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Toxicology Letters 192 (2010) 195–205

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

exachlorobenzene induces cell proliferation and IGF-I signaling pathway in anstrogen receptor �-dependent manner in MCF-7 breast cancer cell line

aría Alejandra Garcíaa, Delfina Penaa, Laura Álvareza, Claudia Coccab, Carolina Pontilloa,osa Bergocb, Diana Kleiman de Pisareva, Andrea Randia,∗

Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, ArgentinaLaboratorio de Radioisótopos, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina

r t i c l e i n f o

rticle history:eceived 7 July 2009eceived in revised form 19 October 2009ccepted 20 October 2009vailable online 30 October 2009

eywords:

a b s t r a c t

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. ER� regu-lates the expression of genes involved in growth and development, and plays an important role in breastcancer. The present study focuses attention on the effect of HCB (0.005, 0.05, 0.5, 5 �M) on cell prolifer-ation in estrogen receptor � (ER�)(+) MCF-7, and ER�(−) MDA-MB-231 breast cancer cell lines. We alsostudied the insulin growth factor-I (IGF-I) signaling pathway in MCF-7 cells. HCB (0.005 and 0.05 �M)stimulated cell proliferation in MCF-7, but not in MDA-MB-231 cells. The pesticide increased apoptosis

exachlorobenzeneGF-I signalingroliferationstrogen Receptor-SrcCF-7

in MCF-7, at HCB (0.5 and 5 �M). At these doses, HCB induced the expression of the aryl hydrocarbonreceptor (AhR)-regulated gene cytochrome P4501A1. MCF-7 cells exposed to HCB (0.005 and 0.05 �M)overexpressed IGF-IR and insulin receptor (IR). IRS-1-phosphotyrosine content was increased in a dosedependent manner. ICI 182,780 prevented HCB-induced cell proliferation and IGF-I signaling in MCF-7cells incubated in phenol-red free media. In addition, HCB (0.005 �M) increased c-Src activation, Tyr537-ER� phosphorylation and ER� down-regulation. Taken together, our data indicate that HCB stimulation

F-I s

of cell proliferation and IG

. Introduction

Several reports have suggested an association between expo-ure to environmental organochlorine compounds and breastancer risk (Aronson et al., 2000). Hexachlorobenzene (HCB) is onef the most widespread environmental pollutants. Although these of HCB was discontinued in most countries, it is still released

nto the environment as a byproduct in several industrial pro-esses (Bailey, 2001). HCB exposure elicits toxic effects in humansnd laboratory animals. Chronic exposure of humans to HCB pro-uces a number of effects, such as triggering of porphyria, increasedynthesis of liver microsomal enzymes, neurological symptoms,mmunological disorders and thyroid dysfunctions (ATSDR, 2002).nvironmental exposure to HCB can occur mainly through the con-

umption of contaminated food, cow milk (Maitre et al., 1994),y prenatal exposure and from breast milk (ATSDR, 2002). Majorxposure can also occur in individuals living near an exposedite, through contaminated soil or air (ATSDR, 2002). HCB has

∗ Corresponding author at: Departamento de Bioquímica Humana, Facultad deedicina, Universidad de Buenos Aires, Paraguay 2155, 5to piso, CP 1121 Buenos

ires, Argentina. Tel.: +54 11 4508 3672; fax: +54 11 4508 3672.E-mail address: andybiol@yahoo.com.ar (A. Randi).

378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2009.10.026

ignaling is ER� dependent in MCF-7 cells.© 2009 Elsevier Ireland Ltd. All rights reserved.

been classified by the International Agency for Research on Can-cer (IARC) as a Group 2B carcinogen (possibly carcinogenic tohumans) (ATSDR, 2002). HCB is known to be a thyroid carcino-gen in hamsters (Cabral and Shubik, 1986), and acts as a tumorpromoter of rat liver foci growth (Ou et al., 2001) and rat mam-mary tumors (Randi et al., 2006). HCB is a weak agonist of thearyl hydrocarbon receptor (AhR) (Hahn et al., 1989), a cytoso-lic ligand-activated transcription factor that binds to promoterregions of AhR target genes such as cytochrome P4501 (CYP1)family members and several drug-metabolizing enzymes. Chem-icals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a strongagonist of the AhR, also induce hypothyroxinemia and CYP1A1in laboratory animals (Gorski and Rozman, 1987; Guruge et al.,2009). We have previously demonstrated that in vivo administra-tion of HCB induces alterations in insulin-like growth factors (IGFs)signaling pathway in mammary gland and mammary tumors inrat (Randi et al., 2006). Several breast cancer cell lines have beenutilized as in vitro models for investigating the effects of environ-mental compounds on the development of breast cancer. Estrogen

receptor � positive (ER�+) MCF-7 is a highly differentiated humanbreast adenocarcinoma cell line, strongly responsive to estradiol,insulin and IGFs, associated with a hormone-dependent stage ofbreast carcinoma (Brooks et al., 1973). Estrogen receptor � nega-tive (ER�−) MDA-MB-231 adenocarcinoma cell line, in contrast, is

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ften used as a model of late-stage, hormone-independent tumorsCailleau et al., 1978). Although both cell lines express AhR, MCF-

has shown to be more responsive to AhR induction (Oenga etl., 2004). The CYP-dependent monooxygenase system catalyzesxidative metabolism of a wide variety of drugs, carcinogens, pes-icides, and steroid hormones. The CYP1 family, which consists oft least three enzymes (CYP1A1, CYP1A2, and CYP1B1), is inducibley polyhalogenated aryl hydrocarbons compounds, such as 2,3,7,8-etrachlorodibenzo-p-dioxin (TCDD) (Guruge et al., 2009).

ER� is involved in proliferation and development of both nor-al and carcinogenic cells in the mammary gland. This receptor

an be activated by estrogens in a ligand-dependent manner,r in a ligand-independent manner by phosphorylation in spe-ific residues (Pearce and Jordan, 2004). c-Src tyrosine kinase haseen identified as a crucial molecule downstream of ER�, which,y physical interaction with ER�, might mediate estrogen rapidction (Cheskis, 2004). In MCF-7 cells, Tyr537 of ER� is basallyhosphorylated in vivo, independent of ligand stimulation. Thishosphorylation is the binding site for the SH2 domain of c-Src tyro-ine kinase and is required for triggering DNA synthesis and tumorrowth (Varricchio et al., 2007). It has been reported that c-Src pro-otes ER� proteolysis in human breast cancer (Chu et al., 2007).

R� expression and function is frequently lost in advanced andighly malignant tumors (Lapidus et al., 1998). Accumulating evi-ence suggests a mechanistic link between growth factor receptorathways and extranuclear ER� in breast cancer cells, whereby ER�inds to the Insulin growth factor-I receptor (IGF-IR) and activates

ts downstream signaling pathways (Knowlden et al., 2005).The IGF-IR is a transmembrane tyrosine kinase that regulates

arious biological processes such as proliferation, survival and dif-erentiation in mammary gland. Recent clinical and experimentalata indicate that IGF-IR is overexpressed in ER�(+) breast cancerells and primary breast tumors (Surmacz, 2000). Insulin is knowno play a role in human mammary gland development and function.t is well established that in vitro, this hormone stimulates breastancer cell metabolism and growth. Insulin action is mediated pre-ominantly by the tyrosine kinase insulin receptor (IR) and partlyy the IGF-IR (Milazzo et al., 1992). After autophosphorylation,

GF-IR and IR bind to the adapter molecules insulin receptor sub-trate type 1 (IRS-1) and Shc, which are in turn phosphorylated onyrosine residues. IRS-1 phosphorylation leads to the activation ofI3K/AKT and Grb-mSos/Ras/Raf/ERK1-2 pathways (White, 1998).n hormone-dependent breast cancer cells, ER� and IGF-IR are co-xpressed, and estradiol acts in synergy with IGF-I to stimulate cellroliferation. Moreover, estradiol up-regulates IGF-IR mRNA androtein levels (Hamelers and Steenbergh, 2003). Studies on breastancer cell lines also established that ER� may regulate cell pro-iferation by controlling IRS-1 expression, thereby amplifying orttenuating signaling through the IGF signal transduction pathwayMolloy et al., 2000).

In the current study, we examined the ability of HCB to pro-ote cell proliferation in MCF-7 and MDA-MB-231 breast cancer

ell lines, and to alter insulin/IGF-I signaling pathway in MCF-7 celline. We sought to evaluate the possible involvement of ER� on HCBctivation of cell proliferation and IGF signaling pathway.

. Materials and methods

.1. Chemicals

Hexachlorobenzene (>99% purity, commercial grade) was obtained from

ldrich-Chemie GmbH & Co. (Steinheim, Germany). Cell culture media Dulbecco’sodified Eagle’s medium (DMEM) with phenol-red and fetal bovine serum were

btained from PAA Laboratories GmbH (Pasching, Austria). DMEM media withouthenol-red, cell culture media supplements and antibiotics l-glutamine, penicillin,treptomycin sulfate, and amphotericin B were all purchased from PAA LaboratoriesmbH (Pasching, Austria).

tters 192 (2010) 195–205

Anti-IGF-IR� and anti-IRS-1 antibodies were purchased from Cell SignalingTechnology, Inc. (Beverly, USA). Anti-phosphotyrosine antibody was purchasedfrom Transduction Laboratories (BD, Biosciences Clontech, Palo Alto, USA); anti-IR and anti-ER� antibodies were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA); anti-c-Src, anti-pTyr416-c-Src and anti-pTyr537-ER� antibodieswere purchased from Abcam Inc. (Cambridge, England). 5-Bromo-2′-deoxyuridine(BrdU, 99%), 17�-estradiol (E2) (>98%), anti-BrdU and anti-�-actin antibodies wereobtained from Sigma Chemical Co. (St. Louis, MO, USA).

TUNEL label mix (Nucleotide mix, containing fluorescein-dUTP and dNTP) waspurchased from Roche (Nutley, NJ, USA). Texas red-conjugated anti-mouse IgG anti-body was from Jackson Immunoresearch Laboratories, Inc. (Baltimore, USA). ICI182,780 was obtained from Tocris Bioscience (MO, USA). The enhanced chemilu-minescence kit (ECL) was from GE Healthcare Life Sciences. CYP1A1 and GAPDHprimers were obtained from Invitrogen Life Technology (Carlsbad, CA). The ran-dom primers were obtained from Biodynamics, Argentina. Enzymes and cofactorsfor reverse transcription (RT) and PCR were purchased from Promega Corporation(Madison, WI). All reagents used were of analytical grade.

2.2. Cell culture and treatment

MCF-7 and MDA-MB-231 cell lines were purchased from American Type CultureCollection (ATCC, USA). Cells were cultured at 37◦ , in a 5% CO2 incubator with DMEMcomplete growth medium that consisted of DMEM supplemented with 10% fetalbovine serum (FBS), 1% antibiotic–antimicotic mixture (10,000 Units/ml penicillin,10 mg/ml streptomycin sulfate, 25 �g/ml amphotericin B) and 1% glutamine. Cellsat 70–80% confluence were treated with HCB dissolved in absolute ethanol. Finalethanol concentration in each treatment was 0.5%. Previous assays showed that thisconcentration of vehicle had no influence on the analyzed parameters. All experi-ments were repeated at least 3 times, and the results shown are representations ofsimilar findings.

2.3. Cell proliferation measured by bromo-2′-deoxyuridine (BrdU) incorporationassay

MCF-7 and MDA-MB-231 cells were seeded on 24-well plates (2 × 104 cells/well)in DMEM complete growth medium followed by overnight incubation to allow thecells to attach. The next day the medium was changed to DMEM 0.1% FBS and 24 hlater cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle, in DMEM 1%FBS for 24 h. For ICI 182,780 treatment, MCF-7 cells were maintained in phenol-redfree DMEM 0.1% charcoal-treated FBS for 24 h and HCB was added to phenol-red freeDMEM 1% charcoal-treated FBS, in the presence or absence of 0.1 nM E2 and/or 10 nMICI 182,780. Twenty �M BrdU were added to the media during the last 2 h, and aftertreatment, cells were washed with phosphate-buffered saline (PBS) and fixed with10% formaldehyde during 15 min. Cells were permeabilized with 6N HCl, 1% TritonX-100 for 15 min, washed with PBS and treated with 0.1 M sodium borate, 1% TritonX-100 for 15 min. Cells were blocked with 5% FBS and incubated with monoclonalanti-BrdU antibody (1:200) overnight at 4 ◦C, and Texas Red-conjugated secondaryantibody (1:250) for 2 h at 37 ◦C. Total nuclei were stained with 3 mg/ml Hoechstdye. Eight to ten random fields were analyzed in each sample under a fluorescencemicroscope and the total number of nuclei for each field was counted. Image-Pro Plusv4.5 (Media Cybernetics Inc., Silver Spring, MD) software was used for image cap-turing and cell counting. Proliferation percentages were calculated as the numberof BrdU positive nuclei over the total number of nuclei.

2.4. Cell proliferation measured by clonogenic method

MCF-7 and MDA-MB-231 cells were seeded on 6-well plates (1.5 × 103 cells) inDMEM complete growth medium followed by overnight incubation to allow cellsto attach. The next day the medium was changed to DMEM 0.1% FBS and 24 h latercells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle, in DMEM 1% FBSduring 7 days. For ICI 182,780 treatment, MCF-7 cells were maintained in phenol-redfree DMEM 0.1% charcoal-treated FBS for 24 h and HCB was added to fresh phenol-red free DMEM 1% charcoal-treated FBS in presence or absence of 0.1 nM E2 and/or10 nM ICI 182,780. After treatment, cells were fixed and stained with 1% toluidineblue and the number of colonies per well was counted using optical microscopy.Results were normalized to arbitrary units, designating a value of 100 to controlassays.

2.5. Cells treatment for receptors studies

MCF-7 cells were seeded in 100 mm dishes (1.5 × 106 cells) in DMEM completegrowth medium. The next day the medium was changed to DMEM 2% FBS and 24 hlater, cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle (Figs. 2 and 3).For experiments of HCB effect on c-Src activation and ER� phosphorylation (Fig. 9),

MCF-7 cells were grown in phenol-red free DMEM 2% charcoal-treated FBS, andexposed to HCB (0.005 and 5 �M) or vehicle. For experiments using ICI 182,780(Figs. 7 and 8), cells were grown in phenol-red free DMEM 2% charcoal-treatedFBS, in the presence or absence of 10 nM ICI 182,780 during 24 h. HCB (0.005 and5 �M) or vehicle was added to the media in the presence or absence of 1 nM E2

and/or 10 nM ICI 182,780. After 24 h of exposure to HCB, cells were washed with PBS

ogy Letters 192 (2010) 195–205 197

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Fig. 1. Effect of HCB on cell proliferation in breast cancer cell lines. (A) BrdU incorpo-ration assay. Cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle during24 h. BrdU (20 �M) was added to the culture media and after 2 h, cells were fixed.Immunohistochemistry assay was performed as described in Section 2. The per-centage of proliferating cells was calculated as the number of BrdU positive nucleiover the total number of nuclei for each field. Values are means ± SD from threeindependent experiments, expressed as percentage of proliferating cells. Asterisksindicate significant differences vs. control (***p < 0.001, ANOVA and Tukey post hoctest). (B) Clonogenic assay. Cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or

M.A. García et al. / Toxicol

nd total cellular protein lysates were obtained by scraping cells with lysis buffer0.1 M Tris–HCl, 1% Triton X-100, 1 mM EGTA, 0.1 mM NaF, 0.02 mg/ml leupeptin,mM Na3VO4, 1 mM PMSF). Samples were centrifuged at 16,000 × g for 10 min and

upernatants were kept at −80 ◦C. The protein concentration of cell lysates wasetermined according to Bradford (1976).

.6. IRS-1 immunoprecipitation

Cell lysates (300–500 �g) suspended in immunoprecipitation buffer (200 mMris, pH 7.4, 1 M NaCl, 100 mM EDTA, 1% IGEPAL CA-630, 2% Triton X-100) containingmM leupeptin, 200 mM PMSF, 1 mg/ml aprotinine, 2 mM Na3VO4 and 1 M NaF,ere incubated with 4 �g of anti-IRS-1 antibody and 15 �l of A/G plus agarose, in anal volume of 0.5 ml for 16–24 h at 4 ◦C under constant shaking. Precipitates wereashed four times with immunoprecipitation buffer, centrifuged at 12,000 × g andenatured in Laemmli buffer at 95 ◦C for 5 min.

.7. Western blotting

Total cellular protein lysates (50–100 �g) or IRS-1 immunoprecipitates werelectrophoresed in 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGE) prior to transfer to polivinylidene difluoride membranes (PVDF) (Mil-ipore, Bedford, MA), and blocked for 1 h in TBS (10 mM Tris, 150 mM NaCl, pH 8)

ith 3% bovine serum albumin (BSA) for anti-phosphotyrosine or with 3% BSA-3%ilk for the remaining antibodies. For immunoprecipitates, PVDF membranes were

ncubated overnight with monoclonal anti-phosphotyrosine antibody (1:1000)nd re-probed using polyclonal anti-IRS-1 (1:200). For protein blottings, incuba-ions were performed using polyclonal anti-IGF-IR� (1:500), polyclonal anti-IR1:200), polyclonal ER� (1:1000), polyclonal c-Src (1:500), polyclonal pTyr416-c-Src1:300), polyclonal pTyr537-ER� (1:800) or monoclonal �-actin (1:2000) antibod-es. The suitable horseradish peroxidase-conjugated anti-species-specific antibodies1:1000) (HRP) were used for protein detection. The immune complexes wereisualized by ECL, and quantified by scanning laser densitometry in a FotodyneFoto/Analyst) Gel-Pro Analyzer 3.1.

.8. In situ detection of DNA fragmentation

Apoptotic nuclei were identified by the detection of the DNA breaks with theerminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-nd labeling technique (TUNEL). In brief, MCF-7 cells were seeded in 24-well plates2 × 104 cells) in DMEM complete growth medium and allowed to adhere overnight.he next day the medium was changed to phenol-red free DMEM 0.1% charcoal-reated FBS and 24 h later cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M)r vehicle, in phenol-red free DMEM 1% charcoal-treated FBS during 24 h. Afterreatment, cells were washed with PBS and fixed with 10% formaldehyde during5 min. Cells were washed and incubated with 20 �g/ml proteinase K in PBS during0 min. After that, cells were washed and permeabilized with 0.1% Triton X-100.ells were incubated with the equilibration buffer and then, the reaction bufferhich contained the TUNEL label mix (nucleotide mix, containing fluorescein-dUTP

nd dNTP), was added to the 3-OH ends of DNA fragments by the enzyme terminaleoxynucleotidyl transferase (TdT) (0.18 U/�l) and incubated during 1 h at 37 ◦C.ell nuclei were stained with Hoescht dye. Later, cells were mounted under glassoverslips, and analyzed under a fluorescence microscope. For negative controls wemitted the TdT reaction step in the TUNEL method. Eight to ten random fieldsere analyzed in each sample and the total number of apoptotic nuclei for eacheld was counted. Image-Pro Plus v4.5 (Media Cybernetics Inc., Silver Spring, MD)oftware was used for image capturing and cell counting. Apoptotic percentagesere calculated as the number of apoptotic nuclei over the total number of nuclei

bserved.

.9. RT-PCR analysis of CYP1A1 mRNA

MCF-7 cells were seeded in 6-well plates (3 × 105 cells) in DMEM completerowth medium. The next day the medium was changed to phenol-red free DMEM% charcoal-treated FBS and 24 h later, cells were exposed to HCB (0.005, 0.05,.5 and 5 �M) or vehicle. After 24 h, cells were washed with PBS and treated withRI-reagent for total RNA extraction. An aliquot of 2 �g of total RNA was used to syn-hesize first-strand cDNA with the random primer, deoxynucleotide triphosphates,ranscriptase reverse, and transcriptase reverse buffer. The RT mixture (1 �l) wasmplified with hot starting PCR in a 50-�l reaction using GoTaq DNA polymerase forYP1A1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control toorrect for differences between lanes in the amount of RNA. PCR was performed asollows: reaction mixture for CYP1A1 amplification was incubated at 95 ◦C for 4 minnd then amplified at 94 ◦C for 1 min, 60 ◦C for 1 min, and 72 ◦C for 1 min. Reactionsere repeated for 38 cycles. Reaction mixture for GAPDH amplification was incu-

ated at 95 ◦C for 4 min and then amplified at 94 ◦C for 1 min, 55 ◦C for 1 min, and2 ◦C for 1 min. Reactions were repeated for 27 cycles. The PCR mixture (50 �l) con-ained GoTaq reaction buffer with 1.5 mM MgCl2, 0.2 mM dNTP’s mix, 1.25 U GoTaqNA polymerase 0.5 �M each forward and reverse primer, and 0.5 �l of RT products.CR products were detected as a single band on 2% agarose gel, containing 0.05%v/v) ethidium bromide. Bands were detected by Fotodyne (Foto/Analyst) equip-

vehicle during 7 days. After treatment, cells were fixed and stained with toluidineblue and the total number of colonies per well was counted. Data are expressed asmeans ± SD of five independent experiments. Asterisks indicate significant differ-ences vs. control (**p < 0.005, ANOVA and Tukey post hoc test).

ment, and intensity was quantified by scanning laser densitometry in a Fotodyne(Foto/Analyst), Gel-Pro Analyzer 3.1.

2.10. Statistical analysis

Homogeneity of variance was tested in each experiment by Bartlett’s test. Datawere evaluated by one-way ANOVA followed by Tukey post hoc test to identify sig-nificant differences between controls and treatments. Differences were consideredsignificant when p-values were <0.05. For each experiment, at least three indepen-dent assays were performed, and final results represent the mean ± SD of n assays.For western blots and RT-PCR, the more representative assay was selected for eachexperiment.

3. Results

3.1. HCB effect on cell proliferation in breast cancer cell lines

Because it has been shown that organochlorine pesticides

or their metabolites could induce cell proliferation in estrogen-sensitive cells, we hypothesized that HCB could increase cellproliferation in MCF-7 cells. The proliferation-inducing proper-ties of HCB were compared using cultured ER�(+) MCF-7 andER�(−) MDA-MB-231 cell lines. Proliferating cells were identified

198 M.A. García et al. / Toxicology Letters 192 (2010) 195–205

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total lysates. Samples were electrophoresed and immunoblottedwith anti-phosphotyrosine and anti-IRS-1 antibodies. Our resultsshowed that HCB increased IRS-1 phosphorylation, and had noeffect in IRS-1 protein levels in MCF-7 cells. The pesticide increased

Fig. 3. Effect of HCB on IRS-1 and IRS-1-phosphotyrosine levels in MCF-7 cell line.Cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle for 24 h. Wholecell lysates were precipitated with anti-IRS-1 antibody and resolved by SDS-PAGE.Membranes were blotted for phosphotyrosine and reblotted for IRS-1 as described

ig. 2. Effect of HCB on IGF-IR and IR levels in MCF-7 cell line. (A) IGF-IR levels. (Bhole cell lysates were prepared, and total protein was resolved by SDS-PAGE and

xperiment is shown in the upper panels. Quantification of IGF-IR and IR levels by dhe lower panels. Values are means ± SD. Asterisks indicate significant differences v

y the incorporation of BrdU, followed by immunocytochemicaletection. As shown in Fig. 1A, cell proliferation was significantly

ncreased (95% and 86%) over control after treatment with HCB0.005 and 0.05 �M, respectively), returning to control values atigher HCB concentrations. Conversely, when the ER�(−) breastancer cell line MDA-MB-231 was treated with HCB, no increase inrdU positive nuclei was observed at any assayed dose. Cell pro-

iferation was also evaluated by clonogenic method. Fig. 1B showshe effect of HCB on MCF-7 and MDA-MB-231 cell proliferation.n MCF-7 cells, HCB (0.005 and 0.05 �M) stimulated cell prolifera-ion (60% and 50%, respectively). At the higher doses, no significantlterations were observed when compared with control cells. InDA-MB-231 cell line, the compound did not affect cell prolifera-

ion at any assayed dose.

.2. IGF-IR and IR levels after HCB treatment in MCF-7 cell line

Because IGF-IR is involved in the development of breast can-er, and the IR content is increased in human breast cancer (Papat al., 1990), we decided to study IGF-IR and IR protein contentn the ER�(+) MCF-7 cell line treated with HCB (0.005, 0.05, 0.5nd 5 �M) or vehicle. Total cell lysates were electrophoresed andmmunoblotted using specific antibodies. As shown in Fig. 2A, HCB0.005 and 0.05 �M) increased IGF-IR levels (65% and 110%, respec-ively) over control. At higher doses HCB did not alter the receptorevels. As shown in Fig. 2B, only 0.005 and 0.05 �M HCB dosesignificantly increased IR levels (70% and 50%, respectively).

.3. HCB effect on IRS-1 content and tyrosine phosphorylation inCF-7 cell line

IRS-1 is the main intracellular substrate activated by IGF-I and

nsulin in human breast cancer cells. In order to study the effectf HCB on insulin/IGF-I signaling pathway activation, we examinedhe ability of HCB to alter IRS-1 content and its tyrosine phospho-ylation levels. Cells were treated with HCB (0.005, 0.05, 0.5 and�M) or vehicle for 24 h, and IRS-1 was immunoprecipitated from

vels. Cells were exposed to HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle during 24 h.d for IGF-IR or IR as described in Section 2. A western blot from one representativemetric scanning of the immunoblots of four independent experiments is shown introl (*p < 0.05, **p < 0.005, ANOVA and Tukey post hoc test).

in Section 2. A western blot from one representative experiment is shown in theupper panel. Quantification of pTyr levels (left axis) and p-Tyr/IRS-1 ratio (rightaxis) by densitometric scanning of the immunoblots are shown in the lower panel.Values are means ± SD of three independent experiments. Asterisks indicate signif-icant differences vs. control (*p < 0.05, and ***p < 0.001, ANOVA and Tukey post hoctest).

M.A. García et al. / Toxicology Letters 192 (2010) 195–205 199

Fig. 4. Effect of ICI 182,780 on HCB-induced cell proliferation in MCF-7 cells. Cells cultured in estrogen-depleted medium were treated with HCB (0.005, 0.05, 0.5 and 5 �M)or vehicle in the absence or presence of 10 nM ICI 182,780 (left panel), or with 0.1 nM E2 or E2 plus HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle, in the presence or absence of10 nM ICI 182,780 (right panel), during 24 h for BrdU incorporation assay and 7 days for the clonogenic assay. (A) BrdU incorporation assay. BrdU (20 �M) was added to themedia, and after 2 h cells were fixed. Immunohistochemistry assay was performed as described in Section 2. The percentage of proliferating cells was calculated as the numberof BrdU labeled cells over total number of cells. Each value represents the mean percentage of proliferating cells (mean ± SD) from three independent experiments. Asterisksi **p < 0a stainea ignificd

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ndicate significant differences vs. its corresponding control (*p < 0.05, **p < 0.005, *nd Tukey post hoc test. (B) Clonogenic assay. After treatment, cells were fixed andre expressed as means ± SD of four independent experiments. Asterisks indicate sifference vs. vehicle treated cells. ANOVA and Tukey post hoc test.

-Tyr/IRS-1 ratio in a dose dependent manner (40%, 57%, 83% and35%, respectively) (Fig. 3).

.4. Role of ER˛ on HCB-induced MCF-7 cell proliferation

As cell proliferation was increased only in MCF-7 cells, weecided to study if HCB-induced cell proliferation was specificallyediated by ER�. Pre-confluent MCF-7 cells, maintained under

strogen-free conditions, were treated with HCB (0.005, 0.05, 0.5nd 5 �M) or vehicle (left panel), or with E2 (0.1 nM) or E2 (0.1 nM)lus HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle (right panel), inoth cases in the presence or absence of ICI 182,780 (10 nM).

reliminary experiments showed that the maximal growth stim-lation of MCF-7 cells was reached at 1 nM E2, and maximum

nhibition of cell proliferation was reached at 10 nM ICI 182,780data not shown). Our results show that HCB (0.005 and 0.05 �M)ncreased incorporation of BrdU (120% and 70% over control,

.001). ###p < 0.001 indicates significant difference vs. vehicle treated cells. ANOVAd with toluidine blue and the total number of colonies per well was counted. Dataant differences vs. control (*p < 005, ***p < 0.001). ###p < 0.001 indicates significant

respectively) (Fig. 4A). Cell proliferation measured by clonogeniccounting was significantly increased by HCB 0.005 �M (200% overcontrol) (Fig. 4B). ICI 182,780 is a specific inhibitor of ER, and, incontrast to partial antagonists such as tamoxifen, completely abro-gates the ability of ER to function as a transcription factor in aligand-dependent or -independent manner (Howell et al., 2000).Interestingly, treatment of cells with this antagonist completelyprevented HCB effect on cell proliferation, evaluated either byBrdU incorporation or clonogenic method. Treatment of cells withE2 (0.1 nM) increased cell proliferation (500%) (Fig. 4A) and 450%(Fig. 4B) when compared with vehicle treated cells. Treatment ofcells with E2 plus HCB (0.005 and 0.05 �M) resulted in a signif-

icant increase of this parameter over E2-treated cells, evaluatedboth with BrdU assay (40% and 30%, Fig. 4A), as well as clono-genic method (37% and 25%, Fig. 4B). In the presence of E2 plus ICI182,780, cell proliferation was similar to the basal levels at all HCBdoses.

200 M.A. García et al. / Toxicology Letters 192 (2010) 195–205

Fig. 5. Detection of in situ DNA breaks by TUNEL in MCF-7 cells. Cells were exposedto HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle and after 24 h cells were fixed. Tuneltwas

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Fig. 6. Analysis of CYP1A1 expression in MCF-7 cells treated with HCB. Cells weretreated with HCB (0.005, 0.05, 0.5 and 5 �M) or vehicle for 24 h. Representativepattern of RT-PCR amplification of both control and HCB-treated cells cDNA, syn-thesized from total RNA, is shown in the upper panel. GAPDH was used as a loadingcontrol. Quantification of cDNA after correction with GAPDH cDNA is shown in thelower panel. Ethidium bromide stained gels were photographed, scanned and the

echnique was performed as described in Section 2. The percentage of apoptotic cellsas calculated as the number of positive fluorescent nuclei over total nuclei. Data

re expressed as means ± SD of three independent experiments. Asterisks indicateignificant differences vs. control (*p < 0.05, ANOVA and Tukey post hoc test).

.5. In situ detection of DNA fragmentation after HCB treatmentn MCF-7

In order to investigate if HCB exerted an apoptotic effect at theigher doses (0.5 and 5 �M), induction of apoptosis was analyzedy immunohistochemical detection of fragmented DNA (TUNEL) inesponse to HCB treatment. We found that the mean percentagef apoptotic cells increased in 114% and 71% in HCB-treated cells0.5 and 5 �M, respectively), compared to control cells. No signifi-ant difference in the apoptotic percentage was observed betweenells treated with HCB at the lower doses (0.005 and 0.05 �M) andontrol (Fig. 5).

.6. Effect of HCB on CYP1A1 mRNA expression in MCF-7

Because it has been demonstrated that the AhR has anti-roliferative activity in MCF-7 cells (Barouki et al., 2007), wexamined the effect of HCB on CYP1A1 mRNA expression. The RT-CR measurements revealed a significant upregulation (175% and30%) of the mRNA content of CYP1A1 after exposure to HCB (0.5nd 5 �M, respectively) (Fig. 6). GAPDH mRNA was used as a loadingontrol.

.7. Effect of ICI on HCB-induced IGF-I signaling pathway inCF-7

In order to determine the contribution of ER� on HCB effectn IGF-I signaling pathway, pre-confluent MCF-7 cells maintainednder estrogen-free conditions were exposed to HCB for 24 h. Thesessays were performed in MCF-7 cells exposed to HCB 0.005 �M,oncentration that induced the highest increase on cell prolifera-ion, and 5 �M, which increased the percentage of apoptotic cellsnd CYP1A1 mRNA levels. MCF-7 cells were incubated with HCB0.005 and 5 �M) or vehicle, in the presence or absence of ICI82,780 (10 nM) (Fig. 7A and C) and with E2 (1 nM) and E2 (1 nM)lus HCB (0.005 and 5 �M) or vehicle, in the presence or absencef ICI 182,780 (10 nM) (Fig. 7B and D). As shown in Fig. 7A, HCB.005 �M increased IGF-IR protein levels (80%), whereas HCB 5 �Mid not alter this parameter. When MCF-7 cells were treated with

CB plus E2, both HCB concentrations induced an increase in IGF-IRrotein levels (Fig. 7B). Co-treatment with ICI 182,780 preventedhe stimulatory effect of HCB in both cases (Fig. 7A and B).

To evaluate the possible involvement of ER� on HCB activationf IGF-I signaling pathway, IRS-1 levels were evaluated in whole

band intensities determined. The values are mean ± SD of three independent experi-ments. Significantly different from control cells (**p < 0.01, ***p < 0.001). ANOVA andTukey post hoc test.

cell lysates precipitated with anti-IRS-1 antibody and blotted withtotal phosphotyrosine and IRS-1 antibodies. HCB (0.005 and 5 �M)significantly increased pTyr/IRS-1 ratio (32% and 60%, respectively)(Fig. 7C). When cells were treated with E2, HCB did not modifyIRS-1 levels; however, p-Tyr/IRS-1 ratio increased (56% and 70%)with HCB 0.005 and 5 �M compared to E2-treated cells. ICI 182,780prevented HCB-inducing effect on p-Tyr/IRS-1 ratio, either in thepresence or absence of estradiol (Fig. 7C and D).

The enhancement of IRS-1 level either at 0.005 or 5 �M (Fig. 7C)in MCF-7 cells cultured in estrogen-depleted medium is in contrastto unchanged values of this parameter in Figs. 3 and 7D. In thelast cases, cells were cultured either in a non-estrogen-depleted,phenol-red containing medium (Fig. 3), or in a phenol-red freeDMEM 2% charcoal-treated FBS supplemented with E2 (Fig. 7D),suggesting that IRS-1 levels were maximally increased in cells cul-tured in the presence of estrogenic compounds.

3.8. HCB effect on ER˛ expression levels in MCF-7

Because HCB showed a potential estrogenic effect on MCF-7cells, we decided to determine if the pesticide altered ER� pro-tein content on this cell line. Our data showed that addition ofHCB 0.005 �M to estrogen-deprived MCF-7 cells decreased 50% ER�protein levels, with no significant changes at HCB 5 �M (Fig. 8A).ER� levels were further decreased (85%) when cells were incubatedin the presence of ICI 182,780. This result was expected, consideringthat ICI 182,780 down-regulates the receptor by mechanisms thatinclude impaired dimerization, increased turnover and disruptednuclear localization (Howell et al., 2000). When HCB and E2 weregiven in combination, the concentration of ER� was not altered

in comparison to that of E2-treated controls, probably becausethe receptor was already down-regulated by E2, as other authorsdemonstrated (Chu et al., 2007). This down-regulation was furtherenhanced by ICI 182,780 treatment (Fig. 8B).

M.A. García et al. / Toxicology Letters 192 (2010) 195–205 201

Fig. 7. Effect of ICI 182,780 on IGF-I signaling pathway in HCB-treated MCF-7 cells. (A) IGF-IR levels. (B) IGF-IR levels in presence of E2. (C) p-Tyr/IRS-1 levels. (D) p-Tyr/IRS-1levels in the presence of E2. Cells were exposed to HCB (0.005 and 5 �M) or vehicle, in the absence or presence of 10 nM ICI 182,780 (A and C), or with 1 nM E2 or E2 plus HCB(0.005, and 5 �M) or vehicle, in the presence or absence of 10 nM ICI 182,780 (B and D), during 24 h. For IGF-IR detection, whole cell lysates were prepared, and total proteinwas resolved by SDS-PAGE and blotted for IGF-IR as described in Section 2. For IRS-1 and p-Tyr detection, whole cell lysates were precipitated with anti-IRS-1 antibodya d rebe r leveo D of th(

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nd resolved by SDS-PAGE. Membranes were blotted for total phosphotyrosine anxperiment is shown in the upper panels. Quantifications of IGF-IR (A and B) or pTyf the immunoblots are shown in the lower panels. Data are expressed as means ± S*p < 0.05, **p < 0.005, ANOVA and Tukey post hoc test).

.9. HCB-induced c-Src activation and ER˛ phosphorylation onyr537 in MCF-7

Under proliferative conditions, E2 activates the signal transduc-

ng c-Src/Ras/Erk pathway, triggering S-phase entry of the cellsMigliaccio et al., 1996). We investigated whether HCB treatmenttimulates c-Src kinase activity, using anti-phosphotyrosine-416ntibody, a specific phosphorylation site for activated c-Src (Frame,002). As shown in Fig. 9A, HCB (0.005 �M) increased Tyr416-c-Src

lotted for IRS-1 as described in Section 2. A western blot from one representativels (left axis) and p-Tyr/IRS-1 ratio (right axis) (C and D) by densitometric scanningree independent experiments. Asterisks indicate significant differences vs. control

phosphorylation (103%) and pTyr416-c-Src/total c-Src ratio (40%).By contrast, HCB (5 �M) decreased pTyr416-c-Src/total c-Src ratio(27%).

Tyrosine 537 is the only tyrosine phosphorylation present in

ER� of MCF-7 cells. This phosphorylation is required for a strongassociation of ER� with the c-Src-SH2 domain (Song et al., 2005).Western blotting of cell lysates after 24 h of exposure to HCB(0.005 �M) shows an increase on Tyr537-ER� phosphorylation(110%), and possibly due to the HCB-induced down-regulation

202 M.A. García et al. / Toxicology Letters 192 (2010) 195–205

Fig. 8. Effect of HCB on ER� protein levels in MCF-7 cells, in the absence (A) or presence (B) of E2. Cells were exposed to HCB (0.005 and 5 �M) or vehicle, in the absenceor presence of 10 nM ICI 182,780 (A), or to 1 nM E2 or E2 plus HCB (0.005 and 5 �M) or vehicle, in the presence or absence of 10 nM ICI 182,780 (B) during 24 h. Whole celllysates were prepared, and total protein was resolved by SDS-PAGE and blotted for ER�, as described in Section 2. A western blot from one representative experiment isshown in the upper panels. Quantification of ER� levels by densitometric scanning of the immunoblots is shown in the lower panels. Data are expressed as means ± SD ofthree independent experiments. Asterisks indicate significant differences vs. control (**p < 0.005, ANOVA and Tukey post hoc test).

Fig. 9. Effect of HCB on Tyr416-c-Src and Tyr537-ER� phosphorylation in MCF-7. Cells were exposed to HCB (0.005 and 5 �M) or vehicle, during 24 h. Whole cell lysateswere resolved by SDS-PAGE. Membranes were blotted for: (A) total c-Src and pTyr416-c-Src antibodies and (B) total ER� and pTyr537-ER� antibodies. Western blots fromone representative experiment are shown in the upper panels. Quantification of: (A) p-c-Src levels (left axis) and p-c-Src/total c-Src ratio (right axis), and (B) p-ER� levels(left axis) and p-ER�/total ER� ratio (right axis) by densitometric scanning of the immunoblots are shown in the lower panels. Data are expressed as means ± SD of threeindependent experiments. Asterisks indicate significant differences vs. control (*p < 0.05, **p < 0.005, ***p < 0.001, ANOVA and Tukey post hoc test).

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f ER� observed, Tyr537-ER�/total ER� rate is further increased170%). On the other hand, HCB 5 �M has no effect on these param-ters (Fig. 9B).

. Discussion

Several epidemiological studies have examined the correlationetween HCB levels in serum or breast tissue samples with can-er risk, although this relationship is controversial (Charlier et al.,003). Dewailly et al. (1994) observed that women with mam-ary adenocarcinomas showed significantly higher levels of HCB

n mammary tissue and serum than women with benign mam-ary tumors. However, the strength of evidence seems to point

o a lack of association between HCB exposure and breast cancern humans (Zheng et al., 1999). In the case of data driven in rats,

e have previously demonstrated that HCB enhances the devel-pment and malignancy of NMU-induced mammary tumors andlters insulin/IGF-I signaling pathway, with regard to IR and IGF-IRxpression as well as IRS-1 content and tyrosine phosphorylationRandi et al., 2006).

In the present study, we investigated the ability of HCB toromote cell proliferation in ER�(+) MCF-7 and ER�(−) MDA-MB-31 cell lines and alter Insulin/IGF-I signaling pathway in ER�(+)CF-7. The highest HCB dose used in the present study is in the

ame range of order as that found in serum from humans fromhighly contaminated population (To-Figueras et al., 1997). Our

esults show that HCB (0.005 and 0.05 �M) is able to significantlyncrease the proliferation of MCF-7 cell line, without effect at HCB.5 and 5 �M. In contrast, cell proliferation was not altered byCB in MDA-MB-231 cells. Increased effects at low HCB dosesnd a decline at higher doses have been observed before in ouraboratory, for EGFR levels and cytosolic protein tyrosine kinasectivity in rat liver (Randi et al., 2003). When cells were grownn an estrogen-depleted medium, HCB also stimulated prolifera-ion of MCF-7 cells only at the lower concentrations. These resultshow that the effect of HCB on cell proliferation was not dose-esponsive. HCB-induced cell proliferation was suppressed by thenti-estrogen ICI 182,780. Taken together, the experimental evi-ence presented in this study shows for the first time that HCBtimulates cell proliferation in estrogen-sensitive cells in an ER�-ependent manner. It has been demonstrated that in addition toormone-dependent ER activation, hormone-independent activa-ion also occurs in human breast cancer cells (Marsaud et al., 2003).dditionally, unlike other estrogenic compounds, HCB does notppear to have a significative affinity for the ER (Noguerol et al.,006). These results suggest that, although HCB induces estrogen-

ike responses, the intracellular pathway mediating these actionsay be different than the one used by endogenous estrogen. In a

imilar way, �-hexachlorocyclohexane induced an ER�-dependentell proliferation and pS2 gene expression in MCF-7 cells, but itas observed that its action is not through the classic pathway of

inding and activating the ER� (Steinmetz et al., 1996). It has alsoeen reported that TCDD inhibited cell growth in MDA-MB-468, anR�(−) and highly AhR-responsive breast cancer cell line (Wang etl., 1997).

Under normal circumstances, tissue homeostasis is a perfectlyhoreographed process balancing prosurvival and death signals.here are different reports demonstrating that agonists of thehR have anti-proliferative activity in MCF-7 cells (Barouki et al.,007). Only the higher doses of HCB used in this study (0.5 and

�M) increased CYP1A1 gene expression and induced apoptosis

n MCF-7 cells, suggesting that the pesticide effect on apoptosis ishR-dependent. It is known that these HCB doses are capable of dis-lacing TCDD from AhR, according to previous studies from Hahn etl. (1989). Recent results from our laboratory have shown that HCB

tters 192 (2010) 195–205 203

(1, 10 and 100 mg/kg) treatment for 30 days, induces mRNA TGF-�1expression and apoptosis in female rat thyroid gland (Chiappini etal., 2009).

Components of the IGF-I signaling pathway have been identifiedas being involved in the regulation of breast cancer cell proliferationin vivo and in vitro (Surmacz, 2000). In our study, we report that IGF-IR and IR were expressed at higher levels in MCF-7 cells exposed toHCB 0.005 and 0.05 �M, that is, HCB doses that were able to inducecell proliferation. We also observed that the pesticide increasesIRS-1 phosphorylation at all assayed doses even in the absenceof alterations in IGF-IR and IR levels. The inducing effect of HCBon IGF-I signaling in MCF-7 cells incubated in estrogen-depletedmedium, was suppressed by ICI 182,780, providing evidence thatER� is involved in HCB activation of IGF-I signaling pathway. Wecould hypothesize that increased IRS-1 phosphorylation at all HCBdoses, may be due to increased tyrosine kinase activity of IGF-IRand IR. On this respect, Tannheimer et al. (1998) found that other‘dioxin-type’ compounds, such as benzopyrene and TCDD, activatedIGF-IR by tyrosine phosphorylation, with subsequent activation ofShc and IRS-1 in the human MCF-10A mammary epithelial cell line.Another possibility is that HCB may induce interleukin-4 (IL-4), apleiotropic cytokine expressed by breast cell lines, which signalsthrough the IRS proteins (Gooch et al., 1998). Because HCB inducesan increase of IL-4 blood levels in patients occupationally exposed(Daniel et al., 2002), we could also hypothesize that HCB (0.5 and5 �M) increases IL-4 expression in MCF-7 cell line, increasing IRS-1tyrosine phosphorylation.

Several crosstalks between ER� and other signaling pathwaysmay influence the estrogenic stimulation of cell growth. Many stud-ies have shown that non-nuclear ER� can exist in complexes withseveral signaling molecules. Estrogen treatment of cells induces theinteraction between ER� and IGF-IR, which activates signaling viaERK1/2. Estrogen also stimulates the association between ER� andthe p85 subunit of PI3K, as well as its interaction with IRS-1, whichthen translocates to the nucleus where it may exist in transcriptioncomplexes. Finally, ER� can also interact with Shc, which links thereceptor to other signaling intermediates in the membrane (Schiffand Osborne, 2005). Recent observations demonstrate that in MCF-7 cells, Tyr537 of ER� is phosphorylated under basal conditions, andthat this generates a binding site for the c-Src SH2 domain (Barlettaet al., 2004).

Our study demonstrates that HCB treatment (0.005 �M) leadsto c-Src activation, ER� phosphorylation on Tyr537 and cell prolif-eration. In a similar manner, we have recently reported that HCBinduced c-Src kinase activity in rat liver and in the WB-F344 rat hep-atic cell line (Randi et al., 2008). Other authors have reported thatER� is also activated by other kinases in different phosphorylationsites (Pearce and Jordan, 2004).

Activation of c-Src induced by binding of TCDD to the AhRis known to involve translocation of c-Src protein from cytosolto the plasma membrane, which apparently triggers the growthfactor signaling cascade (Park et al., 2007). On this respect, wehave recently demonstrated that HCB triggers AhR translocationto the nucleus, c-Src activation and EGFR transactivation in ratliver (Randi et al., 2008). However, in the present study, wehave shown that HCB at low doses (0.005 and 0.05 �M) doesnot induce the expression of CYP1A1 mRNA, an AhR-dependenteffect, showing that the effect of HCB (0.005 �M) on c-Src activa-tion is not AhR-dependent. Therefore, we propose that challengingMCF-7 cells with HCB induces the assembly and activation ofthe mitogenic ER�/c-Src complex in the plasma membrane. We

could consider two possibilities to explain how HCB activates c-Src: (a) by activation of protein tyrosine phosphatase 1B (PTP1B),which is expressed in human breast cancer cell lines, and is capa-ble of dephosphorylating the negative regulatory site of c-Src(Bjorge et al., 2000); (b) by ER� itself, which is known to acti-

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ate c-Src/Shc/Ras/Erk pathway, as suggested by Varricchio et al.2007).

It has been demonstrated that, under proliferative conditions, E2ctivation of ER� by Tyr537 phosphorylation increased expressionf cyclin D1, nuclear exclusion of the CDK inhibitor p27, stimu-ation of G1-S transition and cell apoptosis attenuation of MCF-7ells. The entire network is under the control of c-Src activity andR�/androgen receptor/c-Src association (Varricchio et al., 2007).ue to the fact that in the present study we have demonstrated

hat HCB presents an estrogenic effect, we could speculate thatCB (0.005 and 0.05 �M) may increase cell proliferation stimulat-

ng G1–S transition, as well as cell apoptosis attenuation. Furthertudies are necessary to elucidate if HCB affects these processes.

Different stimuli, in addition to receptor ligands and emanatingr not from cell surface receptors, are able to alter ER� turnover.n the present study, we observe that HCB 0.005 �M induces ER�own-regulation. It has been demonstrated that c-Src stimulatesoth ubiquitination and proteasome-dependent ER� degradationChu et al., 2007). Our results suggest that ER� down-regulation

ay be induced by HCB (0.005 �M) stimulation of Tyr416 c-Srchosphorylation. In this respect, ER� from MCF-7 and progesteroneeceptor from T47-D cells were shown to be degraded through theroteasome pathway after hyperactivation of MAPKs (Brinkmannd El-Ashry, 2009). It has also been reported that, although beingoorly ubiquitinated in MCF-7 cells, ligand-free ER is also likelyo be degraded through the proteasome pathway (Marsaud et al.,003).

According to our results and studies from others authors, wepeculate that the potential estrogenic effect of HCB on MCF-7 celline, may involve a stimulation of c-Src, which in turn would acti-ate ER� by phosphorylation on Tyr 537. Activated ER� could thentimulate proliferation via an IGF signaling pathway on this celline. Another possibility is that HCB mechanism of action on c-Srctimulation could be mediated by ER� itself, which is known toctivate c-Src/Shc/Ras/Erk pathway, as suggested by Varricchio etl. (2007). Finally, HCB treatment of cells may induce the interac-ion between ER� and IGF-IR, which activates signaling via ERK1/2Kahlert et al., 2000). While we speculate that the effects of c-Srcn ER� signaling crosstalk and transcriptional activity may play anmportant role in HCB-induced cell proliferation, further work inhis area is required.

The above studies suggest that HCB has some potential xenoe-trogenic effects, the intracellular mechanism of action of whichs different from the classic ER-mediated pathway used by E2 ornvironmental estrogens such as o,p′-DDT. HCB fails to exhibit sev-ral properties that are characteristic of true estrogens, such asompetition for high-affinity ER binding sites.

In conclusion, our results show for the first time that HCBtimulates IGF-I signaling pathway and cell proliferation in MCF-cells in an ER�-dependent manner. In addition, we show thatCB (0.005 �M) induces c-Src activation and ER� phosphorylation

n Tyr537. Taken together, our results provide a clue to the molecu-ar events involved in the mechanism of action of HCB in mammaryancer development.

onflict of interest statement

The authors declare that there are no conflicts of interest.

cknowledgements

Many thanks are given to Rodolfo Kölliker-Frers for his technicalssistance. This work was supported by grants from the Nationalgency of Scientific and Technological Promotion (PICT 05-25849),ational Council of Scientific and Technological Research (CON-

tters 192 (2010) 195–205

ICET) (PIP6060); and University of Buenos Aires (PIDs M041 andM092). Andrea Randi, Diana Kleiman de Pisarev, Claudia Cocca andRosa Bergoc are established researchers of the CONICET. RodolfoKölliker-Frers is Technical Professional of the CONICET.

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