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TOXLET 8824 1–9 Toxicology Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
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Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3 K/Akt signalling pathways
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Anna Ptak * , Marta Hoffman, Izabella Gruca, Justyna Barc
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Department of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland
H I G H L I G H T S
BPA stimulated OVCAR-3 cell migration. BPA up-regulated the migration-related factors MMP-2, MMP-9 and N-cadherin. Inhibition of ERK1/2 and Akt pathways abolishes BPA-induced cell migration.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online xxx
Bisphenol A (BPA), is present in a multitude of products, including food and water containers, food can linings, dentistry sealants, and thermal paper. BPA can induce the growth of human ovarian cancer cell lines. Reduction of adhesion and the initiation of metastasis are important events in cancer progression; therefore, this study investigated the effects of BPA (0.1–100 nM) on the migration of OVCAR-3 ovarian cancer cells and the expression levels of metalloproteinases (MMPs) and cadherins. The oestrogenic compound 17b-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. BPA stimulated cell migration, and the effect of BPA was similar to that of 17b-estradiol. BPA-induced cell migration was accompanied by up-regulation of the migration-related factors MMP-2, MMP-9 and N-cadherin, but E-cadherin expression and activity was unaffected. The stimulatory effects of BPA on cell migration were abolished by pre-treatment of the cells with inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways (PI3K). In conclusion, the results presented here show that BPA induces OVCAR-3 cells migration by activating MAPK and PI3K/Akt signalling pathways. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Bisphenol A Migration ERK1/2 Akt OVCAR-3
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1. Introduction
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Bisphenol A (BPA) is widely used as an ingredient in industrial and consumer products, including food and water containers, food can linings, dentistry sealants, and thermal paper. Epidemiological studies have reported that up to 95% of adults have detectable levels (nanomolar concentrations) of BPA in their serum, saliva, and urine (see review by Dekant and Volkel, 2008). Furthermore, some studies have reported a correlation between increasing BPA levels in the environment and the incidence of cancer in humans (Keri et al., 2007). Previous studies by our own and other groups
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* Corresponding author. Tel.: +48 12 6645004; fax: +48 12 6645101. E-mail address: [email protected] (A. Ptak).
have identified BPA as a mitogen for hormone-dependent cancers such as ovarian cancer (Park et al., 2009; Ptak et al., 2011, 2013; Ptak and Gregoraszczuk, 2012). BPA exerts its biological activity in ovarian cancer cells through the activation of different signalling pathways, including the mitogen-activated protein kinase (MAPK/ ERK), as well as phosphatidylinositol 3-kinase (PI3K/Akt) pathways (Park et al., 2009; Ptak and Gregoraszczuk, 2012; Ptak et al., 2013). The MAPK/ERK and PI3K/Akt pathways are important in ovarian carcinogenesis (Fresno Vara et al., 2004; Miller et al., 2014). Furthermore, both the MAPK/ERK and PI3K/Akt pathways were involved in ovarian cancer cell migration (Ellerbroek et al., 2001; Jeong et al., 2013). Ovarian cancer is highly lethal because of its aggressive metastasis within the peritoneal cavity and the fact that it is often diagnosed at an advanced stage. When the cancer is confined to
http://dx.doi.org/10.1016/j.toxlet.2014.07.001 0378-4274/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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the ovary, the 5 year survival rate is greater than 90%; however, this rate drops to less than 20% when it is associated with disseminated intra-abdominal disease. Invasion and metastasis are the most important causes of death from cancer; these processes are thought to involve multiple steps that are dependent on the activities of a number of mediators. E-cadherin is a key mediator of cell–cell adhesions and reduction of adhesion plays an important role in metastasis (Cavallaro, 2004; Cavallaro and Christofori, 2004). Alteration in the function of E-cadherin has been implicated in the progression, invasion and metastasis of ovarian cancer. Furthermore, reduced expression of E-cadherin occurs in ovarian carcinomas and is associated with tumour progression (Yuecheng et al., 2006). However, in more advanced poorly differentiated ovarian carcinomas, there are contradictory reports of the absence or presence of E-cadherin expression (Hudson et al., 2008). Switching of the expression of cadherin from E-cadherin to N-cadherin is a key event in cancer progression (Hazan et al., 2000). N-cadherin promotes aggressive behaviour of tumour cells and the protein is overexpressed in invasive and metastatic human ovarian and breast cancers (Hazan et al., 2000; Hudson et al., 2008). Matrix metalloproteinases (MMPs) degrade extracellular matrix and basement membranes and are thus important players in tumour invasion. MMP-2 and MMP-9 (also known as gelatinase A and gelatinase B, respectively) differ from other MMPs because of their unique ability to degrade type IV collagen, which is a major component of the basement membrane. Above all, MMP-2 and MMP-9 have been suggested to be critical for the invasive and metastatic potential in ovarian carcinoma. Elevated expression of MMP-2 and MMP-9 has been detected in ovarian cancer ascites and tissues as well as cancer cells in vitro (Davidson et al., 2002; Symowicz et al., 2007; Kenny and Lengyel, 2009). Moreover, MMP-9 has been associated with E-cadherin cleavage (Symowicz et al., 2007). To our knowledge, no information is currently available regarding the role of BPA in ovarian cancer metastasis. Although all human ovary cell types may undergo neoplastic transformation, the vast majority (80–90%) of tumours are derived from the ovarian surface epithelium. Here, the OVCAR-3 cell line was used as an in vitro model of epithelial ovarian cancer. OVCAR-3 is a highly metastatic, drug-resistant, human ovarian carcinoma cell line; therefore, it is an ideal model to study the effects of and mechanisms involved in tumour cell invasion and metastasis (Hamilton et al., 1983). The effects of BPA on the migration of human OVCAR-3 ovarian cancer cells were examined, and the associated molecular and cellular mechanisms were studied.
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2. Materials and methods
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2.1. Cell culture and chemicals
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The human ovarian carcinoma cell line OVCAR-3 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were routinely cultured in RPMI 1640 medium (without phenol red) supplemented with 50 U/mL penicillin, 50 mg/mL streptomycin, and 10% heat inactivated foetal bovine serum (FBS) (PAA Laboratories GmbH, Colbe, Germany), and were maintained at 37 C in a humidified atmosphere containing 5% CO2. 17bestradiol (E2), the MAPK inhibitor PD098059, and trypan blue were obtained from Sigma (St. Louis, MO, USA). The PI3K inhibitor LY294002 was purchased from Cell Signaling Technology (Danvers, MA, USA). MMP-2 inhibitor I and MMP-9 inhibitor I were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). BPA (AccuStandard, New Haven, CT, USA) was dissolved in absolute ethanol. The final concentration of ethanol in the medium was 0.1%, at which it had no effect on cell migration (data not shown). Control cells were exposed to an equivalent volume of solvent.
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2.2. Cell treatment
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OVCAR-3 cells were seeded into 96-well plates at a density of 2 104 cells/well or 24-well plates at a density of 2 105 cells/well. Twenty-four hours before each experiment, the culture medium was changed to medium without FBS and then cells were treated with vehicle (0.1% ethanol), BPA (0.1, 10, 40, and 100 nM), E2 (40 nM), for a further 3, 6, 24, or 48 h. To investigate the involvement of signal transduction pathways in the effects of BPA, and E2 on cell migration, the cells were pre-treated for 2 h with the MAPK inhibitor PD098059 (Sigma) (100 mM) or the PI3K inhibitor LY294002 (Cell Signaling Technology) (10 mM); these inhibitors were also included in the culture medium during the 24 h exposure of cells to the test compounds. The concentrations of the inhibitors included in the study were chosen based on our preliminary experiments. Doses which had no effect on basal cell migration were chosen.
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2.3. Migration assay
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OVCAR-3 cell migration was measured using the QMC Chemotaxis Cell Migration Assay and 8 mm pore size Boyden chambers (Chemicon International, Temecula, CA, USA), according to the manufacturer’s instructions. The cells were pre-treated with PD098059 (100 mM), LY294002 (10 mM), MMP-2 inhibitor (15 mM), MMP-9 inhibitor (10 mM), or vehicle (RPMI 1640 medium without phenol red or FBS) for 2 h at 37 C. These inhibitors were also included in the culture medium during the 24 h exposure of cells to the test compounds. The cells were then seeded onto the upper layer of a cell-permeable membrane at a density of 2.5 104 cells/well and culture medium containing 10% FBS was placed below the membrane. The amount of fluorescent product was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an FLx800 spectrofluorometer (BioTek Instruments, Winooski, VT, USA). Data were analysed using KC Junior software (BioTek Instruments), normalised to fluorescence levels in vehicle-treated cells, and expressed as relative fluorescence units (RFU).
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2.4. MMP activity
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The activities of MMP-2 and MMP-9 were assessed using SensoLyte 490 Assay Kits (Anaspec, Fremont, CA, USA), according to the manufacturer’s instructions. The supernatant from cells maintained in serum-free RPMI 1640 medium was centrifuged, collected, and stored at 70 C. The amount of fluorescent product was measured at an excitation wavelength of 340 nm and an emission wavelength of 490 nm using an FLx800 spectrofluorometer (BioTek Instruments). Data were recorded every 30 min for 3 h and analysed using KC Junior software (BioTek Instruments), normalised to fluorescence levels in vehicle-treated cells, and expressed as RFU.
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2.5. Real-time PCR analysis
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After treatment of the cells with the test compounds for 3, 6 or 24 h, total RNA isolation and cDNA synthesis were performed using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The lysis solution contained DNase I to remove genomic DNA contamination. The resulting pre-amplified cDNA preparations were analysed by real-time PCR using a StepOnePlus Real-Time PCR System (Applied Biosystems) and TaqMan Gene Expression Assays in combination with TaqMan Gene Expression Master Mix containing ROX (Applied Biosystems). The thermal cycling conditions were as follows: 50 C for 2 min, 95 C for 10 min, and then
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40 cycles of 95 C for 15 s and 60 C for 60 s. Duplicate control samples lacking cDNA were prepared for each gene and showed no DNA contamination. The following TaqMan Gene Expression Assays were used: MMP-2, Hs01548727_m1 (detects transcript variants 1 (NM_004530) and 2 (NM_001127891) of this gene); MMP-9, Hs00234579_m1 (detects NM_004994.2); CDH1, Hs01023894_m1; CDH2, Hs00983056_m1; E-cadherin, Hs01023894_m1 (detects NM_4360.3); and N-cadherin, Hs00983056_m1 (detects NM_1792.3). Expression levels were normalised to those of GAPDH (Hs00234387_ m1) and relative expression was quantified using the 2 DDCt method (Livak and Schmittgen, 2001).
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2.6. Western blot analysis
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After exposure to the test compounds for 24 h, the cells were transferred into ice-cold lysis buffer and stored at 20 C. The protein concentrations of the lysates were determined using a Bradford assay (Bio-Rad Protein, Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 mg) from each treatment group were assayed. Proteins were separated by 12–15% SDS-PAGE and then transferred to PVDF membranes using a Bio-Rad Mini-Protean 3
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system. The blots were blocked for 2 h with 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20, and then incubated overnight at 4 C with antibodies specific for human MMP-2 (#4022), human MMP-9 (#3852), human E-cadherin (#3195), and human N-cadherin (#4061) (Cell Signaling Technology). The membranes were thenwashed three times and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (#7074) (Cell Signaling Technology). b-actin (A5316) (Sigma) was detected as a loading control. Immunopositive bands were visualised using Western Blotting Luminol Reagent (sc-2048) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
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2.7. Statistical analysis
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Fluorometric assays and real-time PCR experiments were repeated three times and each sample was run in quadruplicate. All data are represented as the mean SEM. Western blot experiments were repeated three times. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Data were analysed using a one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference test. P < 0.05 was considered statistically significant.
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Fig. 1. OVCAR-3 cell migration. The effects of exposure of cells for (a) 6 h, and (b) 24 h to various concentration of BPA (0.1–100 nM), and E2 (40 nM). C, control (untreated cells). ***P < 0.001, compared with cells exposed to vehicle.
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3. Results
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3.1. Effects of BPA on cell migration
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The effects of BPA and E2 on the migration ability of OVCAR-3 cells were measured using a transwell migration chamber assay. Basal migration of untreated cells was 1355 133 RFU after incubation for 6 h and 4365 966 RFU after incubation for 24 h. A 6 h treatment of OVCAR-3 cells with BPA (0.1–100 nM) and E2 (40 nM) had no effect on cell migration (Fig. 1a). An increase in cell migration was noted under the influence of 40 nM (9504 322 RFU) and 100 nM (10534 690 RFU) of BPA at 24 h of treatment (P < 0.001; Fig. 1b). The sub-maximal dose of BPA (40 nM) was chosen for the following experiments. Similarly, basal cell migration was significantly increased by 2.47-fold after treatment with E2, at 40 nM, (P < 0.001; Fig. 1b).
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3.2. Effects of BPA on MMP-2 expression and activity
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The effects of BPA and E2 on the expression levels of MMP-2 mRNA and protein were examined using real-time PCR and immunoblotting, respectively. A 3 h treatment of OVCAR-3 cells
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with BPA (40 nM) and E2 (40 nM) had no effect on MMP-2 mRNA levels (Fig. 2a). However, compared with control cells, statistically significant increases in MMP-2 mRNA levels were observed after exposure of the cells for 6 or 24 h to BPA (2.0-fold or 2.7-fold, respectively), (Fig. 2a). E2 had no effects on MMP-2 gene expression (Fig. 2a). MMP-2 protein levels were measured after exposure of cells to the test compounds for 24 h. The results mirrored those of the real-time PCR analyses; cells exposed to BPA had significantly higher MMP-2 protein levels than control cells (Fig. 2b). The effects of the test compounds on the activity of MMP-2 in the culture medium were also determined. After culture of OVCAR3 cells for 24 h, basal MMP-2 activity in the medium was 1554 54 RFU. Exposure of the cells for 24 h to BPA significantly increased MMP-2 activity by 1.43-fold, (P < 0.01; Fig. 2c). E2 had no effect on MMP-2 activity (Fig. 2c). Considering their effects on MMP-2 expression and activity, we then determined whether inhibition of this protein would abrogate the effects of BPA on cell migration. Pre-treatment of the cells with an MMP-2 inhibitor (15 mM) significantly reduced BPA-induced cell migration by 26% (P < 0.001), and had no effect on E2-induced migration (Fig. 2d). Taken together, these data support a role for MMP-2 in BPAinduced OVCAR-3 cell migration.
Fig. 2. Effects of BPA (40 nM), and E2 (40 nM) on MMP-2 expression and activity in OVCAR-3 cells. (a) Real-time PCR analysis of MMP-2 mRNA expression after exposure to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of MMP-2 protein expression after exposure of the cells to the test compounds for 24 h. Expression levels were normalised to those of b-actin and then to those of the control cells. (c) MMP-2 enzyme activity after exposure of the cells to the test compounds for 24 h. Data were normalised to the control cells. (d) Cell migration after exposure of cells pre-treated with or without an MMP-2 inhibitor (15 mM) to the indicated test compounds for 24 h. Data were normalised to the control cells. C, control (untreated cells). *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control cells (a–c) or with the absence of an MMP-2 inhibitor (d).
Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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Fig. 3. Effects of BPA (40 nM), and E2 (40 nM) on MMP-9 expression and activity in OVCAR-3 cells. (a) Real-time PCR analysis of MMP-9 mRNA expression after exposure to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of MMP-9 protein expression after exposure of the cells to the test compounds for 24 h. Expression levels were normalised to those of b-actin and then to those of the control cells. (c) MMP-9 enzyme activity after exposure of the cells to the test compounds for 24 h. Data were normalised to the control cells. (d) Cell migration after exposure of cells pre-treated with or without an MMP-9 inhibitor (10 mM) to the indicated test compounds for 24 h. Data were normalised to the control cells. C, control (untreated cells). *P < 0.05, **P < 0.01, and ***P < 0.001, compared with control cells (a–c) or with the absence of an MMP-9 inhibitor (d).
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3.3. Effects of BPA on MMP-9 expression and activity
3.4. Effects of BPA on E-cadherin expression
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The effects of the test compounds on MMP-9 expression and activity were also examined. None of the compounds affected MMP-9 mRNA levels after exposure of OVCAR-3 cells for 3 h (Fig. 3a); however, a statistically significant increase in MMP-9 gene expression was noted after exposure of the cells for 6 or 24 h to BPA (2.45-fold or 1.7-fold higher than the control cells, respectively); and after exposure of the cells for 6 h to E2 (2.2fold higher than the control cells), (Fig. 3a). The BPA-, and E2induced increases in MMP-9 mRNA levels were mirrored by parallel increases in MMP-9 protein expression (Fig. 3b). After culture of OVCAR-3 cells for 24 h, the basal MMP-9 activity in the medium was 1225 143 RFU. Exposure of the cells to BPA, or E2 increased the basal enzyme activity by 1.64-, or 1.50-fold, respectively (P < 0.01; Fig. 3c). Pre-treatment of cells with an MMP-9 inhibitor (10 mM) completely inhibited BPA-, and E2induced cell migration (P < 0.001; Fig. 3d). These data support a role for MMP-9 in BPA-, and E2-induced OVCAR-3 cell migration.
The effects of BPA, and E2 on E-cadherin gene and protein expression were examined in the OVCAR-3 cell line, which overexpresses E-cadherin. None of the compounds, had any effect on CDH1 gene (Fig. 4a) or protein (Fig. 4b) expression, even after cells were exposed for 24 h.
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3.5. Effects of BPA on N-cadherin expression
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Since the results described above suggested that E-cadherin is not involved in BPA-, and E2-induced cell migration, real-time PCR and immunoblot analyses were performed to examine the potential involvement of N-cadherin. CDH2 gene expression was not affected by exposure of OVCAR-3 cells to any of the compounds for 3 h (Fig. 5a). However, statistically significant increases in mRNA levels were noted after exposure of the cells for 6 or 24 h to BPA (2.2-fold and 1.8-fold higher than the control cells, respectively), or E2 (3.5-fold and 1.5-fold, respectively), (Fig. 5a). Similar
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Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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Fig. 4. Effects of BPA (40 nM), and E2 (40 nM) on E-cadherin gene and protein expression. (a) Real-time PCR analysis of CDH1 mRNA levels after cells were exposed to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of E-cadherin protein levels after cells were exposed to the test compounds for 24 h. b-actin was used as a loading control. C, control (untreated cells). 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281
changes in N-cadherin protein expression were also observed after cells were exposed to BPA, or E2 for 24 h (Fig. 5b). 3.6. Involvement of the ERK1/2 and PI3K pathways in the stimulatory effects of BPA Pre-treatment of OVCAR-3 cells with the ERK1/2 inhibitor PD098059 (100 mM) or the PI3K inhibitor LY294002 (10 mM) for 2 h prior to a 24 h exposure to the test compounds reversed the stimulatory effects of BPA, and E2 on cell migration (Fig. 6a). Furthermore, the BPA-induced increases in MMP-2, MMP-9 and Ncadherin protein expression, and E2-induced MMP-9 and Ncadherin protein expression were reduced to control levels after cells were pre-treated with PD098059 or LY294002 (Fig. 6b). Inhibition of either of these kinases also abolished the BPA-induced increases in MMP-2 activity (Fig. 6c) and the BPA-, and E2-induced increases in MMP-9 activity (Fig. 6d).
4. Discussion
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Invasion and metastasis are major events underlying cancer morbidity and mortality. This study demonstrates that BPA stimulate cell migration in a model of epithelial ovarian cancer. The effect of BPA on OVCAR-3 cell migration was comparable to that of E2, which was used as a positive control. Previous studies have demonstrated that E2 stimulates migration of the human ovarian cancer cell lines OVCAR-5 (Yan et al., 2013) and SKOV3 (Song et al., 2005). The effects of BPA on OVCAR-3 cell migration reported here are also in line with recent data indicating that low nanomolar concentrations of BPA induce migration of human prostate cancer cells (Derouiche et al., 2013) and human uterine myoma mesenchymal stem cells (Wang et al., 2013). To our knowledge, this study is the first report of a stimulatory effect of BPA on ovarian cancer cell migration.
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Fig. 5. Effects of BPA (40 nM), and E2 (40 nM) on N-cadherin gene and protein expression. (a) Real-time PCR analysis of CDH2 mRNA levels after cells were exposed to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of N-cadherin protein levels after cells were exposed to the test compounds for 24 h. b-actin was used as a loading control. C, control (untreated cells). *P < 0.05, **P < 0.001, and ***P < 0.001, compared with control cells.
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Fig. 6. Effects of ERK1/2 and PI3K inhibition on BPA-, and E2-induced changes in (a) cell migration, (b) N-cadherin, MMP-2 and MMP-9 protein expression, (c) MMP-2, and (d) MMP-9 activity in OVCAR-3 cells. The cells were pre-treated for 2 h with the ERK1/2 inhibitor PD098059 (100 mM) or the PI3K inhibitor LY294002 (10 mM), and then exposed to the indicated test compounds (BPA, 40 nM; E2, 40 nM) for a further 24 h (in the presence of the inhibitors). C, control cells (not exposed to a test compound). ***P < 0.001, compared with the absence of an inhibitor. Samples labeled as LY and PD were exposed to the kinase inhibitors only. b-actin was used as a loading control.
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The stimulatory effects of BPA on cell migration were accompanied by up-regulation of the migration-related factors MMP-2 and MMP-9, and inhibitors of these proteins abolished the stimulatory effects of the test compounds, suggesting that MMP-2 and MMP-9 are involved in cell migration induced by BPA. By contrast, E2 up-regulated MMP-9 but had no effect on MMP-2 expression or activity in OVCAR-3 cells, and E2-induced cell migration was attenuated by exposure to the MMP-9 inhibitor but not the MMP-2 inhibitor. MMP-2 and MMP-9 have been linked to gynaecological tumour aggressiveness, including cervical cancer (Rauvala et al., 2006), endometrial cancer (Graesslin et al., 2006), and ovarian cancer (Schmalfeldt et al., 2001; Kenny and Lengyel, 2009; Hu et al., 2012). Up-regulated MMP-2 and MMP-9 are associated with a poor prognosis of ovarian cancer (Schmalfeldt et al., 2001). To our knowledge, only one study describing the effect of BPA on MMPs is currently available; this study demonstrated that BPA stimulates MMP-9 secretion by human granulosa lutein cells (Dominguez et al., 2008), which is in line with our observations. Similarly, E2 stimulates MMP-9 expression in OVCAR-5 human ovarian cancer cells (Yan et al., 2013). Taken together with the results of previous studies, the data presented here suggest that BPA stimulate ovarian cancer cell migration by influencing the functional activities and expression levels of MMP2 and MMP-9. The differential effects of BPA and E2 on the expression levels of individual MMPs suggest that BPA utilises an oestrogen receptor-independent pathway to affect cell migration. In most carcinomas, metastasis is associated with a deregulated adhesion phenotype characterised by loss of epithelial E-cadherin, which generally prevents tumour metastasis and inhibits cancer cell growth (Baranwal and Alahari, 2009). However, E-cadherin expression has been related to tumour development in ovarian
cancers because it is rarely expressed in normal ovaries. E-cadherin is also actively involved in the regulation of proliferation and survival signals (Reddy et al., 2005; Steinberg and McNutt, 1999). Moreover, co-expression of E-cadherin and N-cadherin in human ovarian tumours has been reported previously (Hudson et al., 2008). N-cadherin promotes aggressive behaviour of tumour cells, and the protein is overexpressed in invasive and metastatic human ovarian and breast cancer tumours (Hazan et al., 2000; Hudson et al., 2008). Here, BPA, and E2 had no effects on E-cadherin expression in the OVCAR-3 cell line, but all of these compounds upregulated N-cadherin expression. To our knowledge, this study is the first report of the effect of BPA on cadherin expression in ovarian cancer cells. Previous studies demonstrated that BPA down-regulates E-cadherin expression in X-01 cells derived from human blastocysts of Chinese individuals (Yang et al., 2013) and up-regulates N-cadherin expression in rat testis (Salian et al., 2009). Other studies demonstrated that E2 down-regulates Ecadherin in human epithelial ovarian cancer SKOV3 cells (Ding et al., 2006) but has no effect on its expression levels in oesophageal adenocarcinoma cells (Sukocheva et al., 2013). Our observations are in agreement with the preliminary data presented by Reddy et al. (2005) which showed that E2 does not affect the expression levels of E-cadherin in OVCAR-3 cells. However, some cell lines may respond differently to the same chemical, due to the use of different signalling pathways. In our previous study, we demonstrated that BPA activate key signal transduction pathways associated with cell growth by rapidly inducing the phosphorylation of STAT-3, ERK1/2 and Akt ( Q3 Ptak and Gregoraszczuk, 2012). Accumulating evidence suggests that MAPK/ERK and PI3K/Akt play central roles in the regulation of cancer cell migration (Goetze et al., 2002; Zhang et al., 2012). To
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investigate whether these signalling pathways are involved in BPA-, and E2-induced OVCAR-3 cell migration, the chemical inhibitors PD098059 and LY294002 were used to block the MAPK/ ERK and PI3K/Akt pathways, respectively. Cell migration induced by all of the test compounds was blocked by pre-treatment of cells with either of the kinase inhibitors. These results are consistent with previous reports of the roles of ERK and Akt in migration stimulated by E2 (Li et al., 2010; Zhang et al., 2012). Moreover, inhibition of either signalling pathway attenuated the stimulatory effects of the test compounds on the activities of MMP-2 and MMP-9, and the expression levels of MMP-2, MMP-9 and N-cadherin. These findings suggest that BPA stimulate the release of migration-related factors such as MMP-2, MMP-9 and N-cadherin through the MAPK/ERK and PI3K/Akt signalling pathways. E2 also appears to stimulate cell migration via the MAPK/ERK and PI3K/Akt signalling pathways, although MMP-2 does not seem to be involved in this process. By contrast, a recent report by Zhang et al. (2012) demonstrated that blockage of the PI3K/Akt and MAPK signalling pathway decreases E2-induced MMP-2 and MMP-9 protein expression in Ishikawa cells, suggesting that the effects of this compound on signalling pathways may be cell-type specific. In summary, this study demonstrates that BPA utilise the MAPK/ERK and PI3K/Akt pathways to induce the activity and expression of MMP-2, MMP-9 and N-cadherin, leading to enhanced migration of OVCAR-3 cells. Overall, these observations suggest that BPA function as factors induced ovarian cancer cells migration.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgement
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This work was supported by the Ministry of Science and Higher Education (Republic of Poland) as an Iuventus Plus project (IP2011 044171) from 2011 to 2013.
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Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
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Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3 K/Akt signalling pathways
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Anna Ptak * , Marta Hoffman, Izabella Gruca, Justyna Barc
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Department of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland
H I G H L I G H T S
BPA stimulated OVCAR-3 cell migration. BPA up-regulated the migration-related factors MMP-2, MMP-9 and N-cadherin. Inhibition of ERK1/2 and Akt pathways abolishes BPA-induced cell migration.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online xxx
Bisphenol A (BPA), is present in a multitude of products, including food and water containers, food can linings, dentistry sealants, and thermal paper. BPA can induce the growth of human ovarian cancer cell lines. Reduction of adhesion and the initiation of metastasis are important events in cancer progression; therefore, this study investigated the effects of BPA (0.1–100 nM) on the migration of OVCAR-3 ovarian cancer cells and the expression levels of metalloproteinases (MMPs) and cadherins. The oestrogenic compound 17b-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. BPA stimulated cell migration, and the effect of BPA was similar to that of 17b-estradiol. BPA-induced cell migration was accompanied by up-regulation of the migration-related factors MMP-2, MMP-9 and N-cadherin, but E-cadherin expression and activity was unaffected. The stimulatory effects of BPA on cell migration were abolished by pre-treatment of the cells with inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways (PI3K). In conclusion, the results presented here show that BPA induces OVCAR-3 cells migration by activating MAPK and PI3K/Akt signalling pathways. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Bisphenol A Migration ERK1/2 Akt OVCAR-3
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1. Introduction
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Bisphenol A (BPA) is widely used as an ingredient in industrial and consumer products, including food and water containers, food can linings, dentistry sealants, and thermal paper. Epidemiological studies have reported that up to 95% of adults have detectable levels (nanomolar concentrations) of BPA in their serum, saliva, and urine (see review by Dekant and Volkel, 2008). Furthermore, some studies have reported a correlation between increasing BPA levels in the environment and the incidence of cancer in humans (Keri et al., 2007). Previous studies by our own and other groups
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* Corresponding author. Tel.: +48 12 6645004; fax: +48 12 6645101. E-mail address: [email protected] (A. Ptak).
have identified BPA as a mitogen for hormone-dependent cancers such as ovarian cancer (Park et al., 2009; Ptak et al., 2011, 2013; Ptak and Gregoraszczuk, 2012). BPA exerts its biological activity in ovarian cancer cells through the activation of different signalling pathways, including the mitogen-activated protein kinase (MAPK/ ERK), as well as phosphatidylinositol 3-kinase (PI3K/Akt) pathways (Park et al., 2009; Ptak and Gregoraszczuk, 2012; Ptak et al., 2013). The MAPK/ERK and PI3K/Akt pathways are important in ovarian carcinogenesis (Fresno Vara et al., 2004; Miller et al., 2014). Furthermore, both the MAPK/ERK and PI3K/Akt pathways were involved in ovarian cancer cell migration (Ellerbroek et al., 2001; Jeong et al., 2013). Ovarian cancer is highly lethal because of its aggressive metastasis within the peritoneal cavity and the fact that it is often diagnosed at an advanced stage. When the cancer is confined to
http://dx.doi.org/10.1016/j.toxlet.2014.07.001 0378-4274/ ã 2014 Published by Elsevier Ireland Ltd.
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the ovary, the 5 year survival rate is greater than 90%; however, this rate drops to less than 20% when it is associated with disseminated intra-abdominal disease. Invasion and metastasis are the most important causes of death from cancer; these processes are thought to involve multiple steps that are dependent on the activities of a number of mediators. E-cadherin is a key mediator of cell–cell adhesions and reduction of adhesion plays an important role in metastasis (Cavallaro, 2004; Cavallaro and Christofori, 2004). Alteration in the function of E-cadherin has been implicated in the progression, invasion and metastasis of ovarian cancer. Furthermore, reduced expression of E-cadherin occurs in ovarian carcinomas and is associated with tumour progression (Yuecheng et al., 2006). However, in more advanced poorly differentiated ovarian carcinomas, there are contradictory reports of the absence or presence of E-cadherin expression (Hudson et al., 2008). Switching of the expression of cadherin from E-cadherin to N-cadherin is a key event in cancer progression (Hazan et al., 2000). N-cadherin promotes aggressive behaviour of tumour cells and the protein is overexpressed in invasive and metastatic human ovarian and breast cancers (Hazan et al., 2000; Hudson et al., 2008). Matrix metalloproteinases (MMPs) degrade extracellular matrix and basement membranes and are thus important players in tumour invasion. MMP-2 and MMP-9 (also known as gelatinase A and gelatinase B, respectively) differ from other MMPs because of their unique ability to degrade type IV collagen, which is a major component of the basement membrane. Above all, MMP-2 and MMP-9 have been suggested to be critical for the invasive and metastatic potential in ovarian carcinoma. Elevated expression of MMP-2 and MMP-9 has been detected in ovarian cancer ascites and tissues as well as cancer cells in vitro (Davidson et al., 2002; Symowicz et al., 2007; Kenny and Lengyel, 2009). Moreover, MMP-9 has been associated with E-cadherin cleavage (Symowicz et al., 2007). To our knowledge, no information is currently available regarding the role of BPA in ovarian cancer metastasis. Although all human ovary cell types may undergo neoplastic transformation, the vast majority (80–90%) of tumours are derived from the ovarian surface epithelium. Here, the OVCAR-3 cell line was used as an in vitro model of epithelial ovarian cancer. OVCAR-3 is a highly metastatic, drug-resistant, human ovarian carcinoma cell line; therefore, it is an ideal model to study the effects of and mechanisms involved in tumour cell invasion and metastasis (Hamilton et al., 1983). The effects of BPA on the migration of human OVCAR-3 ovarian cancer cells were examined, and the associated molecular and cellular mechanisms were studied.
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2. Materials and methods
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2.1. Cell culture and chemicals
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The human ovarian carcinoma cell line OVCAR-3 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were routinely cultured in RPMI 1640 medium (without phenol red) supplemented with 50 U/mL penicillin, 50 mg/mL streptomycin, and 10% heat inactivated foetal bovine serum (FBS) (PAA Laboratories GmbH, Colbe, Germany), and were maintained at 37 C in a humidified atmosphere containing 5% CO2. 17bestradiol (E2), the MAPK inhibitor PD098059, and trypan blue were obtained from Sigma (St. Louis, MO, USA). The PI3K inhibitor LY294002 was purchased from Cell Signaling Technology (Danvers, MA, USA). MMP-2 inhibitor I and MMP-9 inhibitor I were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). BPA (AccuStandard, New Haven, CT, USA) was dissolved in absolute ethanol. The final concentration of ethanol in the medium was 0.1%, at which it had no effect on cell migration (data not shown). Control cells were exposed to an equivalent volume of solvent.
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2.2. Cell treatment
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OVCAR-3 cells were seeded into 96-well plates at a density of 2 104 cells/well or 24-well plates at a density of 2 105 cells/well. Twenty-four hours before each experiment, the culture medium was changed to medium without FBS and then cells were treated with vehicle (0.1% ethanol), BPA (0.1, 10, 40, and 100 nM), E2 (40 nM), for a further 3, 6, 24, or 48 h. To investigate the involvement of signal transduction pathways in the effects of BPA, and E2 on cell migration, the cells were pre-treated for 2 h with the MAPK inhibitor PD098059 (Sigma) (100 mM) or the PI3K inhibitor LY294002 (Cell Signaling Technology) (10 mM); these inhibitors were also included in the culture medium during the 24 h exposure of cells to the test compounds. The concentrations of the inhibitors included in the study were chosen based on our preliminary experiments. Doses which had no effect on basal cell migration were chosen.
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2.3. Migration assay
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OVCAR-3 cell migration was measured using the QMC Chemotaxis Cell Migration Assay and 8 mm pore size Boyden chambers (Chemicon International, Temecula, CA, USA), according to the manufacturer’s instructions. The cells were pre-treated with PD098059 (100 mM), LY294002 (10 mM), MMP-2 inhibitor (15 mM), MMP-9 inhibitor (10 mM), or vehicle (RPMI 1640 medium without phenol red or FBS) for 2 h at 37 C. These inhibitors were also included in the culture medium during the 24 h exposure of cells to the test compounds. The cells were then seeded onto the upper layer of a cell-permeable membrane at a density of 2.5 104 cells/well and culture medium containing 10% FBS was placed below the membrane. The amount of fluorescent product was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an FLx800 spectrofluorometer (BioTek Instruments, Winooski, VT, USA). Data were analysed using KC Junior software (BioTek Instruments), normalised to fluorescence levels in vehicle-treated cells, and expressed as relative fluorescence units (RFU).
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2.4. MMP activity
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The activities of MMP-2 and MMP-9 were assessed using SensoLyte 490 Assay Kits (Anaspec, Fremont, CA, USA), according to the manufacturer’s instructions. The supernatant from cells maintained in serum-free RPMI 1640 medium was centrifuged, collected, and stored at 70 C. The amount of fluorescent product was measured at an excitation wavelength of 340 nm and an emission wavelength of 490 nm using an FLx800 spectrofluorometer (BioTek Instruments). Data were recorded every 30 min for 3 h and analysed using KC Junior software (BioTek Instruments), normalised to fluorescence levels in vehicle-treated cells, and expressed as RFU.
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2.5. Real-time PCR analysis
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After treatment of the cells with the test compounds for 3, 6 or 24 h, total RNA isolation and cDNA synthesis were performed using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The lysis solution contained DNase I to remove genomic DNA contamination. The resulting pre-amplified cDNA preparations were analysed by real-time PCR using a StepOnePlus Real-Time PCR System (Applied Biosystems) and TaqMan Gene Expression Assays in combination with TaqMan Gene Expression Master Mix containing ROX (Applied Biosystems). The thermal cycling conditions were as follows: 50 C for 2 min, 95 C for 10 min, and then
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40 cycles of 95 C for 15 s and 60 C for 60 s. Duplicate control samples lacking cDNA were prepared for each gene and showed no DNA contamination. The following TaqMan Gene Expression Assays were used: MMP-2, Hs01548727_m1 (detects transcript variants 1 (NM_004530) and 2 (NM_001127891) of this gene); MMP-9, Hs00234579_m1 (detects NM_004994.2); CDH1, Hs01023894_m1; CDH2, Hs00983056_m1; E-cadherin, Hs01023894_m1 (detects NM_4360.3); and N-cadherin, Hs00983056_m1 (detects NM_1792.3). Expression levels were normalised to those of GAPDH (Hs00234387_ m1) and relative expression was quantified using the 2 DDCt method (Livak and Schmittgen, 2001).
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2.6. Western blot analysis
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After exposure to the test compounds for 24 h, the cells were transferred into ice-cold lysis buffer and stored at 20 C. The protein concentrations of the lysates were determined using a Bradford assay (Bio-Rad Protein, Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 mg) from each treatment group were assayed. Proteins were separated by 12–15% SDS-PAGE and then transferred to PVDF membranes using a Bio-Rad Mini-Protean 3
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system. The blots were blocked for 2 h with 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20, and then incubated overnight at 4 C with antibodies specific for human MMP-2 (#4022), human MMP-9 (#3852), human E-cadherin (#3195), and human N-cadherin (#4061) (Cell Signaling Technology). The membranes were thenwashed three times and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (#7074) (Cell Signaling Technology). b-actin (A5316) (Sigma) was detected as a loading control. Immunopositive bands were visualised using Western Blotting Luminol Reagent (sc-2048) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
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2.7. Statistical analysis
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Fluorometric assays and real-time PCR experiments were repeated three times and each sample was run in quadruplicate. All data are represented as the mean SEM. Western blot experiments were repeated three times. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Data were analysed using a one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference test. P < 0.05 was considered statistically significant.
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Fig. 1. OVCAR-3 cell migration. The effects of exposure of cells for (a) 6 h, and (b) 24 h to various concentration of BPA (0.1–100 nM), and E2 (40 nM). C, control (untreated cells). ***P < 0.001, compared with cells exposed to vehicle.
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3. Results
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3.1. Effects of BPA on cell migration
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The effects of BPA and E2 on the migration ability of OVCAR-3 cells were measured using a transwell migration chamber assay. Basal migration of untreated cells was 1355 133 RFU after incubation for 6 h and 4365 966 RFU after incubation for 24 h. A 6 h treatment of OVCAR-3 cells with BPA (0.1–100 nM) and E2 (40 nM) had no effect on cell migration (Fig. 1a). An increase in cell migration was noted under the influence of 40 nM (9504 322 RFU) and 100 nM (10534 690 RFU) of BPA at 24 h of treatment (P < 0.001; Fig. 1b). The sub-maximal dose of BPA (40 nM) was chosen for the following experiments. Similarly, basal cell migration was significantly increased by 2.47-fold after treatment with E2, at 40 nM, (P < 0.001; Fig. 1b).
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3.2. Effects of BPA on MMP-2 expression and activity
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The effects of BPA and E2 on the expression levels of MMP-2 mRNA and protein were examined using real-time PCR and immunoblotting, respectively. A 3 h treatment of OVCAR-3 cells
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with BPA (40 nM) and E2 (40 nM) had no effect on MMP-2 mRNA levels (Fig. 2a). However, compared with control cells, statistically significant increases in MMP-2 mRNA levels were observed after exposure of the cells for 6 or 24 h to BPA (2.0-fold or 2.7-fold, respectively), (Fig. 2a). E2 had no effects on MMP-2 gene expression (Fig. 2a). MMP-2 protein levels were measured after exposure of cells to the test compounds for 24 h. The results mirrored those of the real-time PCR analyses; cells exposed to BPA had significantly higher MMP-2 protein levels than control cells (Fig. 2b). The effects of the test compounds on the activity of MMP-2 in the culture medium were also determined. After culture of OVCAR3 cells for 24 h, basal MMP-2 activity in the medium was 1554 54 RFU. Exposure of the cells for 24 h to BPA significantly increased MMP-2 activity by 1.43-fold, (P < 0.01; Fig. 2c). E2 had no effect on MMP-2 activity (Fig. 2c). Considering their effects on MMP-2 expression and activity, we then determined whether inhibition of this protein would abrogate the effects of BPA on cell migration. Pre-treatment of the cells with an MMP-2 inhibitor (15 mM) significantly reduced BPA-induced cell migration by 26% (P < 0.001), and had no effect on E2-induced migration (Fig. 2d). Taken together, these data support a role for MMP-2 in BPAinduced OVCAR-3 cell migration.
Fig. 2. Effects of BPA (40 nM), and E2 (40 nM) on MMP-2 expression and activity in OVCAR-3 cells. (a) Real-time PCR analysis of MMP-2 mRNA expression after exposure to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of MMP-2 protein expression after exposure of the cells to the test compounds for 24 h. Expression levels were normalised to those of b-actin and then to those of the control cells. (c) MMP-2 enzyme activity after exposure of the cells to the test compounds for 24 h. Data were normalised to the control cells. (d) Cell migration after exposure of cells pre-treated with or without an MMP-2 inhibitor (15 mM) to the indicated test compounds for 24 h. Data were normalised to the control cells. C, control (untreated cells). *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control cells (a–c) or with the absence of an MMP-2 inhibitor (d).
Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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Fig. 3. Effects of BPA (40 nM), and E2 (40 nM) on MMP-9 expression and activity in OVCAR-3 cells. (a) Real-time PCR analysis of MMP-9 mRNA expression after exposure to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of MMP-9 protein expression after exposure of the cells to the test compounds for 24 h. Expression levels were normalised to those of b-actin and then to those of the control cells. (c) MMP-9 enzyme activity after exposure of the cells to the test compounds for 24 h. Data were normalised to the control cells. (d) Cell migration after exposure of cells pre-treated with or without an MMP-9 inhibitor (10 mM) to the indicated test compounds for 24 h. Data were normalised to the control cells. C, control (untreated cells). *P < 0.05, **P < 0.01, and ***P < 0.001, compared with control cells (a–c) or with the absence of an MMP-9 inhibitor (d).
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3.3. Effects of BPA on MMP-9 expression and activity
3.4. Effects of BPA on E-cadherin expression
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The effects of the test compounds on MMP-9 expression and activity were also examined. None of the compounds affected MMP-9 mRNA levels after exposure of OVCAR-3 cells for 3 h (Fig. 3a); however, a statistically significant increase in MMP-9 gene expression was noted after exposure of the cells for 6 or 24 h to BPA (2.45-fold or 1.7-fold higher than the control cells, respectively); and after exposure of the cells for 6 h to E2 (2.2fold higher than the control cells), (Fig. 3a). The BPA-, and E2induced increases in MMP-9 mRNA levels were mirrored by parallel increases in MMP-9 protein expression (Fig. 3b). After culture of OVCAR-3 cells for 24 h, the basal MMP-9 activity in the medium was 1225 143 RFU. Exposure of the cells to BPA, or E2 increased the basal enzyme activity by 1.64-, or 1.50-fold, respectively (P < 0.01; Fig. 3c). Pre-treatment of cells with an MMP-9 inhibitor (10 mM) completely inhibited BPA-, and E2induced cell migration (P < 0.001; Fig. 3d). These data support a role for MMP-9 in BPA-, and E2-induced OVCAR-3 cell migration.
The effects of BPA, and E2 on E-cadherin gene and protein expression were examined in the OVCAR-3 cell line, which overexpresses E-cadherin. None of the compounds, had any effect on CDH1 gene (Fig. 4a) or protein (Fig. 4b) expression, even after cells were exposed for 24 h.
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3.5. Effects of BPA on N-cadherin expression
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Since the results described above suggested that E-cadherin is not involved in BPA-, and E2-induced cell migration, real-time PCR and immunoblot analyses were performed to examine the potential involvement of N-cadherin. CDH2 gene expression was not affected by exposure of OVCAR-3 cells to any of the compounds for 3 h (Fig. 5a). However, statistically significant increases in mRNA levels were noted after exposure of the cells for 6 or 24 h to BPA (2.2-fold and 1.8-fold higher than the control cells, respectively), or E2 (3.5-fold and 1.5-fold, respectively), (Fig. 5a). Similar
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Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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Fig. 4. Effects of BPA (40 nM), and E2 (40 nM) on E-cadherin gene and protein expression. (a) Real-time PCR analysis of CDH1 mRNA levels after cells were exposed to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of E-cadherin protein levels after cells were exposed to the test compounds for 24 h. b-actin was used as a loading control. C, control (untreated cells). 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281
changes in N-cadherin protein expression were also observed after cells were exposed to BPA, or E2 for 24 h (Fig. 5b). 3.6. Involvement of the ERK1/2 and PI3K pathways in the stimulatory effects of BPA Pre-treatment of OVCAR-3 cells with the ERK1/2 inhibitor PD098059 (100 mM) or the PI3K inhibitor LY294002 (10 mM) for 2 h prior to a 24 h exposure to the test compounds reversed the stimulatory effects of BPA, and E2 on cell migration (Fig. 6a). Furthermore, the BPA-induced increases in MMP-2, MMP-9 and Ncadherin protein expression, and E2-induced MMP-9 and Ncadherin protein expression were reduced to control levels after cells were pre-treated with PD098059 or LY294002 (Fig. 6b). Inhibition of either of these kinases also abolished the BPA-induced increases in MMP-2 activity (Fig. 6c) and the BPA-, and E2-induced increases in MMP-9 activity (Fig. 6d).
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Invasion and metastasis are major events underlying cancer morbidity and mortality. This study demonstrates that BPA stimulate cell migration in a model of epithelial ovarian cancer. The effect of BPA on OVCAR-3 cell migration was comparable to that of E2, which was used as a positive control. Previous studies have demonstrated that E2 stimulates migration of the human ovarian cancer cell lines OVCAR-5 (Yan et al., 2013) and SKOV3 (Song et al., 2005). The effects of BPA on OVCAR-3 cell migration reported here are also in line with recent data indicating that low nanomolar concentrations of BPA induce migration of human prostate cancer cells (Derouiche et al., 2013) and human uterine myoma mesenchymal stem cells (Wang et al., 2013). To our knowledge, this study is the first report of a stimulatory effect of BPA on ovarian cancer cell migration.
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Fig. 5. Effects of BPA (40 nM), and E2 (40 nM) on N-cadherin gene and protein expression. (a) Real-time PCR analysis of CDH2 mRNA levels after cells were exposed to the test compounds for 3, 6 or 24 h. Expression levels were normalised to those of GAPDH and then to the control cells. RQ, relative quantity. Control value = 1.0. (b) Immunoblot analysis of N-cadherin protein levels after cells were exposed to the test compounds for 24 h. b-actin was used as a loading control. C, control (untreated cells). *P < 0.05, **P < 0.001, and ***P < 0.001, compared with control cells.
Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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Fig. 6. Effects of ERK1/2 and PI3K inhibition on BPA-, and E2-induced changes in (a) cell migration, (b) N-cadherin, MMP-2 and MMP-9 protein expression, (c) MMP-2, and (d) MMP-9 activity in OVCAR-3 cells. The cells were pre-treated for 2 h with the ERK1/2 inhibitor PD098059 (100 mM) or the PI3K inhibitor LY294002 (10 mM), and then exposed to the indicated test compounds (BPA, 40 nM; E2, 40 nM) for a further 24 h (in the presence of the inhibitors). C, control cells (not exposed to a test compound). ***P < 0.001, compared with the absence of an inhibitor. Samples labeled as LY and PD were exposed to the kinase inhibitors only. b-actin was used as a loading control.
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The stimulatory effects of BPA on cell migration were accompanied by up-regulation of the migration-related factors MMP-2 and MMP-9, and inhibitors of these proteins abolished the stimulatory effects of the test compounds, suggesting that MMP-2 and MMP-9 are involved in cell migration induced by BPA. By contrast, E2 up-regulated MMP-9 but had no effect on MMP-2 expression or activity in OVCAR-3 cells, and E2-induced cell migration was attenuated by exposure to the MMP-9 inhibitor but not the MMP-2 inhibitor. MMP-2 and MMP-9 have been linked to gynaecological tumour aggressiveness, including cervical cancer (Rauvala et al., 2006), endometrial cancer (Graesslin et al., 2006), and ovarian cancer (Schmalfeldt et al., 2001; Kenny and Lengyel, 2009; Hu et al., 2012). Up-regulated MMP-2 and MMP-9 are associated with a poor prognosis of ovarian cancer (Schmalfeldt et al., 2001). To our knowledge, only one study describing the effect of BPA on MMPs is currently available; this study demonstrated that BPA stimulates MMP-9 secretion by human granulosa lutein cells (Dominguez et al., 2008), which is in line with our observations. Similarly, E2 stimulates MMP-9 expression in OVCAR-5 human ovarian cancer cells (Yan et al., 2013). Taken together with the results of previous studies, the data presented here suggest that BPA stimulate ovarian cancer cell migration by influencing the functional activities and expression levels of MMP2 and MMP-9. The differential effects of BPA and E2 on the expression levels of individual MMPs suggest that BPA utilises an oestrogen receptor-independent pathway to affect cell migration. In most carcinomas, metastasis is associated with a deregulated adhesion phenotype characterised by loss of epithelial E-cadherin, which generally prevents tumour metastasis and inhibits cancer cell growth (Baranwal and Alahari, 2009). However, E-cadherin expression has been related to tumour development in ovarian
cancers because it is rarely expressed in normal ovaries. E-cadherin is also actively involved in the regulation of proliferation and survival signals (Reddy et al., 2005; Steinberg and McNutt, 1999). Moreover, co-expression of E-cadherin and N-cadherin in human ovarian tumours has been reported previously (Hudson et al., 2008). N-cadherin promotes aggressive behaviour of tumour cells, and the protein is overexpressed in invasive and metastatic human ovarian and breast cancer tumours (Hazan et al., 2000; Hudson et al., 2008). Here, BPA, and E2 had no effects on E-cadherin expression in the OVCAR-3 cell line, but all of these compounds upregulated N-cadherin expression. To our knowledge, this study is the first report of the effect of BPA on cadherin expression in ovarian cancer cells. Previous studies demonstrated that BPA down-regulates E-cadherin expression in X-01 cells derived from human blastocysts of Chinese individuals (Yang et al., 2013) and up-regulates N-cadherin expression in rat testis (Salian et al., 2009). Other studies demonstrated that E2 down-regulates Ecadherin in human epithelial ovarian cancer SKOV3 cells (Ding et al., 2006) but has no effect on its expression levels in oesophageal adenocarcinoma cells (Sukocheva et al., 2013). Our observations are in agreement with the preliminary data presented by Reddy et al. (2005) which showed that E2 does not affect the expression levels of E-cadherin in OVCAR-3 cells. However, some cell lines may respond differently to the same chemical, due to the use of different signalling pathways. In our previous study, we demonstrated that BPA activate key signal transduction pathways associated with cell growth by rapidly inducing the phosphorylation of STAT-3, ERK1/2 and Akt ( Q3 Ptak and Gregoraszczuk, 2012). Accumulating evidence suggests that MAPK/ERK and PI3K/Akt play central roles in the regulation of cancer cell migration (Goetze et al., 2002; Zhang et al., 2012). To
Please cite this article in press as: Ptak, A., et al., Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.07.001
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investigate whether these signalling pathways are involved in BPA-, and E2-induced OVCAR-3 cell migration, the chemical inhibitors PD098059 and LY294002 were used to block the MAPK/ ERK and PI3K/Akt pathways, respectively. Cell migration induced by all of the test compounds was blocked by pre-treatment of cells with either of the kinase inhibitors. These results are consistent with previous reports of the roles of ERK and Akt in migration stimulated by E2 (Li et al., 2010; Zhang et al., 2012). Moreover, inhibition of either signalling pathway attenuated the stimulatory effects of the test compounds on the activities of MMP-2 and MMP-9, and the expression levels of MMP-2, MMP-9 and N-cadherin. These findings suggest that BPA stimulate the release of migration-related factors such as MMP-2, MMP-9 and N-cadherin through the MAPK/ERK and PI3K/Akt signalling pathways. E2 also appears to stimulate cell migration via the MAPK/ERK and PI3K/Akt signalling pathways, although MMP-2 does not seem to be involved in this process. By contrast, a recent report by Zhang et al. (2012) demonstrated that blockage of the PI3K/Akt and MAPK signalling pathway decreases E2-induced MMP-2 and MMP-9 protein expression in Ishikawa cells, suggesting that the effects of this compound on signalling pathways may be cell-type specific. In summary, this study demonstrates that BPA utilise the MAPK/ERK and PI3K/Akt pathways to induce the activity and expression of MMP-2, MMP-9 and N-cadherin, leading to enhanced migration of OVCAR-3 cells. Overall, these observations suggest that BPA function as factors induced ovarian cancer cells migration.
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgement
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This work was supported by the Ministry of Science and Higher Education (Republic of Poland) as an Iuventus Plus project (IP2011 044171) from 2011 to 2013.
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