCF-7 cells, whereas stimulation by isoform-selective retinoid receptor ligands AGN195183 (RAR) and AGN194433 (RAR) were relatively low. These data suggest that RAR?-signaling is important for the NIS induction in MCF-7 cells. An RXR-selective agonist AGN194204, which has 10-times higher affinity for all RXR isoforms compared with 9-cis RA (24), modestly induced iodide uptake and NIS mRNA in MCF-7 cells. Because the RXR agonist has no binding affinity and transactivation activity for RARs (24), NIS is likely to be modestly induced by RXR signaling in MCF-7 cells. A previous report has shown additive or synergistic effects of RAR-ligands with RXR ligands on NIS mRNA and iodide uptake induction in MCF7 cells (11). In contrast, we observed that AGN194204 (pan-RXR ligand) did not significantly enhance the iodide uptake induced by tRA, AGN190168 (RAR ?/-ligand), and AGN194433 (RAR-ligand). Further studies are needed to clarify the mechanism of the differential regulation between RAR and ?/.
Although 13-cis RA has a low affinity for RARs (about 10-times lower than tRA and 9-cis RA) and RXRs (about 100-times lower than 9-cis RA) (47), it stimulates transactivation by RAR, likely after isomerization to tRA and 9-cis RA (51). Glutathione S-transferases effectively catalyze isomerization of 13-cis RA to tRA (52). Expression of one of the glutathione S-transferases, which is associated with resistance to doxorubicin, is reduced in wild MCF-7 cells, compared with less-differentiated breast cancer cells (53). The lower induction of iodide uptake in 13-cis RA-treated MCF-7, compared with tRA, might involve the reduced expression of glutathione S-transferase.
Although our study with isoform-specific retinoid receptor ligands suggests an important role of RAR? in the NIS expression in MCF-7 cells, previous reports demonstrated the abundant expression of RAR and and reduced RAR? in MCF-7 cells (14, 54). tRA treatment significantly increases RAR? mRNA in MCF-7 xenograft tumors (14). Our time course study indicated relatively slow induction of NIS mRNA by AGN190168 (RAR ?/-ligand). An RAR-selective ligand AGN195183 and an RXR-selective ligand AGN194204 modestly induced iodide uptake mediated by NIS. During the early phase of the NIS induction by tRA and 9-cis RA before the induction of RAR?, NIS could be modestly induced through RAR signaling, which would not be activated by AGN190168 (RAR ?/-ligand).
Dex and tRA have been shown to synergistically increase the expression of various genes (19, 20, 55). The mouse mammary tumor virus promoter has a retinoic acid response element and a glucocorticoid response element, both of which are located separately but can be synergistically activated by tRA and Dex without direct receptor interaction (19). In other genes, glucocorticoids regulate various genes at the posttranscriptional level by stabilizing mRNA (36, 37, 38, 39, 40). In this study, we showed that Dex stabilized NIS mRNA and synergistically induced iodide uptake and NIS mRNA with tRA in MCF-7 cells. tRA induces NIS mRNA partially at the transcriptional level (9), and the effect of Dex on tRA-induced NIS mRNA is rapidly observed within 3 h after the beginning of the treatment. Further studies with Dex on the NIS transcription may provide additional insight into the role of Dex on the NIS up-regulation.
Although the iodide uptake and NIS mRNA expression in TSH-stimulated FRTL-5 rat thyroid cells is reduced by tRA (33) and Dex (17, 56), our study indicates that these nuclear receptor ligands synergistically increase the NIS expression in MCF-7 breast cancer cells. In addition, T3 (17) and E2 (18) down-regulate the TSH-induced NIS expression in thyroid cells, whereas these hormones, as well as the estrogen receptor antagonist tamoxifen, do not significantly influence tRA-induced iodide uptake in MCF-7 cells. These observations suggest the differential regulatory mechanism of NIS in thyroid tissue and breast cancer.
The absence of an influence of T3 and E2 on RA-induced NIS expression in breast cancer is important for therapeutic applications. To use systemic radioiodide treatment targeted to breast cancer, iodide uptake in the thyroid gland must be blocked to protect the thyroid from the radioiodide and to maximally concentrate radioiodide into breast cancer. Our in vivo study with MCF-7 xenograft model mice demonstrated that both tRA and T4 significantly reduced iodide uptake in the thyroid gland (Ref. 14 and unpublished data), consistent with the previous in vitro studies (17, 33). Dex will likely also reduce iodide uptake into the thyroid. Tamoxifen is a commonly used agent in breast cancer, and it did not significantly affect the tRA-induced iodide uptake in MCF-7 cells. tRA treatment before radioiodide therapy, therefore, could be used with tamoxifen treatment in estrogen receptor-positive breast cancer.
We and other groups have observed tRA stimulation of NIS expression in MCF-7 cells in vitro (9, 11, 57). We have also reported on significant induction of NIS in vivo by systemic tRA treatment in a transgenic mouse model of breast cancer, murine mammary tumor virus-polyoma virus middle T antigen, as well as a MCF-7 xenograft model (14). Only two of eight breast cancer cell lines tested, however, show RA induction of NIS mRNA (11). Approximately 50% of metastatic thyroid cancer concentrates radioiodine with recombinant TSH after total thyroidectomy (44), yet relatively few normal thyroid cell lines (25, 58, 59), and no thyroid cancer cell lines (60, 61, 62), have NIS activity. The disparity between in vitro and in vivo breast cancer models, therefore, is similar to that which is seen in thyroid cancer.
Dex decreased the EC50 of tRA and RAR ?/-ligand AGN190168 for iodide uptake more than 10 times in MCF-7 cells. Indeed, our in vitro clonogenic assay indicated cell-selective cytotoxicity in MCF-7 cells treated with Dex and reduced concentration of tRA. The addition of Dex has a potential to achieve the NIS induction with a lower dose of tRA in vivo. The combination of Dex and the RAR?/-selective agonist AGN190168 provided a means for longer duration and higher induction of iodide uptake in vitro, suggesting higher efficacy of 131I after the combination treatment.
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