Reagents. We purchased bisphenol A (BPA), N-[2-(p-bromocinnamylamino)ethyl]-5-soquinolinesulfonamide dihydrochloride (H89), PD98059 (PD), and arsenic trioxide (As2O3) from Sigma-Aldrich (Milan, Italy); AG1478 (AG) from Biomol Research Laboratories (DBA, Milan, Italy), and 1-(4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)-ethanone (G-1) from Calbiochem (Merck KGaA, Frankfurt, Germany). As2O3 was dissolved in phosphate-buffered saline, and BPA and PD were dissolved in ethanol; AG1478, H89, and G-1 were solubilized in dimethyl sulfoxide (DMSO).
Cell culture. SKBR3 cells. SKBR3 human breast cancer cells were maintained in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were changed to medium without serum the day before experiments for immunoblotting and reverse-transcription polymerase chain reaction (RT-PCR).
CAFs. CAFs were extracted as previously described (Madeo and Maggiolini 2010
). Briefly, breast cancer specimens were collected from primary tumors of patients who had undergone surgery. Signed informed consent was obtained from all the patients and from the institutional review board(s) of the Regional Hospital of Cosenza. Tissues from tumors were cut into smaller pieces (1–2 mm diameter), placed in digestion solution (400 IU collagenase, 100 IU hyaluronidase, and 10% serum, containing antibiotic and antimycotic solution), and incubated overnight at 37°C. The cells were then separated by differential centrifugation at 90 × g
for 2 min. Supernatant containing fibroblasts was centrifuged at 485 × g
for 8 min; the pellet obtained was suspended in fibroblasts growth medium (Medium 199 and Ham’s F12 mixed 1:1 and supplemented with 10% FBS) and cultured at 37°C in 5% CO2
. Primary cells cultures of breast fibroblasts were characterized by immunofluorescence. Briefly cells were incubated with human anti-vimentin (V9) and human anti-cytokeratin 14 (LL001), both from Santa Cruz Biotechnology DBA (Milan, Italy). To assess fibroblasts activation, we used anti-fibroblast activated protein α (FAPα) antibody (H-56; Santa Cruz Biotechnology DBA) (data not shown).
SKBR3 cells and CAFs were grown in 10-cm dishes, exposed to treatments or ethanol (or DMSO), which was used as the vehicle, and then lysed as described previously (Pandey et al. 2009
). Protein concentrations were determined using Bradford reagent (Sigma-Aldrich) according to the manufacturer’s recommendations. Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Milan, Italy). Membranes were probed overnight at 4°C with antibodies against c-Fos (H-125), β-actin (C-2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; E-4), Egr-1 (588), CTGF (L-20), ERK2 (C-14), ERα (F-10), or GPR30 (N-15), all from Santa Cruz Biotechnology, DBA (Milan, Italy), or ERβ from Serotec (Space Import Export, Milan, Italy). Results of densitometric analyses of Western blots, obtained using ImageJ software (Abramoff et al. 2004
), are presented as optical density (OD; expressed in arbitrary units) relative to the control (ERK2 or β-actin).
Plasmids and luciferase assays. The Ctgf luciferase reporter plasmid p(-1999/+36)-luc, which is based on the backbone of vector pGL3-basic (Promega), was a gift from B. Chaqour (Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY, USA). The luciferase reporter plasmid for c-FOS encoding a –2.2-kb 5´ upstream fragment of human c-FOS was a gift from K. Nose (Department of Microbiology, Showa University School of Pharmaceutical Sciences, Hatanodai, Shinagawa-ku, Tokyo, Japan). The EGR-1 luciferase reporter plasmid pEgr-1A, which contains the –600 to +12 5´-flanking sequence from the human EGR-1 gene was a gift from S. Safe (Department of Veterinary Physiology and Pharmacology, Texas A&M University, Houston, TX, USA). For the luciferase assays, cells were transferred into 24-well plates containing 500 μL of regular growth medium per well the day before transfection. On the day of transfection, SKBR3 cell medium was replaced with RPMI without phenol red and serum, and transfection was performed using Fugene6 Reagent (Roche Molecular Biochemicals, Milan, Italy) and a mixture containing 0.5 μg of each reporter plasmid. Renilla luciferase (pRL-CMV; 1 ng) was used as a transfection control. After 5–6 hr, ligand was added and cells were incubated for 16–18 hr. We measured luciferase activity using the Dual Luciferase Kit (Promega, Milan, Italy) according to the manufacturer’s recommendations. Firefly luciferase values generated by the reporter plasmid were normalized to Renilla luciferase values. Normalized values obtained from cells treated with ethanol vehicle were set as 1-fold induction, and the activity induced by treatments was calculated based on this value.
RT-PCR and real-time PCR.
Total RNA was extracted using Trizol commercial kit (Invitrogen, Milan, Italy) according to the manufacturer’s protocol. RNA was quantified spectrophotometrically, and cDNA was synthesized from the RNA by reverse transcription using murine leukemia virus reverse transcriptase (Invitrogen). We quantified the expression of selected genes by real-time PCR using SYBR Green as the detection method and the Step One sequence detection system (Applied Biosystems Inc., Milan, Italy). Gene-specific primers were designed using Primer Express software (version 2.0; Applied Biosystems Inc.). Assays were performed in triplicate. We used mean values to calculate expression levels by the relative standard curve method. For the sequences of primer used, see Supplemental Material, Table S1(http://dx.doi.org/10.1289/ehp.1104526
Gene silencing experiments.
Cells were plated onto 10-cm dishes, maintained in serum-free medium for 24 hr, and then transfected for an additional 24 hr before treatments using Fugene6. The short hairpin (sh) RNA constructs to knock down the expression of GPER
and the unrelated shRNA control construct have been described previously (Pandey et al. 2009
Wound-healing assay. CAFs were seeded into 12-well plates in regular growth medium. When at 70% to 80% confluency, the cells were transfected with shGPER using Fugene6 reagent for 24 hr. Transfected cells were washed once, medium was replaced with 2.5% charcoal-stripped FBS, and cells were treated. We then used a p200 pipette tip to scratch the cell monolayer. In experiments performed using conditioned medium, CAFs were plated into 12-well plates and transfected with 500 ng shRNA control plasmid or shGPER or shCTGF plasmids using Fugene6, as recommended by the manufacturer. After 24 hr, CAFs were treated with 1 μM BPA, and the conditioned medium was collected and filtered through a sterile nonpyrogenic 0.2 μm filter. The conditioned medium obtained was added to subconfluent SKBR3 cells, and a series of scratches were made using a p200 pipette tip. We evaluated cell migration in three independent experiments after 48 hr of treatment; data are expressed as a percentage of cells in the wound area upon treatment compared with cells receiving vehicle.
Proliferation assay. SKBR3 cells and CAFs were seeded in 24-well plates in regular growth medium. After cells attached, they were washed, incubated in medium containing 2.5% charcoal-stripped FBS, and transfected with 500 ng shGPER or control shRNA plasmids using Fugene6 reagent. After 24 hr, cells were treated with 1 μM BPA, and the transfection and treatment were renewed every 2 days. We counted the cells using the COUNTESS automated cell counter (Invitrogen) following the manufacturer’s recommendations.
Statistical analysis. For statistical analysis, we used analysis of variance followed by Newman-Keuls testing to determine differences in means. p-Values < 0.05 are considered statistically significant.