Bisphenol A and Nonylphenol Have the Potential to Stimulate the

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Bisphenol A and nonylphenol have a potential to stimulate the migration of ovarian cancer cells by inducing epithelialmesenchymal transition via an estrogen receptor dependent pathway Ye-Seul Kim, Kyung-A Hwang, Sang-Hwan Hyun, Ki-Hoan Nam, Chang-Kyu Lee, and Kyung-Chul Choi Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500443p • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Chemical Research in Toxicology

Bisphenol A and nonylphenol have a potential to stimulate the migration of ovarian cancer cells by inducing epithelial-mesenchymal transition via an estrogen receptor dependent pathway 1

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Ye-Seul Kim, Kyung-A Hwang, Sang-Hwan Hyun, Ki-Hoan Nam, Chang-Kyu Lee, and Kyung-Chul Choi

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Laboratory of Biochemistry and Immunology, Laboratory of Veterinary Embryology and

Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea;

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Laboratory Animal Resource Center, Korea Research

Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju, Chungbuk, Republic of Korea;

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Department of Agricultural Biotechnology,

Animal Biotechnology Major, and Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea

Running title: Effects of BPA and NP on EMT Keywords: Cancer migration, EMT, bisphenol A, octylphenol, ovarian cancer

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Correspondence: Kyung-Chul Choi, D.V.M., Ph.D.; Laboratory of Biochemistry and

Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea; Phone: +82-43-261-3664; Fax: +82-43-267-3150; Email: [email protected]

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TOC graphic

DMSO

E2

BPA

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N P

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Abstract

Epithelial-mesenchymal transition (EMT) is an important process appeared in embryo development and tumor migration or progression, which is influenced by 17β-estradiol (E2). Bisphenol A (BPA) and nonylphenol (NP) can be suspected as endocrine disrupting chemicals (EDCs) by exerting an estrogenic property. In this study, we examined whether E2, BPA, and NP can lead to EMT process in BG-1 ovarian cancer cells expressing estrogen receptor (ER). To confirm the effect of E2, BPA, and NP, BG-1 cells were cultured under the treatment with E2, BPA, or NP and the alteration of EMT markers such as vimentin was examined at mRNA levels by using real-time and reverse-transcription (RT)-PCR. The expressions of snail, slug, and vimentin were enhanced by the treatment of E2, BPA, or NP compared to a control (DMSO). In protein levels, vimentin protein was increased by E2 and two EDCs, while E-cadherin was decreased. In addition, the expression of snail protein was enhanced by the treatment of E2 and the two EDCs in comparison with the control. Since EMT response in cancer cells can affect metastasis, we also performed a scratch assay and Western blot assay to show the migration ability caused by E2, BPA, or NP. Consequently, E2, BPA, and NP enhanced a migration capability of BG-1 cells and increased the expression of MMP-9 protein. Furthermore, to examine whether EMT and migration of BG-1 cancer cells are induced by BPA or NP via ER dependent pathway, we co-treated the cells with ERantagonist, ICI 182,780, in the presence of E2, BPA, or NP. As results, the expressions of Ecadherin, vimentin, snail, and slug were reversed following treatment with an ER antagonist. Moreover, we confirmed that ICI 182,780 reduced the migration ability of BPA and NP to the control level. Taken together, these results indicate that BPA and NP, the potential EDCs, may have an ability to influence on ovarian cancer metastasis via regulating EMT markers and migration in ER-expressing BG-1 ovarian cancer cells. 3



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Introduction

Ovarian cancer is the most malignant cancer of all gynecology cancers and 5 year survival rate is lower than 30%.1 Because ovarian cancer is asymptomatic in early stage (1 and 2) and has invasion and migration abilities, an early assessment is difficult and cancer treatment is very hard afterward.2 Although the detailed steps related with primary ovarian tumor are revealed, the rate of success in the treatment of metastatic ovarian cancer is low.1 Therefore, more crucial mechanisms regulating progression and metastasis of ovarian cancer need to be identified to enhance therapeutic effects against ovarian cancer. Endocrine-disrupting chemicals (EDCs) are exogenous compounds which have an influence on hormone balance and normal endocrine system.3 Actually, EDCs could threaten health of life by triggering handicapped reproductions, developmental disorders, and cancer development and progression.4,

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Nevertheless, using EDCs in our daily lives is nearly

inevitable because they generally appear from various industrial products such as plastics, pesticides, drugs, detergents, and cosmetics.6 As the exposures to diverse EDCs have been increased, the researches that can inform how EDCs are dangerous to health of human and animal have been performed.3, 7, 8 Bisphenol A (BPA) is a compound of polycarbonates and epoxy resins used in producing plastic products including water containers and dental sealants and as an inner coating material of canned foods. Recently, BPA has been considered as a potent dangerous EDC especially threatening women’s health.9 Nonylphenol (NP) is also well known as an EDC. NP is a degradation product of alkylphenol polyethoxylate. Use of pesticides, polystyrene plastics, and paints including alkylphenol polyethoxylate can lead to the bioaccumulation of NP in the food chain. Accumulation of NP in the body may result in endocrine disruption and immunological and reproduction disorders.9 In general, EDCs have an estrogenic or an androgenic effect by binding to hormone receptors such as estrogen 4


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receptor (ER) and androgen receptor (AR) and thus they may interfere with the actions of endogenous steroid hormones.10 It has been reported that BPA and NP have an estrogenic effect in the body.9,

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17β-estradiol (E2), a potent endogeneous estrogen, can affect the

growth and migration of estrogen dependent cancer cells through diverse functions.12-15 Previous studies also demonstrated the BPA and NP have the cancer proliferation effects in estrogen dependent cancers such as breast and ovarian cancers the way E2 has.9, 16 In this study, we investigated whether the two EDCs, BPA and NP, could affect migration of BG-1 ovarian cancer cells expressing ER in comparison with E2 as a positive control. As a estrogen responsive cancer model, BG-1 ovarian cancer cell line was adopted in this study because ERα and ERβ proteins were identified to be expressed in BG-1 cells, which was already elucidated in our previous study.17

When a migration of cancer cells occurs, an epithelial-mesenchymal transition (EMT) process precedes.18 Because EMT can change morphology of cancer cells more sharply, the cells undergoing EMT can more easily invade other places. During EMT, the expression of cell adhesion proteins such as E-cadherin and desmosome is decreased, while the expression of intermediate filamentous proteins like vimentin and fibronectin is increased.19 In addition, during an EMT process, the expression of transcription factors involved in EMT including E box-binding protein, snail family, ZEB family, and helix-loop-helix (HLH) family are increased.20 Among them, snail is a very important factor because it can block E-cadherin expression and induce an expression of mesenchymal markers.21 Therefore, we examined the alterations in the expression of E-cadherin, vimentin, and snail family to confirm the changes of EMT markers by the treatment of E2, BPA or NP. We also identified the migration ability of BG-1 ovarian cancer cells induced by the treatment of E2, BPA or NP. Furthermore, to 5



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know whether EMT and migration efficacies induced by E2, BPA or NP are mediated by ER dependent signaling pathway, ICI 182,780, an ER-antagonist, was co-treated. This study might help clarify the alterations in EMT and migration of ovarian cancer cells under the influence of BPA and NP and therefore has a significance by additionally displaying their risks to ovarian cancer progression including metastasis via EMT.

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Materials and Methods

Reagents and chemicals E2, BPA, and NP were purchased from Sigma-Aldrich Corp. (St. Louis, Mo, USA). All chemicals were dissolved in 100% dimethyl sulfoxide (DMSO ; Junsei Chemical Co., Tokyo, Japan) and stored as stock solutions at 4℃. DMSO was used as a vehicle for the treatment of E2, BPA, and NP into the cell culture solution.

Cell culture BG-1 human ovarian cancer cells were obtained from Dr. K. S. Korach (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA). The cells were cultured at 37℃ in humidified atmosphere of 5% CO2-95% air. Cell culture medium contains Dulbecco’s modified Eagle’s medium (DMEM; Hyclone Laboratories Inc) supplemented with 10% inactivated fetal bovine serum (FBS; Biological Industries), 1% penicillin G and streptomycin (PAA, Cell Culture Company), and 1% antifungal HEPES (Gibco). Because DMEM and FBS may have estrogenic components, we cultured BG-1 cells in phenol red-free DMEM containing 5% charcoal-dextran treated FBS (CD-FBS) to confirm estrogenicity of BPA and NP as previously described.22, 23 The cells were detached by 0.05% trypsin/ 0.02% EDTA in Mg2+/Ca2+ (Gibco by Life Technologies). To evaluate the effects of E2, BPA, and NP, BG-1 cells were cultured in 100 mm dish under the treatment of DMSO (0.1%), E2 (10-7 M), BPA (10-6 M) or NP (10-6 M). In addition, the cells were treated by the combination of ICI 182,780 (10-6 M) with DMSO (0.1%), E2 (10-7 M), BPA (10-6 M) or NP (10-6 M). 7



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RNA extraction and semi-quantitative RT-PCR Total RNA was extracted by TriZol reagent (Invitrogen Life Technologies) at each time point (0, 6 and 24h) of E2, BPA or NP treatment or each combination with ICI 182, 780 and was dissolved in diethyl pyrocarbonated-deionized (DEPC) water. Concentrations of total RNAs were measured by spectrophotometer (Optizen) at 260/280 nm wavelength. To synthesize cDNA, 1 μg total RNA, MMLV-RT (iNtRON Biotechnology), 200pM random primer (Takara Bio.), dNTP (Bioneer), RNase inhibitor (iNtRON Biotechnology), and RT buffer (iNtRON Biotechnology) were used. After mixing these materials, the mixture was incubated in 37℃ during 1 h and heated in 95℃ during 5 min. To perform PCR, the mixture containing template cDNA, Taq polymerase (iNtRON Biotechnology), PCR buffer (iNtRON Biotechnology), dNTPs (Bioneer), and forward and reverse primers of genes for GAPDH and vimentin was made. PCR process was conducted as previously described.24 Table 1 shows each primer sequence and size. The PCR products were separated by using 1.5% agarose gel and 100bp ladders were used to estimate each band size. Quantitative analysis was performed by using Gel Doc 2000 (Bio-Rad Laboratories, Inc.). The expression level of vimentin mRNA was normalized by GAPDH mRNA.

Real-time PCR A real time PCR was performed by using 2X SYBR premix (Takara Bio.) according to the company’s protocol. Primers used are represented in Table 1. The process of reaction contained denaturation at 95℃ for 15 sec, annealing at 58℃ for 20 sec, and elongation at 72℃ for 15 sec and this reaction was replicated until 40 cycles. The real time PCR experiment was 8


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performed in triplicate. The expression levels of snail and slug mRNAs were normalized by GAPDH mRNA. The results were calculated using relative quantitative methods (2-delta delta Ct).

Western blot analysis Proteins were extracted at various time points (0, 24 and 48h) following treatments with E2, BPA or NP in the absence or presence of ICI 182, 780 by using 1x RIPA lysis buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS). Concentration of the proteins was quantified by using bicinchoninc acid (BCA; SigmaAldrich Co) method. The proteins were separated on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.). Membranes were blocked by 5% skim-milk (Bio-Rad Laboratories Inc) for 2 h in room temperature and incubated with mouse monoclonal anti-GAPDH antibody (Abcam plc.), mouse monoclonal anti-vimentin antibody (Abcam plc.), rabbit polyclonal anti-E-cadherin antibody (Abcam plc.), mouse monoclonal anti-snail antibody (Cell Signaling Technology, Inc), and rabbit monoclonal anti-MMP-9 antibody (Abcam plc.) for 2 h at room temperature. Additionally membranes were incubated with anti-mouse IgG-horse raddish peroxidase (HRP) conjugated secondary antibody (Santa Cruz Biotechnology) and anti-rabbit IgG-HRP conjugated secondary antibody (Santa Cruz Biotechnology). Proteins attached on membranes were visualized by using ECL chemical luminescent system (GenDEPOT). Band density was estimated by using Gel Doc 2000. Protein expression levels of vimentin, E-cadherin, and snail were normalized by GAPDH protein.

Scratch assay 9



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BG-1 cells were cultured at 8x105 cells/well of 6-well plates for 2 days. The cells were scratched and washed with PBS to remove cell debris. The cells were then treated with 5% CD-FBS phenol free media supplemented with DMSO (0.1%), E2 (10-7M), BPA (10-6M), and NP (10-6M). In another experiment, the cells were treated with the combination of ICI 182,780(10-6M) and DMSO (0.1%), E2 (10-7M), BPA (10-6M), and NP (10-6M). The pictures were taken with a microscope under 4x magnification at 0, 48 and 96 h after scratch. The percentage of unrecovered wound area was calculated by dividing the uncovered area at 48 and 96 h with the initial wound area at time zero.

Statistics analysis All experiments were repeated at least 3 times and data were presented as mean ± S.D. Statistical analysis was conducted by using one-way ANOVA test, followed by Dunnett’s multiple comparison test. Differences with P-value

Bisphenol A and Nonylphenol Have the Potential to Stimulate the

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