Bisphenol A stimulates human prostate cancer cell migration via remodelling of calcium signalling
© Derouiche et al.; licensee Springer. 2013
Received: 19 October 2012
Accepted: 4 February 2013
Published: 15 February 2013
Bisphenol A (BPA), the principal constituent of reusable water bottles, metal cans, and plastic food containers, has been shown to be involved in human prostate cancer (PCa) cell proliferation. The aim of the present study was to explore the effect of BPA on PCa cell migration and the pathways involved in these processes. Using the transwell technique, we clearly show for the first time that the pre-treatment of the cells with BPA (1–10 nM) induces human PCa cell migration. Using a calcium imaging technique, we show that BPA pre-treatment induces an amplification of Store-Operated Calcium Entry (SOCE) in LNCaP cells. RT-PCR and Western blot experiments allowed the identification of the ion channel proteins which are up-regulated by BPA pre-treatments. These include the Orai1 protein, which is known as an important SOCE actor in various cell systems, including human PCa cells. Using a siRNA strategy, we observed that BPA-induced amplification of SOCE was Orai1-dependent. Interestingly, the BPA-induced PCa cell migration was suppressed when the calcium entry was impaired by the use of SOCE inhibitors (SKF96365, BTP2), or when the extracellular calcium was chelated. Taken together, the results presented here show that BPA induces PCa cells migration via a modulation of the ion channel protein expression involved in calcium entry and in cancer cell migration. The present data provide novel insights into the molecular mechanisms involved in the effects of an environmental factor on cancer cells and suggest both the necessity of preventive measures and the possibility of targeting ion channels in the treatment of PCa cell metastasis.
Prostate cancer (PCa) is the most common non-cutaneous malignancy diagnosed in men and the metastatic PCa forms represent the second cause of mortality ([Gronberg 2003]; Jemal et al. ). Early PCa requires androgen to survive and to proliferate; this dependence is exploited in the treatment of a disseminated disease, where androgen ablation is the first line of therapeutic intervention. Although these regimens are initially effective, tumors ultimately recur due to reactivation of Androgen Receptor (AR) signalling, causing treatment failure and patient morbidity. Despite the importance of understanding androgen action in the prostate, little is understood about the mechanisms underlying androgen independence, and the means by which the androgen requirement is bypassed in relapsed tumors. As such, identifying the factors affecting the efficacy of androgen deprivation therapy is essential in order to improve the outcome of PCa treatment and thereby to increase patient survival.
Accruing evidence indicates that exposure to environmental compounds, “endocrine disrupting compounds”, or EDCs, may adversely impact human health through multiple mechanisms, including alterations to the hormone receptor function ([Henley and Korach 2006]; Welshons et al. ). In humans, a putative link has been established between an increased abundance of EDCs in the environment and rising hormone-dependent cancer incidence (Huff et al. ). Thus, recent investigations have placed particular emphasis on delineating the consequence of EDC exposure for various tissues including the reproductive tissues.
One such environmental factor is bisphenol A (BPA), a non-planar plasticizer leached in microgram quantities from polycarbonate plastics and epoxy resins into food and water supplies (Welshons et al. ). Studies showed that up to 95% of adults have detectable BPA in their urine (Calafat et al. ), with adult serum concentrations reported to range in nanomolar concentrations [reviewed by (Welshons et al. ). Further, BPA has been shown to be involved in prostate carcinogenesis. A recent animal studies showed that perinatal exposure to BPA at low doses results in increased sensitivity to estrogen as the male animal ages, and to an increased risk of developing PCa (Ho et al. ). At environmentally relevant levels (1nM), BPA has also been identified as a mitogen for a subset of PCa cell lines (Wetherill et al. ; Wetherill et al. ; Wetherill et al. ), in addition to accelerating tumor growth after androgen ablation (Wetherill et al. ). BPA was also shown to induce the growth and resistance to apoptosis of human breast cancer cells, suggesting that hormone-dependent tissues are affected by this environment factor (LaPensee et al. ; Pupo et al. ).
Another aspect of tumor cell evolution is their metastasis, which is the major cause of death from PCa. Invasion of surrounding tissue and vasculature by cancer cells is an initial step in tumor metastasis. Environmental factors such as BPA could impact PCa metastasis by inducing cell migration. However, data on the effects of BPA on human cancer cell migration are lacking.
Accumulating data show that cell proliferation, apoptosis and migration are paralleled by an altered function and/or expression of ion channels involved in the signalling of fundamental cellular mechanisms (Lang et al. ; Prevarskaya et al. ). A ubiquitous Ca2+ influx pathway that is activated by intracellular Ca2+ store depletion is store-operated Ca2+ entry (SOCE), which is activated through a complex interplay between a Ca2+ channel at the cell membrane, Orai1, and a Ca2+ sensor located in the endoplasmic reticulum, STIM1 ([Courjaret and Machaca 2012]). Recently, a number of known molecular players in cellular Ca2 homeostasis, including Orai1, STIM1 and transient receptor potential (TRP) channels have been implicated in tumor cell migration and the metastatic cell phenotype (for review see Prevarskaya et al. ). In this context, we previously showed that TRPC1, TRPV6 and Orai1 are the main actors in SOCE in human PCa cells LNCaP (Flourakis et al. ; Vanden Abeele et al. [2003a,, b]).
Here, for the first time, we investigated the impact of BPA on human PCa cell migration and the mechanisms involved in the effects of BPA in these cells. In the latter context, we studied the impact of BPA on calcium signalling, on the expression of ion channels involved in SOCE and human PCa cell migration in the absence of androgens.
BPA increases the migration of prostate cancer cells
Effects of BPA on calcium signalling in prostate cancer cells
We further examined the modification of the calcium signalling and remodelling of the expression of the ion channels in PCa cells after a pre-treatment with BPA. To verify the possible modification of the calcium signalling in BPA-treated cells, a test, as described in the following section, was used to compare the rate of calcium entry in control and in BPA-treated cells.
Ca2+ entry, but not Ca2+ release, is increased in BPA- treated LNCaP cells
We previously showed that the application of the store-depleting SERCA inhibitor thapsigargin (TG) induces a calcium mobilization from intracellular stores and a calcium entry due to SOCE in human PCa cells (Lallet-Daher et al. ).
The two phases of free intracellular calcium concentration ([Ca2+]i) changes were separated using a Ca2+ add-back protocol. The addition of the store-depleting SERCA inhibitor thapsigargin (TG, 200 nM) in nominally Ca2+-free solution was followed by rapid, transient increases in [Ca2+]i, as measured by calcium imaging (Figure 2B) These increases are due to the mobilization of Ca2+ from internal stores. A subsequent addition of Ca2+ to the extracellular bath resulted in a rapid and sustained increase in cytosolic Ca2+ due to SOCE. Analyses were also performed on both the rate of the TG-induced calcium mobilization and the amplitude of SOCE in CTL cells versus those treated with different concentrations of BPA for 24 and 48 h. In these analyses, the amplitude and the decay of the calcium mobilization were not significantly different between CTL and treated cells (Figure 2B, 2D and 2F). Interestingly, in these experiments, we observed that when the cells were cultured in a steroid-free medium (CS-RPMI), the amplitude of the SOCE was significantly lower (40 to 50%) than that developed in cells cultured in normal RPMI containing steroids (data not shown). TG-mediated Ca2+ signalling was then studied in LNCaP treated with BPA for 24, and 48 h. At 24 h (Figure 2B), the amplitude of the Ca2+ entry (SOCE) was 40 ± 15% higher than that developed in the cells cultured for the same periods in CS-RPMI alone (CTL). These effects of BPA reached 85 ± 19% and 122 ± 19% when the cells were pre-treated for 48 h by BPA 1 nM (Figure 2C and 2D) and 10 nM (Figure 2E and 2F) respectively. Similar SOCE amplifications were observed for LNCaP-C4.2, a more invasive cell line derived from LNCaP cells (Figure 3B and 3C). These observations suggest that BPA modulates the expression of the ion channels involved in the SOCE in human PCa cells. The identification of these ion channels could allow a schematization of the mechanism by which BPA modulates these cancer cells migrations.
BPA up-regulated ion channels expression in prostate cancer cell lines
Our previous studies showed that of several ion channels, it was mainly the calcium channels (Orai1/STIM1, TRPV6) and potassium channels (IKCa1, BKCa) that were involved in the generation of the SOCE in human PCa cells (Flourakis et al. ; Lallet-Daher et al. ; Vanden Abeele et al. [2003a,, b]). Thus, experiments were designed to explore the impact of a 48 h exposure of the cells to BPA on the expression of these calcium and potassium channels involved in the SOCE. In the present study, in order to eliminate the impact of steroids, and androgens in particular, the experiments were performed in Phenol red-free RPMI 1640 containing charcoal-stripped Foetal Calf Serum (FCS) (CS-RPMI or -ST), a steroid-deprived medium. We have previously shown that the expression of Orai1 was at least decreased by 90% in this steroïds-free medium (Flourakis et al. ).
Involvement of Orai1 in BPA-induced modification of calcium signalling
Pharmacological tools were also used to study the involvement of Orai1 in BPA-induced SOCE amplification. LNCaP cells were treated with BPA (1nM, 48h) and then the TG-induced SOCE was studied in the presence or absence of the inhibitors. When the experiments were performed using a pyrazole derivative, BTP2 (2 μM), known to inhibit calcium channels involved in SOCE including Orai1 (Eltit et al. ), the TG-induced SOCE amplitude in BPA-treated cells was inhibited by at least 40% (Figure 6E). In the same manner, when the experiments were performed in the presence of an inhibitor of store-operated Ca2+ entry (SKF96365, 10 μM), the TG-induced SOCE was almost completely inhibited in BPA-treated cells (Figure 6F). Taken together, these data suggest that the up-regulation of Orai1 is involved in the amplification of the calcium entry induced by BPA.
Involvement of calcium entry in BPA-induced cell migration
BPA increases the migration of androgen-independent prostate cancer cells
It has been suggested that the environmental factor BPA may play an important role in the initiation (Ho et al. ) and progression of PCa and in hormonal therapy bypass (Wetherill et al. ). At the level of PCa cells, BPA was able to induce androgen-independent tumor cell proliferation and reduced therapeutic efficacy in xenograft models (Wetherill et al. ). While these data point toward the potential for BPA to assist tumor cells in escaping therapy, the molecular mechanisms of this process were not well-known.
This report demonstrates for the first time that the environmentally relevant concentrations of BPA (1–10 nM) induce cell migration by modulating the cell calcium signalling. These data highlight the previously unrecognized action of BPA in the progression of human PCa, thereby providing strong support for the growing recognition of the adverse effects of BPA on human health.
Invasion and metastasis are major events underlying cancer morbidity and mortality (Molloy and Van ’t [Veer 2008]). Because of the widespread metastasis in advanced cancer patients, where a resistance is observed to conventional therapies, the mortality rate remains extremely high and warrants new strategies to intervene in the metastatic cascade. Thus, an enhanced understanding of the molecular events in the pathogenesis of PCa will offer improved diagnosis, prognosis, therapy and prevention measures of the disease, that will ultimately help us to eliminate PCa metastasis. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. One approach could be to focus on the intracellular signalling pathways underlying the metastatic process. Several data have clearly shown the involvement of calcium entry in cancer and non-cancer cell migration (Bisaillon et al. ; Li et al. ; Schaff et al. ; Yang et al. ).
In the present work, we present conclusive evidence for the first time that the pre-treatment of human PCa cells with environmentally relevant concentrations of BPA (1–10 nM) induces their migration (Figure 1). By calcium imaging technique, we show that BPA pre-treatment induces an amplification of Store-Operated Calcium Entry (SOCE) in LNCaP cells (Figure 2). RT-PCR and Western blot experiments allowed us to identify those ion channel proteins up-regulated by BPA pre-treatments. These channels include Orai1, a protein known to constitute an important actor in SOCE in various cell systems including human PCa cells (Figure 4). Meanwhile, in our studies, we failed to observe any direct effect of BPA on the rate of basal calcium (Figure 2) whereas in other cell systems, BPA or its derivatives induced a calcium increase. In TM4 Sertoli cells, a direct application of a derivative compound of BPA, Tetrabromobisphenol A (TBBPA), a commonly used brominated flame retardant (BFR), induced an increase in the basal free calcium rate originating from internal stores (Ogunbayo et al. ). In pituitary tumor cells (GH3/B6/F10 rat Somatomammotropes), Kochukov et al. () showed that BPA at a 1 nM concentration induced a great increase in Ca2+ oscillation frequency, the activation of MAPK pathways (ERK1/2) and subsequently a PRL release (Kochukov et al. ). Similar results were reported by Bulayeva et al. () and Wozniak et al. () where the authors demonstrated that the BPA-induced Ca2+ influx was strictly dependent on membrane Estrogen Receptor (mER-α) and mediated by L-type voltage-gated Ca2+ channels in pancreatic β cells (Bulayeva et al. ; Wozniak et al. ). The LNCaP cells used in our present work do not express L-type voltage-gated Ca2+channels (non-excitable cells). This is probably the reason why a direct application of BPA on LNCaP and LNCaP C4.2 cells failed to induce a direct calcium response.
In the present work, when the human PCa cells were pre-treated with BPA (1–10 nM), an increase in SOCE and a remodelling of ion channel expression was observed. The alterations of the ion channel expression could be mediated by a stimulation of a signal transduction pathways leading to the activation of nuclear transcription factors. Published data suggest several transduction pathways activated by BPA. In PCa cells, BPA has been shown to be an agonist for mutant androgen receptor (AR-T877A) expressed in recurrent PCa (Wetherill et al. [2002,, 2005,, 2006]), and in the LNCaP cell line used in our studies. According to the authors, BPA induces cell proliferation in cells expressing the mutated AR. The clinical ramifications of BPA activating tumor-derived mutant ARs and inducing androgen-independent tumor cell proliferation may be substantial, as BPA can reduce therapeutic efficacy in xenograft models (Wetherill et al. ). In our experiments, the DHT induced the expression of Orai1 (Figure 4C) and BPA appears to mimic these effects on the canonical AR ligand (DHT). However, all the effects of BPA could not be mediated by the activation of AR. In a recent work, Hess-Wilson et al. () showed clearly that BPA and DHT elicited distinct transcriptional signatures in PCa cells expressing the BPA-responsive mutant AR-T877A, even if some common genes were activated by both DHT and BPA in LNCaP cells (Hess-Wilson et al. ). These observations could explain the cell migration and Orai1 expression induced by BPA in androgen-independent human PCa cells PC-3 (Figure 8), where the AR is absent. BPA could thus activate other signal transduction pathways than the AR activation, to induce the effects observed in our studies on androgen-insensitive PCa cells. The study of the involvement of other transduction pathways than those involving the AR receptor (growth factor signalling pathways, …) in the effects of BPA in androgen-dependent and androgen-independent cells needs further extensive investigations in the future. In this context, BPA was reported to induce the phosphorylation of extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear translocation of the nuclear factor (NF)-κ B, in mouse hippocampal HT-22 cells (Lee et al. ). Interestingly, functional NF-κ B-binding sites in promoter regions of STIM1 and Orai1 have been identified and the expression of the Orai1 calcium channel was reported to be positively modulated by NF-κB (Eylenstein et al. ). Subsequently, the store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52, and decreased by treatment with the NF-κ B inhibitor, Wogonin. BPA could thus interfere with the growth factors' signal transduction to activate the PI3K/AkT pathway and thereby induce the activation of the NF-k B transcription factor, leading in turn to an up-regulation of the expression of ion channels including Orai1. In this context, authors have shown that the activation of mER-α induces the activation of the PI3K/Akt signalling cascade (Simoncini et al. ; Stirone et al. ) and BPA is shown to activate mER-α (Bouskine et al. ; Quesada et al. ). The activation of the mER-α in androgen-dependent (LNCaP) and androgen-independent (PC-3) PCa cells may thus induce the PI3K/Akt signalling cascade which leads to the activation of the NF-kB transcription factor and Orai1 gene expression. Several works have also demonstrated the stimulation of the PKA/CREB pathways by nanomolar concentrations of BPA through the activation of mER-α (Bouskine et al. ; Quesada et al. ). However, the involvement of this pathway in the expression of ion channels needs further investigation. As shown in Figure 5, BPA pre-treatment induced an increase in BKCa and IKCa1 Ca2+-activated potassium channel expression in LNCaP cells. We previously showed that the IKCa1 Ca2+-activated potassium channels are involved in SOCE in LNCaP and PC-3 human PCa cells (Lallet-Daher et al. ). These potassium channels could constitute a functional complex with Orai1 protein to promote calcium entry and cell migration. A study is in progress in our lab to show the functional up-regulation and involvement of these Ca2+-activated potassium channels (IKCa1 and BKCa) in cell migration in the BPA-treated LNCaP and PC-3 cancer cells.
Receptor-mediated activation of phospholipases C by the factors present in the serum leads to IP3-mediated depletion of Ca2+ from the ER, which in turn stimulates Ca2+ influx through the plasma membrane involving Orai1/STIM1 complex formation (Varnai et al. ). Recent works have elegantly demonstrated that STIM1, Orai1, and SOCE play critical roles in the migration of a number of cell types in cancer and non-cancer cells (Bisaillon et al. ; Li et al. ). These observations support our data where BPA, by up-regulating the ion channels expression increases the SOCE developed by PCa cells in response to factors present in serum.
Our data show clearly that the BPA-induced cell migration is dependent on the calcium entry and the use of pharmacological tools suggests the involvement of SOCE channels in the effects of BPA on cell migration (Figure 7). Increase in cytoplasmic calcium induced by BPA may have several types of impacts which trigger cell migration, including the induction of the up-regulation of their gene and protein expression, secretion and the activation of the enzymes such as metalloproteinases (MMP2, MMP9), which are involved in cell migration. The MMP proteins are clearly shown to be dependent on calcium for their expression (calcium/Calcineurin/NFAT, …), their processing and their activity (Collier et al. ; Mukhopadhyay et al. ; Stetler-Stevenson et al. ). These observations suggest that the increase in calcium entry induced by BPA pre-treatment could promote all these processes leading to cell migration.
For the first time, we also demonstrate the expression of Orai1 proteins in human PCa tissues (Figure 4F). As shown, a strong immuno-staining of Orai1 protein was found in epithelial cells of the acini and also in the stromal cells. Thus, stromal cells could also be influenced by BPA impregnation. Given the importance of the epithelium-stroma (reactive stroma) in the progression of cancer, the potential effects of BPA on calcium signalling and on the secretion of growth factors by these cells need further investigation.
BPA is consistently detected in almost all individuals in developed nations (Welshons et al.), suggesting that humans are continuously exposed to BPA. In addition, the rapid metabolic clearance of BPA and its detectable levels in human blood and urine suggest that the intake of BPA may be higher than indicated by diverse studies and that long-term daily intake may lead to its bioaccumulation, leading to adverse effects on human health and on cancer progression.
These observations suggest that the BPA concentrations used in the present study are attainable in humans. The present data provide novel insights into the way in which the molecular mechanisms involved in the effects of environmental factors can promote the progression of the cancer in an androgen-independent manner. Our work also highlights the urgency of taking preventive measures and suggests some potential therapeutic opportunities of targeting the ion channels involved in SOCE (Orai1) in order to prevent the PCa cell growth and metastasis.
Chemicals and antibodies
Bisphenol A (BPA) was obtained from Sigma-Aldrich and dissolved in DMSO. Antibodies raised against human ion channel proteins were obtained from commercial sources as follows: Rabbit anti-Orai1 (ProSci Inc.), Rabbit anti-STIM1 (ProSci Inc.), Rabbit anti-TRPC1 (Alomone Labs), Rabbit anti-TRPV6 (Santa Cruz Biotechnology) and Rabbit anti-β-actin (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).
LNCaP, LNCaP-C4.2 and PC-3 PCa cell lines, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in RPMI 1640 and serum as described by Gackière et al. (Gackiere et al. ). For BPA experiments, the cells were treated with Phenol red-free RPMI 1640 containing charcoal-stripped Foetal Calf Serum (FCS) (CS-RPMI). In order to avoid the interference between BPA added for our studies and those leached from RPMI 1640 commercialized in polycarbonate bottles, the medium was prepared in glass bottles using RPMI 1640 powder commercialized by SIGMA (L’Isle d’Abeau, France) in ultrapure water and then filtered on 0.2 μm filters (Thermo Scientific Nalgene, Fontenay-sous-Bois France).
RT–PCR analysis of mRNA expression
Total RNA isolation and RT-PCR experiments were performed as described earlier (Roudbaraki et al. ). The PCR primers (Orai1: 5’-CTTCTTCGACCTCGTCCTCCT-3’ and 5’-CGTAAGGCCAAAGCATGGAA-3’; β-actin : 5’-CAGAGCAAGAGAGGCATCCT-3’ and 5’-GTTGAAGGTCTCAAACATGATC-3’ used in this study were designed on the basis of established GenBank sequences and synthesized by Invitrogen (Carlsbad, CA, USA). The amplified PCR products were of 406 and 212 bp respectively.
For siRNA experiments, equal numbers of cells from the same culture were seeded, transfected overnight with 20 nM of control siRNA (targeting Luciferase mRNA) (Eurogentec, Belgium), or raised against Orai1 mRNA (siOrai1) (5′-UGAGCAACGUGCACAAUCU (dTdT)-3′), using Hyperfect transfection reagent (Qiagen Inc., Courtaboeuf, France) in CS-RPMI containing 10% SVF, according to the manufacturer's instructions. Medium was changed after 24 h and cells were incubated for a further 48h with or without BPA, before performing calcium imaging experiments. We previously showed the efficiency of the siOrai1 used in the present study, in down-regulating the expression of the Orai1 protein in LNCaP cells (Flourakis et al. ).
Orai1 immunofluorescence studies
The protein expression studies of the ion channels in PCa cells were determined by indirect immunofluorecence analysis performed on acetone-fixed cells. Cells grown on glass cloverslips were incubated with PBS containing 0.2% BSA, 0.1% TritonX-100 and 5% donkey serum, for 30 min at room temperature, in order to block the non-specific bindings and to permeabilize the cells. They were then incubated overnight at 4°C with PBS/5% non-immunized serum containing a 1:50e dilution of the primary affinity-purified rabbit anti-Orai1 polyclonal antibody. Cells were then washed with PBS and were incubated with the secondary Alexa fluor 488-labeled anti-rabbit IgG (A-21206; Molecular Probes; dilution 1:2000e) diluted in PBS for 1 h at room temperature. After rinsing three times in PBS, the slides were mounted with Mowiol and the distribution of the labelled proteins was analysed by confocal immunofluoresccence microscopy (Zeiss LSM 510; acquisition parameters: objective 40x/1.3; thickness of confocal slide, 1 μm).
For the immunofluorescence studies of Orai1 protein in human PCa, tissues were obtained from consenting patients following local ethical considerations. The tissues were diagnosed as cancerous or not by anatomopathological examinations. Tissues were from patients prior to any anticancer therapy (chemotherapy, radiotherapy) and were obtained following an office procedure, frozen in liquid nitrogen-cooled isopentane and kept in “Tissue-Tek®” at −80°C before 10 μm cryosections were carried out at −20°C with a cryostat and mounted on glass slides for immunofluorescence studies. All experiments involving patient tissues were carried out under approval number “CP 01/33”, issued by the “Comité Consultatif de Protection des Personnes dans la Recherche. The immunofluorescence experiments for the detection of Orai1 on 7 μm cryosections was carried out following the same procedure as for the PCa cell lines, using anti-Orai 1 antibody and analysed by confocal microscopy.
Western blot assay
Cells cultured at 80% confluence were harvested and total proteins extracted. 40 micrograms of each sample were analysed by SDS-PAGE on 10% acrylamide and processed for western-blotting using antibody as described by (Vanoverberghe et al. ) using BKca (Alomone, 1:500e), TRPV6 (Alomone, 1:500e), TRPC1 (Alomone, 1:500e), Orai1 (ProSci 1:500e), STIM1 (ProSci, 1:500e), IKCa1 (Santa Cruz, 1:200e). Western blotting was performed with an ECL chemiluminescence kit (Millipore). Quantitative evaluation of protein expression was performed using ImageJ software.
Cells were grown on glass coverslips for [Ca2+]i imaging experiments and, before each experiment, the cells were loaded with Fura-2, by adding 2 μM Fura-2 AM (Fura-2 Acetoxymethyl esther) (Calbiochem, Meudon, France) to the culture medium for 45 min at 37°C. The cells cells were then washed three times in HBSS (Hanks Balanced Salt Solution; 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 340 μM Na2PO4, 440 μM KH2PO4, 10 mM Hepes, 5.6 mM glucose and buffered to pH 7.4). When a Ca2+-free medium was required, CaCl2 was omitted and replaced by equimolar MgCl2. The fluorescent intensity of Fura-2 in each cell was monitored and recorded at 340 and 380 nm. To represent the variation in the intracellular free calcium concentration, either the fluorescence intensity ratio represented by F340/F380 was used as an indicator of changes in cytosolic Ca2+ concentrations, or a calibration was used to represent such variations in nM. All measurements shown are averages of 35–45 cells from a minimum of four experiments on different cell cultures.
Cell migration assays
Cell migration assays were performed in duplicate in modified Boyden chambers. These assays consisted in counting cells migrating through a porous membrane with 8 μm pores (BD Biosciences, Oxford Science Park, Oxford, UK). After trypsinisation, cells in suspension (1 × 105) were loaded into the upper chamber in phenol-red RPMI without FCS. The lower chamber contained RPMI and 10% charcoal-stripped (CS)-FCS (CS-RPMI). The upper and lower chambers contained the same concentration of BPA when tested. After 16 h to 24 h at 37°C in a 5% CO2 incubator, cells that had attached but not migrated were scraped from the upper surface, the membranes were fixed in 70% methanol at −20°C and the migrated cells were stained for nuclei with Hoechst 33342 dye (1 μg/mL) (blue fluorescent) and evaluated by counting cell nuclei in 10 randomly chosen fields under fluorescence microscopy. The results are presented as a percentage of control (CTL), where cells were incubated in CS-RPMI culture medium alone. Alternatively, the Wound Healing Assay was also used to study PCa cell migration. The cells were seeded in a 12-well plate (15 × 104). After the cells formed a confluent mono-layer, scratches were performed using a 100 μl tip. The wells were washed with PBS followed by the addition of BPA at different concentrations in CS-RPMI. The closure of scratch was analyzed under the microscope and images were captured 1 and 15 or 24 h after incubation in the presence or absence of BPA.
Plots were produced using Origin 5.0 (Microcal Software, Inc., Northampton, MA). Results are expressed as mean ± S.E. Statistical analysis was performed using unpaired t tests or ANOVA tests followed by either Dunnett (for multiple control versus test comparisons), or Student-Newman-Keuls post-tests (for multiple comparisons). The Student’s t-test was used for statistical comparison of the differences and p < 0.05 was considered significant.
We would like to thank E Richard (BICel – IFR 147) for the technical assistance in image analysis by confocal microscopy, M Masurelle for assistance in the preparation of the manuscript and H Selliez for reading and language corrections of the manuscript.
This work was supported by grants from the Région Nord Pas-de-Calais, INSERM, the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, La Ligue Nationale Contre le Cancer. S. Derouiche was supported by the Région Nord Pas-de-Calais and Association pour la Recherche sur les Tumeurs de la Prostate (ARTP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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