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Research Article

Calcitriol inhibits Ether-à go-go potassium channel expressionand cell proliferation in human breast cancer cells

Rocío García-Becerraa, Lorenza Díaza,⁎, Javier Camachob, David Barreraa,David Ordaz-Rosadoa, Angélica Moralesa, Cindy Sharon Ortizc, Euclides Avilaa,Enrique Bargallod, Myrna Arrecillase, Ali Halhalia, Fernando Larreaa

aDepartment of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15,Tlalpan 14000 México, D.F., MexicobDepartment of Pharmacology, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional,Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco 07360, México, D.F., MexicocDepartment of Pathology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15,Tlalpan 14000 México, D.F., MexicodDepartment of Breast Tumors, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Tlalpan 14080, México, D.F., MexicoeDepartment of Pathology, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Tlalpan 14080, México, D.F., Mexico

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +52 5 556 55 98E-mail address: lorenzadiaz@gmail.com (L.Abbreviations: DCIS, ductal carcinoma in sit

permeation; VDR, vitamin D receptor

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.11.008

A B S T R A C T

Article Chronology:

Received 29 June 2009Revised version received10 November 2009Accepted 12 November 2009Available online 20 November 2009

Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is

known regarding the molecular basis of this process in breast carcinoma. Ether-à-go-go (Eag1)potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Sincecalcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factorsantagonically regulate cell cycle progression, we investigated a possible regulatory effect ofcalcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferativeactivity of this hormone in human breast tumor-derived cells. RT real-time PCR andimmunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor(VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation,which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistryand Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductalcarcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal

epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferativeeffects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment andprevention.

© 2009 Elsevier Inc. All rights reserved.

Keywords:

VDRTEI-9647Breast cancerVitamin DK+ channelsAstemizole

59.Díaz).u; Eag1, Ether à go-go potassium channel 1; IDC, invasive ductal carcinoma; LP, lymphovascular

r Inc. All rights reserved.

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Introduction

Normal mammary gland functionally expresses the enzymesinvolved in generation and degradation of calcitriol (CYP27B1and CYP24A1, respectively) and the vitamin D receptor (VDR),which acts as a ligand-dependent transcription factor [1,2].Disruption of VDR signaling in the breast gland is associatedwith increased incidence of preneoplastic lesions and acceleratedbreast tumor development [3,4]. In addition, epidemiological,clinical and animal studies have suggested that adequate vitaminD status is important for breast cancer prevention [5-8]. Calcitriol,the most active metabolite of vitamin D, exerts antiproliferativeeffects associated with an increase in G0/G1 arrest, induction ofapoptosis and promotion of cell differentiation [9,10]. Moleculartargets of calcitriol are still under intense investigation and at thepresent time clinical relevant biomarkers known to be modulatedby this hormone in human breast cancer cells include BRCA1, p21,p53, c-Myc, and cyclin D1 [11]. The effects of calcitriol are notrestricted to estrogen-dependent cells, suggesting that otherfactors may be involved in calcitriol-mediated antiproliferativeeffects [12]. Considering the latter and that calcitriol displaysantineoplastic effects, while the potassium channel Ether-à-go-go(Eag1) promotes proliferation and tumor progression [13], wehypothesized that Eag1 might be a potential target for calcitriol. Infact, calcitriol and Eag1 show antagonistic effects upon cell cycleprogression and oxygen homeostasis regulation [14,15]. Expres-sion of Eag1 shows restricted distribution in healthy tissues, but itis abundantly expressed in many cancer cell lines and primarytumors [16-18]. Moreover, its expression has been related tooncogenesis and proliferation [13], and transfection of Eag1 intomammalian cells confers them a transformed phenotype, withgrowth abilities in the absence of serum, no contact inhibition, andaggressive behavior when injected into immunodepressed mice[17]. Therefore, the aim of the present study was to investigate theeffects of calcitriol upon Eag1 expression, as a mean to uncovernew molecular events involved in the antiproliferative activity ofcalcitriol in primary cell cultures obtained from breast tumors. Inaddition, immunohistochemistry of both VDR and Eag1 wasperformed in different kinds of breast tumors in order to showan overview of their distribution and abundance.

In this study we used a mixed cell population directly derivedfrom primary breast tumors in order to keep as close as possiblethe tumoral in vivomicroenvironment, as well as to avoid artifactsdue to genotypic and phenotypic drifts usually seen in establishedcell lines [19]. In addition, this approach allowed us to compareresults obtained from benign fibroepithelial tumors, cancer tumorsand a cell line.

Materials and methods

Reagents

Culture media, fetal bovine serum (FBS), Trizol and the oligonu-cleotides for real-time polymerase chain reaction (PCR) were fromInvitrogen (CA, USA). The TaqMan Master reaction, TaqManprobes, capillaries, probes, reverse transcription (RT) system andthe cell proliferation assay (XTT) were from Roche (Roche AppliedScience, IN, USA). The VDR antagonist (23S)-25-dehydro-1-

hydroxyvitamin D3-26,23-lactone (TEI-9647) and 1α,25-dihy-droxycholecalciferol (calcitriol) were kindly donated from TeijinPharma Limited (Tokyo, Japan) and Hoffmann-La Roche Ltd.(Basel, Switzerland), respectively. Astemizole was obtained fromSigma Chemical Co. (MO, USA). The antibodies were purchasedfrom Santa Cruz Biotechnology Inc., (CA, USA), Invitrogen, Dako(CA, USA), and Cell Marque (TX, USA). The two antibodies for Eag1were kindly provided by Dr. Walter Stühmer (Max-PlanckInstitute, Göttingen, Germany). 3,3′-Diaminobenzidine tetrahy-drochloride substrate kit (DAB) was from Dako.

Human tissues

Sampleswere obtained at the Instituto Nacional de Cancerología inMexico City. The protocol was approved by the institutionalHuman Research Ethics Committee and written informed consentwas obtained from each donor. Needle biopsies were obtainedfrom female patients with palpable breast lumps. A fraction of thespecimens taken during diagnostic procedures was used for cellculturing, a part was frozen at - 70 °C and the rest was processedfor histopathological diagnosis. Human estrogen receptor (ER)negative cells isolated from pleural effusions of breast adenocar-cinoma (MDA-MB-231) were also used in this study.

Cell culture

Primary cell cultures were derived from breast tumors asdescribed previously with some modifications [20,21]. Mixedcell populations (stromal and epithelial) were allowed to growout from cultured explants in supplemented DMEM-HG medium(100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/mlFungizone), containing 5% heat-inactivated-FBS and 1×10-9 M17β-estradiol. Incubations were performed in humidified 5%CO2–95% air at 37 °C. Once 70% confluence was reached,subculturing was performed by tripsinization (trypsin/EDTA0.25%/0. 2 g/L). Culture media was changed every 3 days. Afterapproximately eight passages cells were characterized byWestern blots and immunocytochemistry. Experimental proce-dures were performed in supplemented DMEM-F12 mediumconditioned with 5% charcoal-stripped heat-inactivated FBSwithout 17β-estradiol.

Western blot

Confluent cells were trypsinized, pelleted and lysed with RIPAbuffer (9. 1 mM dibasic sodium phosphate, 1. 7 mM monobasicsodium phosphate, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, pH7.4) in the presence of a protease inhibitor cocktail. Frozen tissuewas homogenized with RIPA buffer using a glass-teflon homoge-nizer. Both total cell lysates and processed tissue were separatedon 10% SDS-PAGE, transferred to nitrocellulose membranes, andblocked with 5% skim milk. Membranes were incubated withrespective antibodies at appropriate dilutions: VDR (D-6 sc-13133,1:200), ERα (F-10, sc-8002, 1:200), ERβ (1531, sc-53494, 1:200),epidermal growth factor receptor 2 (Her-2/neu) (C-18, sc-284,1:200), progesterone receptor (PR) (C-19, sc-538, 1:200), cytoker-atin-7 (CK-7) (N-20, sc-17116, 1:500), Eag1 (62.mAb, 1:200) andtubulin (D66, T0198, 1:50,000). For visualization, membraneswere incubated with horseradish peroxidase-conjugated second-ary antibodies (sc-2004 or sc-2031, 1:2000) and were processed

Table 1 – Primers and probes used in RT real-time PCR analysis.

Gene/access number Sense primer Anti-sense primer Amplicon (nt) Probe no.

Eag1 AF078741.1 CCT GGA GGT GAT CCA AGA TG CCA AAC ACG TCT CCT TTT CC 60 49CYP24A1 NM_000782.3 CAT CAT GGC CAT CAA AAC AA GCA GCT CGA CTG GAG TGA C 65 88GAPDH AF261085.1 AGC CAC ATC GCT GAG ACA C GCC CAA TAC GAC CAA ATC C 66 60

Table 2 – Patient age and histopathological diagnosis.

No. Patient age(years)

Histopathologicaldiagnosis

ERαstatus

1 78 IDC SBR 9 +++2 37 IDC 40%, SBR 8; DCIS 60%, LP +++3 32 IDC, SBR 9 -4 36 Fibroadenoma +5 65 IDC SBR 6, LP -6 61 IDC, SBR 9 +++7 17 Fibroadenoma +8 40 Fibroadenoma ND9 42 Fibroadenoma ND

Abbreviations: IDC: invasive ductal carcinoma; DCIS: ductal carcinomain situ; ERα: estrogen receptor alpha; SBR: Scarff–Bloom–Richardsongrading system; LP: lymphovascular permeation; ND: not determined.

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with the ECL detection system (Amersham Pharmacia UK).Densitometric analysis of resulting bands was performed byusing ImageJ software (NIH, USA).

Histopathology and Immunochemistry

A fragment of each biopsy was fixed in buffered formalin andparaffin-embedded. Sections were cut at 5 μm and placed onpositively charged slides. One slide was stainedwith hematoxylin–eosin for analysis by a specialized pathologist using rutinarynuclear and histologic guidelines, in accordance with the Scarff–Bloom–Richardson (SBR) grading system. The other unstainedsections were de-paraffinized and used for immunohistochemis-try according to conventional procedures. On the other hand,cultured cells were grown on glass coverslips and fixated with96% ethanol. In both cases, antigen retrieval was done byautoclaving in EDTA (0.01 mol/L, pH 9.0) during 30 min. Slideswere blocked with 0.3% H2O2 and incubated in the presence of thefollowing primary antibodies: VDR (1:400), ERα (1:100), ERβ(1:100), Her-2/neu (prediluted), Eag1 (62.mAb, 1:100), PR(1:75), vimentin (1:100) and CK-7 (1:200) during 60 min. Afterwashing, the slides were sequentially incubated with MACH 4mouse probe and MACH 4-HRP polymer (Biocare Medical, CA,USA) during 10 min each. Staining was completed with DAB and0.04% H2O2. In the case of Eag1 for tissue sections, we used asingle chain antibody (33.mAb) coupled to alkaline phosphatase;therefore after antigen retrieval endogenous alkaline phosphataseactivity was blocked with dual enzyme-blocking reagent (Dako).Anti-Eag1 (1:50) was incubated during 60 min and an enhancerof alkaline phosphatase activity (Ventana, AZ, USA) was used. Thereaction was revealed with fast red and α-naphtol (Ventana).Slides were counterstained with hematoxylin.

In order to analyze calcitriol inhibitory action upon Eag1 at theprotein level, cells grown on glass coverslips were cultured in thepresence of calcitriol (1×10-7 M) or its vehicle during 48 h.Afterwards cells were fixated and processed as described above forimmunocytochemistry. Quantitative analysis of Eag1 immunos-taining was performed in photographs by using ImageJ software.Nuclei and cytoplasm were evaluated densitometrically. Fourdifferent biopsies were analyzed, 10 photographs per treatmentfor each biopsy were taken and 3 measurements in each picturewere performed (n=120 observations per treatment).

Real-time PCR

Cells were incubated in the presence of different calcitriolconcentrations or its vehicle (0.1% ethanol) during 24 h. Inhibitoryconcentration (EC50) values were obtained by non-linear regres-sion analysis using sigmoidal fitting with a sigmoidal dose–response curve by means of a scientific graphing software. Theeffects of the VDR antagonist TEI-9647 (1×10-7 M) upon calcitrioleffects were tested by coincubating both factors. Afterwards

mediumwas aspirated andRNAwas extracted using Trizol reagent.Two micrograms of total RNA were reverse transcribed with thetranscriptor RT system. Real-time PCR was carried out using theLightCycler® 2.0 from Roche (Roche Diagnostics, Mannheim,Germany), according to the following protocol: activation of TaqDNA polymerase and DNA denaturation at 95 °C for 10 min,proceeded by 45 amplification cycles consisting of 10 s at 95 °C,30 s at 60 °C, and 1 s at 72 °C. Primers sequences, correspondingprobe numbers and sizes of resulting amplicons are given in Table 1.The expression of CYP24A1 was used as a control for calcitriolbiological activity and was also useful to assess VDR functionality.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was ampli-fied as an internal control. In addition, a previously establishedhuman breast cancer cell line (MDA-MB-231) was used tocorroborate calcitriol effects upon Eag1 mRNA. Same treatments asdescribed before were given to MDA-MB-231 cells.

Proliferation studies

Cell proliferation assays were performed in sextuplicate. In eachwell 1000 cells were seeded and incubated in the presence ofcalcitriol alone or with TEI-9647 during 3 and 6 days. Coincuba-tions with astemizole, a widely used nonspecific Eag1 inhibitor,were also performed. Afterwards cell proliferation was measuredby using the colorimetric XTT assay following manufacturer'sinstructions, and absorbance in each well was determined at492 nm in a Multiskan spectrophotometer (Labsystems Inc.,Canada). EC50 values were obtained as described above.

Statistical analysis

Data are expressed as mean±standard deviation (S.D.). Statisticalanalysis was done using one-way ANOVA and all pairwisemultiplecomparison were determined by Holm-Sidak method. Differenceswere considered significant at P<0.05.

Fig. 1 –Western blot analysis of breast tumor-derived cells. Celllysates (biopsies 1–7) were separated by SDS–PAGE andtransferred to nitrocellulose membranes. Membranes wereincubated with designated antibodies. As positive control (C), ahomogenate of SKBR-3 breast cancer cells was used for Her-2/neu (∼185 kDa), HeLa cells for CK-7 (∼54 kDa) and PR (∼116 and∼81 kDa), while for VDR HeLa cells transfected with a vectorencoding for VDR was used (∼51 kDa). Tubulin represents theloading control (∼50 kDa).

Fig. 2 – Immunohistochemical analysis of different cell markers intissue sections. Eag1 (A, B, C, red staining) was located in the nucleepithelial cells of fibroadenomas, as indicated in all cases by arrowmembrane and cytoplasm of all malignant ductal cells, as well as emarker CK-7 (brown staining) is shown in G, H and I. Representati

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Results

Histopathology and immunocharacterization

Biopsies from nine patients were obtained. The results of thehistopathological analysis and the ERα status are shown in Table 2.Positive diagnosis for invasive ductal carcinoma (IDC) was madefor five samples; IDC and ductal carcinoma in situ (DCIS) werediagnosed in one of these samples. Four additional cases withdiagnosis of fibroadenoma were included.

Seven biopsies were used for cell culturing. Approximately2 weeks after primary cell culture, multiple foci with heterogeneouscell populations were observed around the explants. Cells wereallowed to grow by rounds of trypsinization, and after passage 8 thecells were characterized by Western blot analysis. As depicted inFig. 1, all cultured cells expressed VDR, CK-7 and PR-B; while PR-Aand Her-2/neu were positive only in some of them. Cultured cellswere estrogen receptor negative (ERα and ERβ), but positive forvimentin (data not shown). All our cell cultures displayed a finitemitotic lifespan, and after approximately 15 passages they becamesenescent.

DCIS (A, D, G), fibroadenoma (B, E, H), and IDC (C, F, I) in breastar envelope, nucleus and nucleolus of ductal tumoral cells ands. VDR (D, E and F, brown staining) was strongly stained in thepithelial cells in fibroadenomas (arrows). The luminal/ductalve pictures are displayed (40 X).

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Immunohistochemical analysis of different cell markers in DCIS(Figs. 2A, D, G), fibroadenoma (Figs. 2B, E, H), and IDC (Figs. 2C, F, I)are shown in Fig. 2. Eag1 was found predominantly in the nucleus,nuclear envelope and the nucleolus of ductal epithelial cells,independently of the final diagnosis (Figs. 2A–C). VDRwas detectedin the plasmamembrane, cytoplasm and nuclear envelope of ductalepithelial cells with a strong staining pattern, and to a lesser extentin the nucleus of some stromal cells (Figs. 2D–F). Eag1 and VDRwere predominantly present in CK-7 positive cells (Figs. 2G–I).Immunohistochemical studies revealed that VDR and Eag1 abun-dancewasmore evident in IDC compared to fibroadenomas (Fig. 2);therefore, in order to ascertain this observation we performedWestern blot analysis of frozen breast tissue samples obtained fromwomen with any of these two pathologies (Fig. 3). For Eag1 twodistinct bands were detected in these preparations, with electro-phoretic mobilities corresponding to ∼110 and ∼130 kDa. The∼51 kDa band of VDRwas not detected in rat brain, whichwas usedas positive control for Eag1 (Fig. 3A). Western blots showed thatEag1 and VDR proteins were higher in IDC than in fibroadenomas(Figs. 3A and B), which was further corroborated with densitome-trical analysis of corresponding bands after tubulin normalization(Figs. 3C and D, respectively).

Calcitriol inhibited Eag1 expression in breasttumor-derived cells

Since our hypothesis was that Eag1 might be a potential target forcalcitriol, cells originated from different breast tumors (IDC andfibroadenoma) were incubated in the presence of calcitriol, and

Fig. 3 –Western blot analysis of Eag1 and VDR in invasive ductal carcbreast tumor tissue homogenates (30 μg) were loaded per lane asAdditional samples are shown in B. Two bands of 110 and 130 kDawas used as a loading control for normalization. Optical density (Orespectively. Bars represent the mean±SD of n=4. ⁎P<0.05 vs. ID

Eag1 gene expressionwas studied. The results showed a significantcalcitriol concentration-dependent inhibition of Eag1 gene ex-pression (Fig. 4A). EC50 values of calcitriol on suppressing Eag1mRNA were 1.08×10-9 M. This effect appeared to be mediated bythe VDR, since coincubation of both calcitriol and the VDRantagonist TEI-9647 had no effect upon Eag1 expression (Fig.4B). Indeed, the inhibitory action of calcitriol (1×10-9 M) uponEag1 gene transcription was blocked by TEI-9647 (1×10-7 M);however, a higher concentration of TEI-9647 (1×10-6 M) resultedin agonistic effects, while lower concentrations were not sufficientenough to abolish calcitriol inhibitory actions (data not shown).The well-known up-regulation of CYP24A1 gene expression bycalcitriol was used as a positive control, including the VDR-drivenTEI-9647 antagonistic effects. Calcitriol upregulated CYP24A1 genetranscription 3332 and 23,433 folds over control with 1×10-9 Mand 1×10-8 M, respectively; indicating the presence of a functionalVDR. The effects of calcitriol upon CYP24A1 were attenuated in70% when coincubated with TEI-9647 (data not shown).

In order to further corroborate our results, we also used MDA-MB-231 cells. Calcitriol inhibited Eag1 gene expression in MDA-MB-231 cells, but to a lesser extent of that seen in breast tumor-derived cells (Fig. 4C). Interestingly, basal expression of thecalcitriol-degradating enzyme CYP24A1 was 14-folds higher inMDA-MB-231 cells than in breast-tumor derived cells (Fig. 4D).

Inhibition of the expression of Eag1 by calcitriol was alsodemonstrated at the protein level by immunocytochemistry. Asignificant reduction of Eag1 protein was apparent after 48 h ofcalcitriol exposure. As shown in Fig. 5, immunoreactive Eag1decreased 32.3% in calcitriol vs. vehicle-treated cells (P<0.001).

inoma (IDC) and fibroadenoma (F). (A) One sample of IDC and Findicated. For Eag1 rat brain was used as positive control (C).of Eag1 were detected, and for VDR ∼51 kDa. Tubulin (∼50 kDa).D.) of Eag1 and VDR versus tubulin is shown in C and D,C.

Fig. 4 – Calcitriol inhibits Eag1 gene expression by a VDR-mediated mechanism. (A) Breast tumor-derived cells (BTD) incubated for24 h in the presence of different calcitriol concentrations or its vehicle (V) were harvested for RNA extraction and real-time PCR.Results shown are representative of seven independent experiments from different biopsies and are expressed as the mean±S.D. ofEag1/GAPDH mRNA normalized ratio. Data were normalized setting a value of 1 for vehicle-treated cells. ⁎P<0.05 vs. vehicle. (B)Effects of TEI-9647 upon calcitriol-mediated inhibition of Eag1 gene expression. BTD cells were incubated in the absence or presenceof calcitriol (1×10-9 M), without (-) or with (+) the VDR antagonist TEI-9647 (1×10-7 M). After 24 h RNA was extracted andreal-time PCR analyses were performed. Results are expressed as percent change of vehicle-treated cells, whichwas set to 100%. Barsrepresent the mean±S.D. of three experiments. ⁎P<0.05 vs. vehicle and TEI-9647-treated cells. (C) Calcitriol effects upon Eag1 geneexpression in MDA-MB-231. Cells were subjected to similar treatments as described before. Results shown as the mean±S.D. ofEag1/GAPDH mRNA normalized ratio. ⁎P<0.05 vs. vehicle. (D) CYP24A1 basal gene expression in BTD and MDA-MB-231 cells. Thecalcitriol-degradating enzyme was 14-folds higher in MDA-MB-231 cells than in BTD cells, which was given a value of one. Barsrepresent the mean±S.D. (n=6) ⁎P<0.05 vs. BTD.

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Calcitriol inhibited proliferation of breasttumor-derived cells

In order to study calcitriol ability to inhibit proliferation in ourcultured breast tumor-derived cells, incubations were performedin the presence of this secosteroid. Calcitriol significantly inhibitedcell proliferation in a concentration-dependent manner (Fig. 6A).Similar results were obtained using distinct cell lines, includingthose derived from fibroadenomas. EC50 values of calcitriol onsuppressing cell proliferation were 8.57×10-10 M. As depicted inFig. 6A, vehicle-treated cells duplicated every third day, which wasreflected by increasedmetabolic activity. However, in the presence

of increasing concentrations of calcitriol cell growth was inhibitedand completely abolished between days 3 and 6 for theconcentrations 1×10-8 M and 1×10-7 M. Fig. 6B shows thatantiproliferative actions of calcitriol (1×10-9) were prevented bythe VDR antagonist TEI-9647.

With the purpose of analyzing a possible synergistic effectupon proliferation by inhibiting Eag1 expression and channelactivity, we incubated our cells in the presence of calcitriol andthe Eag1 blocker astemizole. As seen in Fig. 6C, coincubation ofboth calcitriol and astemizole resulted in significant furtherreduction in cell proliferation. Different calcitriol and astemizoledoses were tested obtaining similar results (data not shown).

Fig. 5 – Calcitriol inhibits Eag1 protein expression. Immunocytochemical analysis and quantification of immunoreactive Eag1 incells treatedwith calcitriol or its vehicle was performed as described under Materials andmethods. (A) Negative control was carriedout in the absence of primary antibody. (B) Vehicle-treated cells. (C) Cells incubated in the presence of calcitriol (1×10-7 M)during 48 h. (D) Densitometric analysis showed significant reduction of Eag1 protein in calcitriol vs. vehicle-treated cells (P<0.001).Bars represent the mean±S.D., n=120 observations per treatment in four different biopsies. Eag1 was located in cytoplasmic,nuclear and perinuclear region (brown staining). Representative pictures are shown (40 X).

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Discussion

Calcitriol antiproliferative activity has been widely studied, anddefining specific mechanisms of this hormone to inhibit cellgrowth is of particular interest in breast cancer since in these cellsVDR is overexpressed, as shown in this study. Herein, wedemonstrated a consistent and significant negative regulation ofEag1 expression by calcitriol, an event that was accompanied byinhibition of cell proliferation. These calcitriol effects weremediated by the VDR, as judged by studies in the presence of aspecific VDR antagonist. Inhibition of Eag1 mRNA expression bycalcitriol was also corroborated in MDA-MB-231 cells, whichfurther supported our observations in tumor-derived cells.However, calcitriol effects upon Eag1 in MDA-MB-231 were lessevident and required higher concentrations of the hormone inorder to reach statistical significance. This could be the result ofhigh basal levels of CYP24A1mRNA expression found in MDA-MB-231, which may contribute to rapid calcitriol inactivation, or bedue to alterations of this established cell line as reportedelsewhere [19,22].

The use of Eag1 blockers leads to reduction of cell proliferation.In this study we tested a possible synergistic effect of theantihistamine astemizole, which inhibits Eag1 currents by selec-tively binding to open channels; and calcitriol, which inhibits Eag1expression. With the experimental approach that we used, the

degree of inhibition of the metabolic activity when co-incubatingthe cells with both factors indicated independence rather thansynergy. Indeed, different concentrations of the compounds weretested obtaining similar results, suggesting independent jointaction that further reduced cell proliferation by inhibiting Eag1expression and activity. Calcitriol reduced Eag1 protein expressionby 32.3%; therefore, blocking the remaining channels activity withastemizole may account for the combined effect that we observedwhen coincubating both factors. It is noteworthy to mention thatthe assay used to asses proliferation gives an idea of the number ofmetabolically active (viable) cells, but also may reflect bothproliferation and apoptosis; therefore, a proapoptotic effect ofcalcitriol and astemizole should be taken into consideration anddeserves further investigation. Acting through different mechan-isms, both astemizole and calcitriol have proven to promote cellcycle arrest and apoptosis. Inhibition of proliferation by K+

channels blockers is due to membrane depolarization [23], whileantiproliferative effects of calcitriol have been linked to suppres-sion of growth stimulatory signals and potentiation of growthinhibitory signals, leading to changes in cell cycle regulators suchas p21 and p27, cyclins and retinoblastoma [24]. Results presentedherein together with previous findings showing that silencingEag1 expression with siRNA reduced cell proliferation [25],reinforce the concept that in addition to the well known inhibitorypathways, antiproliferative effects of calcitriol also involve Eag1suppression.

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Considering that Eag1 has been associated with malignanttransformation and hyperproliferative disorders, the findingsreported herein provide new insights into the mechanismswhereby calcitriol exerts antineoplastic activity, highlighting itspossible therapeutic use for Eag1 inhibition in tumors over-expressing this channel. Recent studies have established that Eag1K+ channels are important for breast cancer cell proliferation andcell cycle progression [23,26] and that, independently of itsprimary function as an ion gate, Eag1 contributes to tumorprogression increasing tumor vascularization [14]. In an oppositemanner, calcitriol has shown to restrain angiogenesis in humancancer cells [15], a mechanism that collectively with its ability toinhibit Eag1; as demonstrated herein, may potentiate its anti-tumoral activity.

Fig. 6 – Calcitriol antiproliferative effects in cultured breasttumor-derived cells. (A) Cells were incubated in the presence ofdifferent calcitriol concentrations or its vehicle (V) during 0, 3or 6 days, and cell growth was assayed by the XTT colorimetricmethod. Calcitriol significantly inhibited cell proliferation in aconcentration-dependent manner. Results are expressed as themean±S.D. (n=6); different letters indicate statisticalsignificance (P<0.05). Similar results were obtained using cellsderived from different biopsies. (B) The antiproliferativeactions of calcitriol were prevented by the VDR antagonistTEI-9647. Cells were incubated with calcitriol (1×10-9 M)during 3 days, in the absence (-) or presence (+) of TEI-9647(1×10-7 M), and cell growth was studied as previouslydescribed. Results are expressed as percent change ofvehicle-treated cells, which was set to 100%. Bars represent themean±S.D. (n=6), ⁎P<0.05 vs. vehicle and TEI-9647-treatedcells. (C) Antiproliferative activity of calcitriol was potentiatedby coincubation with astemizole. Cells were incubated withcalcitriol (1×10-7 M) in the absence (-) or presence (+) ofastemizole (3 μM) during 7 days. Bars represent themean±S.D.(n=6), ⁎P<0.05 vs. vehicle-treated cells. ⁎⁎P<0.05 vs. calcitrioland astemizole-treated cells.

Contrary to wild-type, VDR knockout mice carcinogen-induced tumors are unresponsive to calcitriol, which providedevidence for the role of VDR in mediating genomic antiproli-ferative effects of calcitriol [27]. Consequently, corroborating thepresence and functionality of VDR in our tumor-derived cellswas central. In this study, activity of VDR was demonstrated byinhibition of proliferation and up-regulation of CYP24A1 geneexpression in cells exposed to calcitriol. Moreover, immunohis-tochemical and Western blot studies confirmed the presence ofVDR in all samples studied. Membrane and cytoplasmiclocalization of VDR suggested the presence of VDR in itsunbound form, since in ligand-depleted conditions the majorityof VDR is mainly found in the cytoplasmic fraction [28]. Anotherplausible explanation for extranuclear VDR immunolocalizationcould be that prolactin derived from tumoral epithelial cells maybe blocking VDR nuclear translocation, as has been previouslyshown in other cell types [29].

VDR was colocalized in the same tumor cells that stronglyexpressed Eag1, the latter being found preferentially in the nucleusand perinuclear region. This unique cellular distribution of Eag1has been reported previously in other cells andmay be the result ofseveral putative nuclear localization signals in its carboxyl-terminal domain [18,30,31]. The physiological role of Eag1 nuclearlocalization remains unclear; however, in its N-terminus Eag1contains a stretch of amino acids that form a PER-ARNT-SIM (PAS)dimerization domain [32], which is particularly unusual for an ionchannel because nearly all eukaryote proteins that contain a PASdomain are transcription factors that reside within the nucleus orshuttle between the nucleus and cytoplasm [33]. Otherwise, it hasrecently been shown that Eag1 transcripts can be alternativelyspliced to produce a soluble protein able to enter the nucleus andactivate specific signaling pathways [31]. If Eag1 indeed could actas a transcription factor, our observation that calcitriol reducesEag1 abundance suggests that it may also impair Eag1 signaling,which deserves further investigation.

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Interestingly, in the immunohistochemical analysis all samplesstudied showed increased Eag1 and VDR abundance in IDCcompared to fibroadenomas, an observation that was furthercorroborated by Western blots. These results were expectedconsidering that Eag1 and VDR are preferentially expressed byepithelial cells, as shown herein; and that IDC arises frommalignant epithelial cells. These observations strongly suggest apossible link between Eag1, VDR and breast cancer development,and encourages studying this relation in tumors of different origin.

Eag1 immunoreactivity was also found in ductal epithelial cellsof benign breast tissue, although to a lesser extent when comparedto IDC, supporting previous findings showing Eag1 in the normalmammary gland [18]. Considering its location in non-canceroustissue, it is possible that Eag1 may participate in ductalmorphogenesis and other normal functions of the gland. Theseeffects would be of particular relevance during puberty, pregnancyand lactation, when the gland undergoes extensive ductalelongation and branching, with increased proliferative activity. Itwould be of great interest to investigate the participation of Eag1in these physiological events and the interconnections betweenthe hormonal milieu and Eag1 expression and activity. In thisregard, we have recently shown that Eag1 gene expression isincreased by estrogens and antiestrogens in a cell type-dependentmanner [30]. This issue has not yet been addressed in breast tissueand deserves further studies.

The fact that Eag1 colocalized with VDR in the same cell typeand that they exert antagonistic biological activities suggested thatboth factors may be complementary in their functions. Morespecifically and knowing that CYP27B1 is expressed in mammaryepithelial cells [2], we believe that calcitriol may act as anautocrine-negative growth regulator of Eag1-dependent cellproliferation in breast tissue. In fact, previous reports showedthat proliferation and cell cycle progression in breast cancer cellsdepends on Eag1 expression [23,25], while VDR is an importantnegative growth regulatory factor in the mammary gland [5,34].Furthermore, VDR knockout mice have increased incidence of pre-neoplastic lesions, mammary tumors and ductal thickening [3,4].

We used early passage primary breast tumor-derived culturesin an effort to obtain enriched cell populations that are morerepresentative of the original tumor microenvironment. Immuno-characterization of cells showed distinct molecular signatures: ERswere negative while PR and both luminal/epithelial (CK-7) andmesenchymal (vimentin) markers were positive. Interestingly,vimentin has proved to be an important diagnostic marker forbasal-like carcinoma in ER-negative tumors [35,36]. Additionally,overexpression of the oncogene Her-2/neu was detected in somecell lines, including one derived from a benign breast biopsy. Thisfeature has been associated with increased risk of subsequentbreast cancer development [37]. Considering the overall data, ourcell cultures representedmixed cell populations. It should be takeninto consideration that in vitro conditions may influence the cellphenotype and that isolated cells may behave differently in cultureas compared with their response in a tissue/organ context.

Conclusions

In summary, a new mechanism implicated in the antiproliferativeactivity of calcitriol has been described. In fact, this is the first timethat inhibition of the oncogenic potassium channel Eag1 expres-

sion by calcitriol is reported. This process was mediated through aVDR-dependent signaling pathway and was accompanied byreduced cell proliferation, reinforcing the concept of an implica-tion of Eag1 in tumor cell growth. Our findings justify furtherstudies to qualify VDR as a target for clinical applications in a broadspectrum of neoplasms that overexpress Eag1. In addition, Eag1and VDR colocalized in the same cell type, suggesting autocrineregulation of these antagonic factors, which together with strongerimmunoreactivity of both Eag1 and VDR in malignant cells,highlights calcitriol and its analogs as a therapeutic aid for breastcancer treatment and prevention. The latter is particularlyimportant in high risk groups such as post-menopausal andelderly women, where low vitamin D status have been consis-tently reported [38,39].

Acknowledgments

We thank Teijin Pharma Limited (Japan) for TEI-9647 donation,Hoffmann-La Roche Ltd. (Switzerland) for calcitriol donation andDr.Walter Stühmer (Max-Planck Institute, Germany) for providingEag1 antibodies. We thank MVZ. Silvia Reyes Maya and Dr. AndresCastell for technical assistance (Departamentos de Embriología yBiología Celular y Tisular, Facultad de Medicina, UNAM, México).This work was partially supported by a grant from CONACyT,México.

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