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Clinical Cancer Research Vol. 12, 2038-2048, April 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Physiologic Levels of 2-Methoxyestradiol Interfere with Nongenomic Signaling of 17ß-Estradiol in Human Breast Cancer Cells

Veena Vijayanathan1, Sripriya Venkiteswaran1, Sandhya K. Nair1, Arti Verma1, T.J. Thomas1, Bao Ting Zhu3 and Thresia Thomas2

Authors' Affiliations: Departments of 1 Medicine and 2 Environmental and Occupational Medicine, Environmental and Occupational Health Sciences Institute and The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, and 3 Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina

Requests for reprints: Thresia Thomas, Department of Environmental and Occupational Medicine, 125 Paterson Street, Clinical Academic Building, Room 7092, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903. Phone: 732-235-8458; Fax: 732-235-8473; E-mail: thomasth{at}umdnj.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The purpose of this investigation is to determine the effects of physiologic levels (10-50 nmol/L) of 2-methoxyestradiol (2ME) on the growth of estrogen receptor (ER)–positive breast cancer cells and provide insights into its mechanism(s) of action.

Experimental Design: Using the ER{alpha}-positive breast cancer cells, we studied the effects of 2ME on cell proliferation and cell signaling. Our hypothesis is that 17ß-estradiol (E2) and 2ME can affect shared cell signaling pathways, leading to different outcomes in cell proliferation, depending on the absence/presence of E2.

Results: E2 stimulated the growth of MCF-7 and T-47 D cells and induced Akt phosphorylation, a nongenomic signaling pathway. In the absence of E2, 10 to 50 nmol/L of 2ME enhanced cell growth and Akt phosphorylation. However, in the presence of E2, 2ME inhibited E2-induced cell growth and prevented E2-induced Akt phosphorylation. Confocal microscopic studies showed that 2ME inhibited subcellular distribution of ER{alpha} in response to E2 in MCF-7 and T-47D cells. 2ME also down-regulated E2-induced increases in cyclic AMP and ornithine decarboxylase activity. In addition, treatment of MCF-7 cells with 2ME in the presence of E2 resulted in a decrease in ER{alpha} level by 72 hours. Accelerated down-regulation of ER{alpha} may contribute to growth inhibition in the presence of E2/2ME combinations. In contrast, a concentration of up to 2.5 µmol/L 2ME had no effect on the growth of ER-negative SK-BR-3 cells, either in the presence or absence of E2.

Conclusions: Our results provide evidence for the nongenomic action of 2ME in ER-positive cells. In the presence of E2, 2ME suppressed E2-induced cell growth, Akt signaling, and generation of cyclic AMP, whereas it acted as an estrogen in the absence of E2. The intriguing growth-stimulatory and growth-inhibitory effects of 2ME on breast cancer cells suggests the need for its selective use in patients.

17ß-Estradiol (E2), estrone (E1), and some of its biologically active metabolites not only regulate the physiology of the mammary glands, but also affect the development of breast cancer. Enzymes involved in multiple pathways of biosynthesis and the metabolism of endogenous estrogens contribute to the etiology of human breast cancer (1, 2). Distinct metabolic pathways are responsible for conversion of E2 or E1 to derivatives hydroxylated at the C-2 or C-4 positions (catechol estrogens) or to the 16{alpha} position (16{alpha}-hydroxy-E1; refs. 3, 4). Catechol estrogens are further converted to methoxyestrogens by catechol-O-methyltransferase–mediated methylation. Increased production of 2-methoxyestradiol (2ME) compared with 16{alpha}-hydroxy-E1 has been linked to lower breast cancer risk in humans (5). Consequently, 2ME has been widely viewed as a "good estrogen," exerting growth-inhibitory effects on breast cancer (4). Although serum concentrations of 2ME (25-55 pg/mL) in women are comparable to that of E2 (40-80 pg/mL), as much as 4,000 pg/mL of 2ME has been reported in pregnant women (6).

Studies on the pharmacologic concentrations (1-10 µmol/L) of 2-ME have revealed its antitumor and antiangiogenic effects in preclinical cancer models (7–12). 2ME seems to have increased sensitivity toward rapidly proliferating cancer cells compared with normal cells (13). Antiproliferative and apoptotic effects of 2ME have led to phase I and phase II clinical trials with promising beneficial effects (7, 11). Several mechanism(s), including tubulin depolymerization, up-regulation of p53, up-regulation of death receptor, production of reactive oxygen species, and inhibition of superoxide dismutase have been proposed for its action (7–12). However, pathways such as inhibition of superoxide dismutase remain controversial (14, 15). Dose-dependent mechanistic differences, such as G1 arrest or G2-M arrest of cancer cells have also been observed with 2ME (16). Overall, widely different effects of 2ME do not provide a common pathway or a framework for the action of the compound. In contrast to its antitumor effects, recent studies also indicate that 2ME could act as an estrogen receptor (ER) agonist, stimulating the growth of ER-positive breast tumors (8, 17). This result has fueled a controversy on the "potential and suitability" of 2ME in cancer therapy (18).

The hormonal responses of estrogens are generally mediated by ER{alpha} and ERß, the ligand activated transcription factors that facilitate cell cycle progression and cell proliferation (19). The classical mechanism of action of estrogens is described by ligand-induced conformational changes of ERs, followed by dimerization and increased binding to regulatory regions of estrogen responsive genes. However, recent studies indicate that this classical paradigm of estrogenic action is incomplete and that estrogenic action might involve both genomic and nongenomic components (20, 21). The nongenomic action of E2 involves its interaction with membrane ER, followed by the activation of kinase cascades, either through G proteins or through the coupling of ER with growth factor receptor pathways (20–24). It is possible that the interaction of estrogens with the membrane ERs may not follow the predictions of binding affinity measurements. Although human ER{alpha} and ERß have low binding affinities for 2ME compared with E2, 2ME seems to have antiestrogenic effects on MCF-7 cells (8, 12, 25).

In the present study, we examined the effect of 10 to 50 nmol/L 2ME on several cell signaling variables in ER-positive MCF-7 and T-47D cell lines. For comparison, we also tested the sensitivity of 2ME on an ER-negative cell line, SK-BR-3 (22). As indicators of nongenomic signaling, we examined the effect of 2ME on Akt signaling and cyclic AMP (cAMP) production. Previous studies have reported Akt phosphorylation and increases in the levels of cAMP in response to E2 treatment of breast cancer cells (26–28). As markers of cell proliferation, we examined the effect of 2ME on the activity of ornithine decarboxylase (ODC) and increases in polyamine levels. ODC gene expression and enzyme activity are increased by growth-stimulatory hormones, carcinogens, and tumor promoters (29–31). ODC converts ornithine to putrescine, which is then converted to spermidine and spermine. These polyamines regulate macromolecular functions by virtue of their binding to negatively charged sites, in addition to binding to specific sites in nucleic acids and proteins (29–31). Increases in ODC and polyamine levels are associated with estrogenic action in breast cancer cells (32, 33).

Our results show that 2ME induced Akt phosphorylation in both MCF-7 and T-47D cells in the absence of E2, suggesting its estrogenic activity. However, in the presence of E2, 2ME inhibited Akt phosphorylation in these cells. Confocal microscopic studies showed that the E2-induced pattern of subcellular distribution of ER{alpha} in both cell lines is disrupted by 2ME. Furthermore, 2ME did not have any significant effect on the growth of SK-BR-3 cells at 10 nmol/L to 2.5 µmol/L concentrations, either in the presence or absence of E2.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. MCF-7, T-47D, and SK-BR-3 cell lines were obtained from American Type Culture Collection (Manassas, VA). E2 and 2ME were purchased from Steraloids, Inc. (Wilton, NH). 2ME was further purified by a high-pressure liquid chromatography method (34). The high-pressure liquid chromatography analysis (with UV detection) of the repurified 2ME showed a purity of >99%, and no E2, E1, or other metabolite was detected. In addition, gas chromatography-mass spectroscopy analysis of the trimethylsilylated derivatives of the purified 2ME confirmed the structural identity of 2ME and the absence of E2, E1, or any of the estrogen metabolites/derivatives, after comparing against a library of >60 estrogen derivatives (34). Putrescine, spermidine, spermine, DMEM, phenol red–free DMEM, fetal bovine serum, and anti-ß-actin antibody were obtained from Sigma Chemical Co. (St. Louis, MO). Antibiotics, trypsin, and other additives for cell culture medium were purchased from Invitrogen (Carlsbad, CA). Anti-phospho-Akt and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA). Anti-ER{alpha} antibody was from Santa Cruz Biotechnology, (Santa Cruz, CA). The ERE-linked firefly luciferase reporter gene containing four tandem repeats of 38-mer consensus ERE in pGL3 plasmid (35, 36) was kindly provided by Dr. Carolyn M. Klinge of University of Kentucky. Renilla luciferase plasmid, pGL3 control plasmid, and dual luciferase assay system were purchased from Promega, Madison, WI.

Cell culture. MCF-7 cells were maintained in DMEM containing 10% fetal bovine serum, supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 40 µg/mL gentamicin, 2 µg/mL insulin, 0.5 mmol/L sodium pyruvate, 10 mmol/L nonessential amino acids, and 2 mmol/L L-glutamine. SK-BR-3 cells were maintained in McCoy 5A medium supplemented with 10% fetal bovine serum and 2 mmol/L glutamine. T-47D cells were maintained in RPMI 1640 with serum and antibiotic supplements. All cell lines were grown in phenol red–free DMEM for 7 days prior to experiments (12, 33). This medium contained 10% fetal bovine serum pretreated with dextran-coated charcoal (0.5% Norit A and 0.05% Dextran T-70) to avoid the effects of serum-derived estrogenic compounds.

Cell proliferation. MCF-7 and T-47D cells were seeded in triplicates at a density of 5 x 104 cells/mL/well in 24-well plates. SK-BR-3 cells were seeded at a density of 3 x 104 cells/mL/well. Cells were dosed 24 hours after plating, with required concentrations of E2 and/or 2ME. Cells were re-dosed at 48-hour intervals. After the appropriate treatment periods, live cells were counted using the trypan blue exclusion method, using a hemocytometer.

Western blot analysis. Cells (2.5 x 106/100 mm dish) were washed twice with ice-cold PBS and lysed with the addition of ice-cold lysis buffer [150 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Nonidet P-40, 2 mmol/L EDTA, 50 mmol/L sodium fluoride, 0.2% SDS, 1 mmol/L sodium vanadate, and 1x concentration of a protease inhibitor cocktail (Calbiochem, San Diego, CA)]. Thirty micrograms of protein (determined by the Bradford protein assay) was diluted in 2x SDS-PAGE Laemmli buffer [150 mmol/L Tris-HCl (pH 6.8), 30% glycerol, 4% SDS, 7.5 mmol/L DTT, 0.01% bromophenol blue] and separated on a 10% SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride Polyscreen membrane. After blocking with 5% nonfat milk, membrane was immunoblotted with a 1:100 dilution of the primary antibody. Protein bands were visualized using horseradish peroxidase–conjugated secondary antibody with a chemiluminescence based detection system. To verify equal protein loading, membranes were stripped and reblotted with anti-ß-actin monoclonal antibody. The blots were developed using Kodak XAR Biomax film. Lightly exposed films were scanned using an Epson B4 Scanner and band intensities quantified using the NIH Image J 1.34S program.

Confocal microscopic analysis of ER{alpha}. MCF-7 or T-47 D cells were seeded 5 x 104 cells/well in Labtek four-well slide chamber. After treatment, cells were fixed in 4% paraformaldehyde for 20 minutes at 22°C, rinsed with PBS, permeabilized with 0.1% Triton for 5 minutes. Cells were then blocked in normal goat serum (5%) in PBS followed by incubation with anti-ER{alpha} antibody, and diluted in 2.5% normal goat serum in PBS for 2 hours. Cells were rinsed with PBS and incubated with secondary antibody for 1 hour at 22°C. Cells were then rinsed with PBS and actin was stained by a 2.5% solution of Alexa 546 conjugated to phalloidin in PBS for 20 minutes. Nuclei were stained with 4',6-diamidino-2-phenylindole (~1 nmol/L) solution for 5 minutes. Primary antibody was rabbit-anti-ER{alpha} (sc-7207, Santa Cruz Biotechnology) at 1:100 dilution (24). Antirabbit secondary antibody conjugated to Alexa 488 (green; Molecular Probes/Invitrogen, Carlsbad, CA) was used to detect ER{alpha}. Images were recorded using a Zeiss 510 Laser scanning microscope with a 60x objective at identical intensity settings for all treatment groups. To evaluate nonspecific binding, cells were examined after treatment with fluorescence-labeled secondary antibody alone. No fluorescence was detected in this case.

Measurement of cAMP. MCF-7 cells were plated at a density of 2.5 x 106 cells/100 mm dish. After 24 hours, cells were pretreated with 1 mmol/L 3-isobutyl-1-methylxanthine for 30 minutes and then treated with different concentrations of E2, 2ME, or their combination. Cells were harvested in 500 µL of 0.1 N HCl and cAMP measured according to the manufacturer's instructions (Biomol, Plymouth, PA). cAMP values were normalized against protein levels determined by the Bradford assay.

ODC activity assay. MCF-7 cells (3 x 106 cells/dish) were plated in 100 mm dishes. Cells were treated with 10 nmol/L E2, 2ME, or their combination in triplicate. The ODC assay was done as described previously (30, 33). Briefly, cell pellets were sonicated on ice for 10 seconds in 1 mL of Tris buffer [10 mmol/L Tris-HCl (pH 7.4), 2.5 mmol/L DTT]. After centrifugation, supernatant was used for the assay. Ornithine mix containing 6.5 µCi/mL of 14C-ornithine and 22 mmol/L unlabeled ornithine in 10 mmol/L Tris-HCl (pH 7.4) and pyridoxal-5-phosphate (2 mmol/L) were added to tubes fitted with rubber stoppers with center wells (Kontess Glass Company, Vineland, NJ). Hyamine hydroxide was used to trap 14CO2 and radioactivity determined using a scintillation counter. ODC activity was calculated as nmol/mg protein/h.

Polyamine assay. Polyamine levels were quantified by a previously described procedure using a Hitachi L-7000 high-pressure liquid chromatography unit (30). MCF-7 cells were plated at a density of 3 x 106 cells/100 mm dish, treated with E2 or 2ME for 24 or 48 hours. Cells were homogenized in 8% sulfosalicylic acid and centrifuged. Polyamines were converted to dansyl derivatives and separated on a C18 analytic column with an acetonitrile-water gradient, and quantified by a fluorescence detector. 1,6-Diaminohexane was used as the internal standard.

Transient transfections. Cells (1.75 x 105) were seeded in 24-well plates. After 16 hours, cells were transfected with 0.5 µg of 4-ERE-linked firefly luciferase reporter plasmid and 0.05 µg of renilla plasmid in 100 µL of transfection solution containing LipofectAMINE (37). Transfection solution was removed after 5 hours, and cells allowed to recover for 18 to 24 hours. Cells were then stimulated with E2, 2ME, or their combination. Cells were harvested at 6 or 24 hours after treatment, and luciferase activity assayed with a dual luciferase reporter assay system using a TD-20/20 Luminometer. Reporter activity was normalized for each sample. Normalized luciferase activity = firefly luciferase activity / renilla luciferase activity.

Statistical analysis. All experiments were repeated at least thrice. All blots shown are representative of three separate experiments with mean of fold changes in intensities reported in the text. Statistical difference between control and treatment groups was determined by one-way ANOVA followed by Dunnet's test (GraphPad Prism Software program, San Diego, CA). P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of 2ME on the growth of MCF-7, T-47D, and SK-BR-3 cells in the absence or presence of E2. Figure 1A and B shows the effects of different concentrations of 2ME on MCF-7 and T-47D cell growth in the presence or absence of 10 nmol/L E2. In Fig. 1, 100% control in each graph represents two sets of control cells: one with E2 (for the determination of the effect of 2ME in the presence of E2) and the other with vehicle alone (for the determination of effect of 2ME in the absence of E2). In other words, 100% control in the presence of E2 includes growth stimulation in the presence of E2. E2 stimulated the growth of MCF-7 and T-47D cells by 2.4-fold and 2.1-fold, respectively. Figure 1A and B (insets) show expanded versions of the low-concentration effect of 2ME. In the absence of E2, 10 to 500 nmol/L concentrations of 2ME showed a proliferation effect, whereas higher concentrations showed growth inhibition. However, all 2ME concentrations inhibited cell growth in the presence of 10 nmol/L E2. In the absence of E2, the IC50 (concentration required for 50% of growth inhibition) of 2ME was 1,120 ± 70 nmol/L, whereas it was 180 ± 30 nmol/L in the presence of E2. For the T-47D cells, IC50 of 2ME was 1,950 ± 150 nmol/L in the absence of E2, and 450 ± 75 nmol/L in the presence of E2. Thus, at physiologically relevant concentrations, 2ME showed a significant growth-stimulatory effect in the absence of E2, and a growth-inhibitory effect in the presence of E2.


Figure 1
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  		Fig. 1. Effect of 2ME on the growth of ER-positive (MCF-7 and T-47D) and ER-negative (SK-BR-3) breast cancer cells. MCF-7 cells (A), T-47D (B), and SK-BR-3 (C) were plated in 24-well culture plates in phenol red–free DMEM. After 24 hours, cells were treated with different concentrations of 2ME with ({circ}) or without (bullet) 10 nmol/L E2. Cells were re-dosed at 48 hours and counted after 4 days of treatment, using a hemocytometer with trypan blue exclusion. Insets, expanded plots of the effects of 2ME at physiologically relevant concentrations. Note that 100% control in each graph represents two sets of controls: one with E2 (for the determination of the effect of 2ME in the presence of E2) and the other with vehicle alone (for the determination of effect of 2ME in the absence of E2). Points, mean from three triplicate experiments; bars, ± SD; *, statistically significant compared with the control (P < 0.05).

 
Figure 1C shows the effects of 2ME on the growth of SK-BR-3 cells. For these experiments, SK-BR-3 cells were grown under the same growth conditions as that used for MCF-7 cells. 2ME did not have a significant effect on the growth of these cells up to 2.5 µmol/L concentration, either in the presence or absence of E2.

In other experiments, we tested whether antiproliferative effects observed in the presence of E2 + 2ME is similar to the effect of a high dose of E2. We therefore compared the effect of 10 and 60 nmol/L E2 (equivalent to 10 nmol/L E2 + 50 nmol/L 2ME) on MCF-7 cell growth. We found that both 10 and 60 nmol/L E2 exerted a similar growth stimulation (2.3 ± 0.1-fold increase compared with the control) on day 4 of treatment. This is not surprising because the antiproliferative effect of high-dose E2 is generally observed at micromolar concentrations (38).

Effect of 2ME on Akt phosphorylation in the absence or presence of E2. Because the Akt-mediated phosphorylation pathway plays a key role in cell growth regulation, we determined the effects of E2 on Akt phosphorylation in MCF-7 cells. Sixteen hours after seeding, cells were treated with 10 nmol/L E2 and harvested at 3, 5, 15, 30, and 60 minutes after treatment. Cellular extract was analyzed by Western blots using an antibody specific to phospho-Akt Ser473. Subsequently, blots were stripped and reblotted with an antibody that recognized total Akt. Our results (Fig. 2A and B ) showed a significant (2.7-fold) increase in Ser473 phosphorylation of Akt in cells treated with E2 for 5 minutes, compared with the control. Phospho-Akt levels decreased after 30 minutes of E2 treatment. However, there was no significant change in the level of total Akt under this treatment condition. The blot was stripped further and probed with an anti-ß-actin antibody to ensure equal loading.


Figure 2
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  		Fig. 2. Effects of E2 on the phosphorylation of Akt at Ser473 site. MCF-7 cells were treated with 10 nmol/L E2 for different time periods. Cells were harvested and cellular extract analyzed by Western immunoblotting using an anti-phospho-Akt antibody, specific to Ser473 phosphorylation (A). Densitometric quantification is given in (B). Similar results were obtained in three separate experiments. *, statistical significance compared with the control. Error bars represent ± SD.

 
We also examined the effect of E2 on Akt phosphorylation in MCF-7 cells using an antibody that recognized Thr308 phosphorylation site. Thr308 phosphorylation also showed similar kinetics with a maximal induction of 2.4-fold at 5 minutes of E2 treatment and a significant decrease from the maximal levels by 30 minutes of treatment (results not shown).

We next examined the effect of 2ME on Akt phosphorylation using the antibody that recognized the Ser473 site. MCF-7 cells were treated with 10 nmol/L E2 in combination with 10, 25, or 50 nmol/L 2ME for 5 minutes. Our results showed that 10 nmol/L of E2 induced a 3-fold increase in phospho-Akt levels (Fig. 3A ). 2ME caused a dose-dependent decrease in E2-induced phospho-Akt level. In the presence of 50 nmol/L 2ME, the phospho-Akt band intensity was reduced to a level similar to that of the control. In contrast, total Akt level remained unchanged under all treatment conditions, as determined by stripping the blot and reprobing it with an antibody that recognized total Akt. Thus, 2ME disrupted the E2-mediated signaling process, leading to a decrease in Akt phosphorylation.


Figure 3
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  		Fig. 3. Effects of 2ME on Akt phosphorylation in the presence and absence of E2. Cells were treated with E2 + 2ME (A) or 2ME alone (B) for 5 minutes. Cells were harvested and analyzed by Western blotting using an anti-phospho-Akt antibody, specific for Ser473 phosphorylation, followed by an antibody specific to total Akt. Stripping and reblotting with an anti-ß-actin antibody showed equal loading and transfer.

 
We next examined the effect of 2ME on Akt phosphorylation in MCF-7 cells in the absence of E2. Cells were treated with 10, 25, and 50 nmol/L 2ME and were harvested 5 minutes after treatment. Our results showed that 2ME treatment caused a dose-dependent increase in Akt phosphorylation (Fig. 3B). Thus, 2ME could initiate nongenomic signaling in the absence of E2. However, this effect was not efficient at 10 nmol/L 2ME, but was effective at 50 nmol/L, resulting in an Akt phosphorylation level 2.5-fold higher than that of control (P < 0.05). In the case of E2, even 0.1 nmol/L E2 showed increased Akt phosphorylation (results not shown). These results showed that the ability of 2ME to initiate membrane signaling was lower than that of E2.

Figure 4 shows the effects of E2, 2ME or their combinations on Akt phosphorylation in T-47D cells. E2 induced Akt phosphorylation by 2.5-fold, 5 minutes after its addition (Fig. 4A). Phospho-Akt levels decreased by 60 minutes of E2 treatment. E2-induced Akt phosphorylation was reduced by cotreatment of cells with E2 and 2ME (Fig. 4B). There was a dose-dependent decrease in phospho-Akt levels when E2 was present with 10, 25, and 50 nmol/L of 2ME. In contrast, there was an increase in Akt phosphorylation when cells were treated with 2ME alone, particularly at the 50 nmol/L concentration (Fig. 4C).


Figure 4
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  		Fig. 4. Effects of E2, 2ME or their combinations on Akt phosphorylation in T-47D cells. A, effect of E2 on Akt phosphorylation. Cells were treated with 10 nmol/L E2 for 3, 5, 10, 15, 30, and 60 minutes. Cells were harvested and analyzed by sequential Western blots, using antibodies specific to phospho-Akt, total Akt, and ß-actin. B, cells were treated with 10 nmol/L of E2 and 10, 25, and 50 nmol/L 2ME for 5 minutes. Cell lysate was analyzed by Western blots using antibodies specific to phospho-Akt, Akt, and ß-actin. C, cells were treated with 10, 25, and 50 nmol/L 2ME for 5 minutes and the cell lysate was analyzed by Western blots.

 
Subcellular localization of ER{alpha} by confocal microscopy. Because Akt phosphorylation by E2 and 2ME is likely to be mediated by membrane ER, we next examined the intracellular distribution of ER{alpha} in MCF-7 and T-47D cells and changes in its distribution in response to E2, 2ME, or their combination. Time course experiments at 5, 15, 30, 60, and 120 minutes showed that E2 treatment produced major changes in the distribution of ER{alpha} after 30 and 60 minutes of treatment. Figure 5A shows representative results of the effects of 2ME on ER{alpha} distribution in the absence or presence of E2 in MCF-7 cells. The majority of ER{alpha} in untreated cells seemed to be evenly distributed in the nucleus, with mild cytoplasmic and membrane staining. Arrows point to regions that are magnified in the side panels and illustrate changes in ER{alpha} membrane staining. At 30 minutes of E2 treatment, there was a marked redistribution of ER{alpha} with focal accumulation in specific sites in the nucleus. In contrast, cells treated with 10 nmol/L of 2ME alone showed intense and even distribution of ER{alpha} in the nucleus. In cells treated with E2 + 10 nmol/L 2ME, ER{alpha} remained intensely stained in the nucleus and showed a punctate pattern without focal accumulation in nuclear sites. Similar results, showing the inhibition of the subcellular distribution of ER{alpha} were obtained when E2 was administered in combination with 25 nmol/L 2ME, although 25 nmol/L 2ME alone showed focal accumulation comparable to that of 10 nmol/L E2-treated cells. In contrast, cells treated with 10 nmol/L E2 + 25 nmol/L 2ME showed diffuse and even ER{alpha} nuclear staining pattern. These results show that that 2ME could disrupt the E2-induced subcellular distribution of ER{alpha}.


Figure 5
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  		Fig. 5. Effects of E2 and 2ME on the distribution of ER{alpha} in MCF-7 and T-47D cells. A, MCF-7 cells were treated with E2, 2ME, or their combinations as indicated in the ligand. ER{alpha} was detected by immunocytochemical staining, using an anti-ER{alpha} antibody, followed by a secondary antibody labeled with Alexa 488. Phalloidin staining of filamentous actin shows cytoskeletal margins. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Controls without primary antibody did not show any staining. Similar results were obtained in three separate experiments in triplicate. Images were recorded using a Zeiss 510 laser scanning microscope with 150 x magnification. Arrows, regions magnified in the side panels illustrating changes in membrane distribution of ER{alpha}. B, effects of E2, 2ME, or their combinations on the distribution of ER{alpha} in T-47D cells. Experiments were conducted as described for MCF-7 cells, although actin was better stained in the T-47D cells. Membrane ER{alpha} was visible as yellow by the merging of ER{alpha} (green) and actin (red) at the cell periphery, in E2-treated samples, but not in samples treated with E2 and 2ME. Representative results from three separate experiments are presented.

 
Figure 5B shows the confocal microscopic images of the intracellular distribution of ER{alpha} in T-47D cells in the presence of E2 and 2ME as single agents and in combination. The results of this study showed cytoplasmic and nuclear ER{alpha} in the absence of E2. A redistribution of ER{alpha} including membrane localization, was found after cells were treated with E2. When 2ME was present with E2, ER{alpha} localized in the nucleus, and less frequently on the membrane sites. Because MCF-7 and T-47D cell lines behaved similarly in growth response, Akt phosphorylation and ER{alpha} distribution, we next focused our studies on the MCF-7 cell line.

Effect of E2 and 2ME on the generation of cAMP in MCF-7 cells. We tested the ability of E2 to induce cAMP in MCF-7 cells and the effect of 2ME on E2-mediated cAMP production. Cells were treated with E2 or E2 + 2ME for different time periods and were harvested. Cells were lysed and assayed using a cAMP enzyme immunoassay kit. A 3-fold increase in cAMP was detected 5 minutes after the addition of E2 (Fig. 6 ). The maximal value of cAMP was ~ 200 pmol/mg protein in the presence of E2. The presence of 10 nmol/L 2ME inhibited E2-induced increase in cAMP at 5 minutes of treatment. The increase in cAMP levels after E2 treatment decreased by 30 minutes, but remained higher than that of the control. However, 10 nmol/L 2ME alone did not have a significant effect on cAMP levels at any time point. On the other hand, 50 nmol/L of 2ME increased cAMP levels by 2-fold at 5 minutes after treatment (results not shown). These results further showed nongenomic signaling by E2 and its inhibition by 2ME.


Figure 6
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  		Fig. 6. Effects of 2ME on the generation of cAMP. Cells were treated with 10 nmol/L E2, 10 nmol/L E2 + 10 nmol/L 2ME, or 10 nmol/L 2ME for different time periods. Cells were harvested and cAMP extracted and measured as per the manufacturer's protocol, using a cAMP measurement kit from Biomol. Columns, mean from three triplicate experiments; bars, ±SE. *, P < 0.01 compared with the controls; **, P < 0.01 compared with E2-treated samples.

 
Effects of E2 and 2ME on ODC activity in MCF-7 cells. Figure 7 shows the effects of E2 and 2ME on ODC activity in MCF-7 cells, 24 hours after treatment. E2-induced a 2-fold increase in ODC activity and this increase was down-regulated by cotreatment with 10 and 50 nmol/L of 2ME. In the absence of E2, 50 nmol/L of 2ME significantly increased ODC activity. These results showed that E2-induced ODC activity was suppressed by 10 to 50 nmol/L of 2ME, although 2ME could increase ODC activity in the absence of E2. We also determined the activity of other enzymes in the polyamine biosynthetic and catabolic pathways in MCF-7 cells treated with 2ME in the presence and absence of E2. Our results showed that 2ME had no effect on S-adenosylmethionine decarboxylase and spermine/spermidine acetyl transferase activities.


Figure 7
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  		Fig. 7. Effect of 2ME on ODC activity in the presence and absence of E2. Cells were plated in 100 mm dishes and treated with different concentrations of 2ME. Cells were harvested and ODC activity determined by measuring the release of CO2 during the decarboxylation of 14C-ornithine. Columns, mean; bars, ±SE from three triplicate experiments. *, P < 0.01 compared with the controls; #, P < 0.01 compared with the E2-treated group.

 
Effects of E2 and 2ME on polyamine levels in MCF-7 cells. Because E2-induced ODC activity was down-regulated by 2ME, we examined whether 2ME altered polyamine levels in MCF-7 cells. Cells were harvested at 24 and 48 hours of treatment. Table 1 shows polyamine levels in the presence and absence of E2. E2 induced a significant increase in the levels of putrescine, spermidine, and spermine both at 24- and 48-hour time points. In cells treated with E2 + 10 nmol/L 2ME, there was a trend toward decreases in putrescine, spermidine, and spermine levels at the 24-hour time point, compared with E2-treated samples. In cells treated with E2 + 50 nmol/L 2ME, there was a significant down-regulation of all three polyamines, both at the 24- and 48-hour time points, compared with E2-treated samples. These results show that the antiproliferative effect of 2ME involves a general down-regulation of polyamine levels. In the absence of E2, 50 nmol/L of 2ME caused a significant increase in putrescine levels. In contrast, there was no significant increase in spermidine and spermine levels due to 2ME treatment.


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  		Table 1. Effect of 2ME on polyamine levels in MCF-7 cells

 
Effect of E2 and 2ME on ER{alpha} levels. Ligands such as E2, tamoxifen, and ICI 182780 are known to modulate the steady-state levels of ER{alpha}, due to alterations in the degradation of ER{alpha} (39, 40). Therefore, we examined whether the down-regulation of ER{alpha} levels was associated with antiproliferative effects of 2ME in the presence of E2. Cells were treated with E2, 2ME, or their combination and harvested at 24, 48, and 72 hours. ER{alpha} levels were determined by Western blot analysis using an anti-ER{alpha} antibody. Our results showed a marginal change in ER{alpha} levels after 24 hours of treatment with E2, 2ME, or their combination (Fig. 8 ). However, at 48 hours of treatment, there was a 50% reduction in ER{alpha} in cells treated with E2, whereas in cells treated with 2ME alone, ER{alpha} levels remained similar to that of untreated controls. In contrast, in cells treated with E2 + 2ME, there was a decrease in ER{alpha} levels from 24 to 72 hours. By 72 hours of treatment, ER{alpha} level was reduced by 95% compared with the control. E2-induced down-regulation of ER{alpha} is believed to counter the estrogenic response, providing a feedback control (39, 40). However, when E2 is combined with 2ME, the degradation of ER{alpha} seems to be enhanced. Increased down-regulation of ER{alpha} may contribute to growth inhibition in the presence of E2/2ME combinations.


Figure 8
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  		Fig. 8. Effect of E2 and 2ME on ER{alpha} levels as single agents and in combination. Cells were treated with E2, 2ME, or their combinations, and changes in ER{alpha} levels were determined by Western blot analysis, using an anti-ER{alpha} antibody. Cells were harvested for Western blots after 24, 48, or 72 hours of treatment. After probing with anti-ER{alpha} antibody, membranes were stripped and reprobed with anti-ß-actin antibody to ensure equal loading and transfer.

 
Effects of E2 and 2ME on ERE-reporter activity. We next examined whether genomic responses such as E2-induced ERE reporter gene activation was affected by E2, 2ME, or their combination. Cells were plated in 24-well plates and transfected with 0.5 µg of ERE-linked firefly luciferase plasmid and 0.05 µg of renilla luciferase, using LipofectAMINE as the transfection agent. Cells were 80% to 90% confluent at the time of transfection and maintained in the transfection medium for 5 hours. Cells were stimulated with E2, 2ME, or their combination 24 hours after changing the transfection medium. Cells were harvested and reporter gene activity was assayed at 6 and 24 hours after treatments. Results are presented in terms of fold induction of luciferase activity compared with samples not treated with E2 (Fig. 9 ). Our results show that E2 stimulated ERE reporter gene activity 2- to 3-fold higher than that of the control. The presence of 2ME suppressed E2-induced reporter gene activity by 38% (24 hours), compared with E2-treated group. However, 2ME was only mildly (1.4-fold) active in stimulating ERE reporter gene activity compared with the control. These results show that 2ME can suppress the genomic responses of E2, although this suppression is not as effective as the nongenomic responses.


Figure 9
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  		Fig. 9. Effects of E2, 2ME, or their combinations on the genomic effect of reporter gene activity. Cells were cotransfected with ERE-linked firefly luciferase plasmid and a renilla luciferase plasmid. Subsequently, cells were treated with E2, 2ME or their combinations. Cells were harvested after 6 and 24 hours of treatment and luciferase activity was determined by dual luciferase assay system. A control plasmid pGL3 was also used to examine E2 response. There was no change in fold induction of luciferase activity due to treatments when cells were transfected with pGL3 control plasmid and renilla luciferase plasmid. Columns, mean; bars, ±SD from three separate experiments.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Results presented in this report show that 2ME could induce contrasting estrogenic or antiestrogenic behavior in ER-positive MCF-7 and T-47D cell lines, depending on the absence/presence of E2 in the growth medium. In the absence of E2, physiologic concentrations of 2ME enhanced nongenomic signaling events, such as the phosphorylation of Akt. However, E2-induced phosphorylation of Akt was suppressed by 2ME, contributing to its antiestrogenic effect. Further analysis of the effect of 2ME on E2-induced increase in cAMP levels in MCF-7 cells showed that 2ME suppressed the increase in cAMP, providing support for the idea that nongenomic responses of E2 are suppressed by 2ME. Consistent with the growth-stimulatory effect of 2ME, ODC activity was increased by 2ME alone. In contrast, E2-induced ODC activity was suppressed by 2ME. Polyamine levels showed a varied response; putrescine levels showing an increase in the presence of 50 nmol/L 2ME, whereas spermidine and spermine levels were either not affected or decreased. E2-induced increases in polyamine levels were down-regulated by 10 to 50 nmol/L of 2ME, consistent with the growth-inhibitory effects of 2ME under these conditions. Furthermore, 2ME suppressed E2-induced genomic estrogenic response of ERE reporter gene activation, 24 hours after treatment. Long-term treatment (72 hours) showed that 2ME reduced steady-state levels of ER{alpha} in the presence of E2. Thus, the estrogenic and antiestrogenic effects of 2ME have multiple components, resulting in growth stimulation or growth inhibition under different treatment conditions. It is also important to note here that the studies of Liu and Zhu (9) showed that 2ME was not transformed to estrogenic metabolites in MCF-7 cells. The antiestrogenic effect of 2ME is also consistent with our recent findings on the effect of 2ME on the phosphorylation of activating transcription factor-2, which is involved in E2-induced transcription of cyclin D1 (12).

Our confocal microscopic results show the ability of 2ME to alter E2-induced shuttling of ER{alpha} between different sites within the cell. After 30 minutes of E2-treatment, there was a movement of ER{alpha} to the periphery of the cells and a focal accumulation in specific regions of the nucleus. Ligand-induced redistribution of ER{alpha} and other nuclear receptors has been reported previously (41). This redistribution is believed to be due to the dissociation of native complexes and formation of new complexes pertinent to transcriptional activation, receptor recycling, and degradation. The combination of E2 + 2ME blocked this redistribution. Confocal microscopic studies of Song et al. (24) showed that E2 induced shuttling of ER{alpha} from the nucleus to the membrane as a part of the association of ER{alpha} with growth factor receptors and adaptor proteins. Membrane ER{alpha} level is estimated to be between 2% and 20% of the total ER{alpha} (20, 42) and changes in this distribution may affect the signaling pathways initiated by E2.

Current evidence suggests the participation of membrane ERs, G proteins such as GPR30, cell surface growth factor receptors, as well as the modulator of nongenomic activity of ER (also known as PELP1, for proline-glutamic acid– and leucine-rich protein 1) in nongenomic E2 signaling (20–24). Although the measured relative binding affinity of 2ME for ER{alpha} is only ~1% to 2% of that of E2, correlating to transcriptional effects (9, 10), this may not represent the true affinity of the compound for a multiprotein complex on the cell surface. The conformational flexibility of ER{alpha} in the presence of different ligands and EREs (43, 44) suggests that it can easily assume different conformations; perhaps an agonist-like conformation in the presence of 2ME alone, and an antagonist conformation in the presence of E2/2ME combinations. For example, a novel mode of binding of ER{alpha} has been reported for a series of estrogenic ligands with low relative binding affinities, but potent biological activity (45). It is possible that the conformation of ER{alpha} provoked by E2/2ME combination might be amenable to facile degradation compared with the conformation induced by 2ME alone.

2ME is capable of initiating cell signaling processes including Akt phosphorylation, ODC activation, and production of cAMP in MCF-7 cells. However, not all the variables follow a similar dose-response, suggesting that multiple pathways might be involved. 2ME (10 nmol/L) inhibited E2-induced DNA synthesis and down-regulated cyclin D1 (12). E2-induced cAMP was also down-regulated by 10 nmol/L 2ME. However, maximal down-regulation of Akt phosphorylation was only found at 50 nmol/L 2ME. It seems that E2 signaling is inhibited by 2ME as it encounters the multiprotein receptor system containing E2, ER{alpha}, and other proteins. GPR30 may not be sufficient to support the action of 2ME because significant growth-stimulatory effects were not observed in our studies of SK-BR-3 cells, although it is reported to express this G protein (23).

Whereas the generation of cAMP on MCF-7 cells has been associated with growth stimulation and growth inhibition, these effects vary with the intensity and duration of cAMP response (46). In the current studies, E2-induced cAMP was suppressed by 2ME under conditions of growth inhibition. Although it is well known that cAMP levels regulate cell growth, the pathways affected in breast cancer cells are not clear. One possibility is that cAMP induced the up-regulation of amphiregulin, an epidermal growth factor–like growth factor, that binds and activates the EGF receptor (47). This pathway includes the activation of protein kinase A and the phosphorylation of cAMP-responsive element binding proteins. A decrease in cAMP levels could therefore lead to a decrease in cAMP-responsive element binding protein activation, associated with the down-regulation of cyclin D1 and cell growth inhibition (48).

E2-induced Akt phosphorylation is reported to be due to the activation of phosphoinositide-3-kinase in breast cancer cells (26, 49). The mechanism of activation of the phosphoinositide-3-kinase pathway may involve E2-induced Ras signaling and/or direct interaction of ER{alpha} with the p85 subunit of phosphoinositide-3-kinase (49). Akt activation is reported to stimulate multiple cell survival pathways, including the phosphorylation of BAD, preventing apoptotic signaling and contributing to cell proliferation response (26, 49). Akt-mediated phosphorylation of GSK-3ß is also known to regulate cyclin D1 stability and nuclear localization (50). E2 is also known to enhance the activity of other kinase cascades (51) and characterization of E2-induced genomic and nongenomic responses contributing to cell growth regulation remains far from complete. It is also interesting to note that other compounds, such as resveratrol, induced the expression of progesterone receptor gene in MCF-7 cells when used as a single agent, but the expression of the progesterone receptor gene was suppressed when resveratrol was used in combination with E2. The mixed agonist/antagonist action of resveratrol was also observed in vivo in mammary tissues of animal models of breast cancer (52).

A recent study showed that E2-induced generation of cAMP was involved in E2-induced activation of ODC gene expression (53). Previous studies have shown that E2 increased ODC mRNA expression at 8 hours of treatment (30). Thus, E2 may have genomic and nongenomic effects on the expression of ODC gene and ODC activity. Although 50 nmol/L of 2ME increased ODC activity and enhanced putrescine levels, whereas spermidine and spermine levels were not enhanced. This result indicates a branching point in E2-induced signaling compared with 2ME-induced signaling. Polyamines are regulated by transcriptional and posttranscriptional regulation of biosynthetic and catabolic enzymes as well as membrane-mediated influx and efflux pathways (29, 54). A microarray study on multiple myeloma cell lines showed a decrease in spermidine synthase in cells treated with 2ME (55). However, these down-regulatory responses of 2ME might have been compensated in the long-term, resulting in cell proliferation after treatment with 10 to 50 nmol/L of 2ME for 4 days.

The results of the present study might have implications on the clinical trials for developing 2ME as a potential therapeutic agent for breast cancer. The mixed agonist/antagonist activity of 2ME in ER-positive cells may also account for the discrepancy between different studies contributing to the ongoing controversy on the potential clinical usefulness of 2ME (18). Investigators have usually focused on either the agonist activity in the absence of E2 or the effects of 2ME at micromolar concentrations where apoptotic action is the dominant effect (7, 14, 17). The observation of growth-stimulatory effects of 2ME in MCF-7 nude mice xenografts in the absence of E2 has led Sutherland and collaborators to conclude that 2ME is inappropriate for the evaluation for breast cancer therapy (17). This conclusion was questioned by investigators involved in the development of 2ME for clinical use in breast cancer (18). A large body of data including clinical studies on 170 patients indicates the safety of the compound in humans (56). 2ME also showed synergistic growth-inhibitory action on breast cancer cells when combined with microtubule-disrupting agents, paclitaxel, or vinorelbine (57).

In summary, our results show that 2ME could exert estrogenic or antiestrogen-like action in the same cell line depending on the absence/presence of E2. 2ME inhibited E2-induced nongenomic responses such as Akt phosphorylation and cAMP signaling as well as E2-induced transcription of the ERE-linked reporter gene. The ER{alpha} protein level was also altered in the presence of E2/2ME combinations. These different effects might contribute to the ability of 2ME to suppress E2-induced cell growth. The reduction of ODC activity and polyamine levels following 2ME treatment may also contribute to the antiproliferative activity of 2ME in the presence of E2. In contrast, MCF-7 and T-47D cells responded to 2ME like an estrogen under conditions of estrogen deprivation. Confocal microscopic studies further support the differential effects of 2ME and E2 + 2ME on subcellular distribution of ER{alpha} in these cells. Because 2ME is in clinical trials, our results call for caution in the administration of 2ME to patients with breast cancer, and yet offer the possibility that 2ME might have beneficial anticancer effects in subsets of patients.


   Acknowledgments
 
We thank Dr. Carolyn M. Klinge of the University of Kentucky (Lexington, KY) for generously providing the ERE-luciferase plasmid construct and Dr. Zui Pan of Robert Wood Johnson Medical School (Piscataway, NJ) for her help in the confocal microscopy studies.


   Footnotes
 
Grant support: NIH grants CA42439, CA80163, and CA73058 from the National Cancer Institute, and ES05022 from the National Institute of Environmental Health Sciences (NIEHS Center of Excellence).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: V. Vijayanathan and S. Venkiteswaran contributed equally to this work.

Received 10/ 4/05; revised 1/26/06; accepted 2/ 1/06.


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 Materials and Methods
 Results
 Discussion
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