Toxicology Letters 229 (2014) 118–125

Perfluorooctanoic acid stimulates breast cancer cells invasion and up-regulates matrix metalloproteinase-2/-9 expression mediated byactivating NF-kB

Weidong Zhang b, 1, Fengliang Wang c, 1, Pengfei Xu a, Chen Miao d, Xin Zeng a,Xianwei Cui a, Cheng Lu c, Hui Xie c, Hong Yin c, Fei Chen c, Jingjing Ma c, Sheng Gao c,Ziyi Fu a,*aNanjing Maternal and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing 210004,ChinabDepartment of Ophthalmology, The First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, ChinacDepartment of Breast Surgery, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing 210004, ChinadDepartment of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China

H I G H L I G H T S

� PFOA exposure (�5 nM) evidently enhanced the invasion ability of the breast cancer cells MDA-MB-231.� MMP-2/-9 of MDA-MB-231 cells were increased with PFOA treatment.� PFOA could activate nuclear factor kappaB (NF-kB) by accelerating NF-kB translocation into the nucleus.� NF-kB inhibitor could reverse PFOA-induced breast cancer cells invasiveness enhancement and MMP-2/-9 overexpression.� PFOA can stimulate breast cancer cells invasion and up-regulates matrix metalloproteinase-2/-9 expression mediated by activating NF-kB, whichdeserves more environmental health concerns.

A R T I C L E I N F O

Article history:Received 6 November 2013Received in revised form 25 May 2014Accepted 1 June 2014Available online 21 June 2014

Keywords:Perfluorooctanoic acidBreast cancerInvasion

A B S T R A C T

Perfluorooctanoic acid (PFOA) is widely used because of its stain-resistant and water-repellantproperties. This study aimed to explore the molecular mechanisms undergoing the stimulation effects ofPFOA on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Trans-well filter assayshowed that PFOA exposure (�5 nM) evidently enhanced the invasion ability of the breast cancer cellsMDA-MB-231. Luciferase reporter assay, quantitative real-time PCR, western blotting and gelatinzymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in thecells after PFOA treatment (P < 0.05 each). Western blotting revealed that PFOA could activate nuclearfactor kappaB (NF-kB) by accelerating NF-kB translocation into the nucleus. Furthermore, addition of NF-kB inhibitor in culture medium could suppress the breast cancer cells invasiveness enhancement andMMP-2/-9 overexpression. This study indicates that PFOA can stimulate breast cancer cells invasion andup-regulate matrix metalloproteinase-2/-9 expression mediated by activating NF-kB, which deservesmore environmental health concerns.

ã 2014 Elsevier Ireland Ltd. All rights reserved.

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Toxicology Letters

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* Corresponding author. Nanjing Maternal and Child Health Medical Institute,Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University,123 Mochou Rd., Nanjing 210004, China. Tel.: +86 25 52226159; fax: +86 2552226159.

E-mail address: fzyzzu@hotmail.com (Z. Fu).1 Weidong Zhang and Fengliang Wang have contributed equally to this work.

http://dx.doi.org/10.1016/j.toxlet.2014.06.0040378-4274/ã 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Perfluorooctanoic acid (PFOA), a man-made chemical, isfrequently used in industrial and consumer products, correspond-ing to its stain-resistant and water-repellant characteristics. So far,it has been detected in everything surrounding us, such as non-

W. Zhang et al. / Toxicology Letters 229 (2014) 118–125 119

stick cookware, cosmetics, upholstery, bio-accumulate in foodchains (Bartell et al., 2010; Fraser et al., 2012), even in drinkingwater (Shin et al., 2011). As known, the concentration of PFOAexisted in drinking water varied from picogram per liter tomicrogram per liter (Rayne and Forest, 2009), meanwhile,regression analysis indicated that PFOA contamination increasingon an average of 7–11% per year during past decades (Holmstromet al., 2005). More importantly, several reports demonstrated thatPFOA contamination of human serum attained on an average of28.2 ng/ml (approximately 68.1 nM) (Bartell et al., 2010). Becauseof its widespread occurrence, it has attracted much concentrationsince 2001. Recent epidemiological studies have linked the toxicityof PFOA to tumorigenesis, such as pancreatic cancer, liver cancer,testicular cancer (Lau et al., 2007; Vieira et al., 2013), and probablybreast cancer (Bonefeld-Jorgensen et al., 2011). However, informa-tion about the relationship between PFOA exposure levels andtumor metastasis is omitted, although tumor metastasis is animportant section during carcinoma progression, which mightcause the majority of cancer treatment failure and poor prognosis(Chambers et al., 2002; Siegel et al., 2013).

Cancer metastasis undergoes a series of steps including vesselformation, cell attachment, invasion and cell proliferation(Chambers et al., 2002). Cancer cells must migrate through anddigest surrounding tissue barriers to escape from the primary siteand form the distant organ colonization, so the degradation ofbasement membranes and extracellular matrix (ECM) is a crucialstep for tumor metastasis (Geiger and Peeper, 2009). Thisprocedure requires different cellular proteolytic enzymes, in-cluding matrix metalloproteinases (MMPs) family, which is one ofthe important families of proteinases responsible for the ECMdestruction and rebuilding. The MMPs are a family of 24 humanzinc-binding endopeptidases which can degrade virtually allextracellular matrix (ECM) components (Overall and Dean, 2006).These MMPs are synthesized in most cells and immediatelysecreted into the ECM (Visse and Nagase, 2003). They play animportant role in various cellular processes including embryo-genesis, inflammation, wound healing, arthritis, cardiovasculardiseases and cancer invasion (Egeblad and Werb, 2002). In thoseMMPs, MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelati-nase), which can efficiently degrade native collagen types I/IV,fibronectin, entactin, and elastin, are well known to play criticalroles in cancer progress, such as invasion and distant metastasisof breast cancer as well (Deryugina and Quigley, 2006).Meanwhile, MMP-2/-9 overexpression is closely related to poorprognosis in patients.

MMP-2/-9 expression could be regulated by nuclear factorkappaB (NF-kB) (Adya et al., 2008). NF-kB is an importanttranscription factor that controls several cellular responsesincluding invasion, inflammation (Karin and Greten, 2005),apoptosis, and survival (Karin and Lin, 2002). Once the activatedNF-kB complex translocates from cytoplasm to nuclei and bind totarget genes, it could initiate the genes transcription (Karin andBen-Neriah, 2000). Corsini et al. found that PFOA could regulateLPS-induced MMP-9 release (Corsini et al., 2011), and previousstudies have indicated that NF-kB activation contributes to theenhanced invasiveness of carcinoma cells by regulating MMP-9and MMP-2 expression (Adya et al., 2008; Cheng et al., 2006),while the effects of environmental pollutants on cancer cellinvasion via this pathway are still unknown. In this study, we havedemonstrated that low concentration of PFOA contributed toenhancing MDA-MB-231 breast cancer cells invasiveness, so wefurther explored the role of NF-kB in the underlying molecularmechanism mediating the overexpression of MMP-2/-9 andenhancement of invasiveness of the breast cancer cells exposed toPFOA.

2. Materials and methods

2.1. Reagents and antibodies

The PFOA (purity � 95%, by HPLC) was purchased from Sigma(USA). MDA-MB-231 human breast cancer cell line was obtainedfrom the American Type Culture Collection. Cell culture mediumDMEM was purchased from Gibco (Invitrogen China Limited,China). All antibodies used in this study were provided by CellSignaling Technology (USA). Other reagents were provided bySigma–Aldrich Inc. (USA) unless otherwise mentioned.

2.2. Cell culture and PFOA exposure

Human breast cancer cells MDA-MB-231 were maintained inDMEM medium containing 100 U/ml streptomycin and 100 U/mlpenicillin supplemented with 10% fetal bovine serum (FBS). Cellswere culturedina humidifiedatmosphere of5%carbondioxide(CO2)at 37 �C. PFOA was dissolved in sterile water to make a 2 mM stocksolution and added to the media to reach different doses (0, 0.1, 1, 5,10, and 50 nM). In order to explore the role of NF-kB in the molecularpathway mediating the promoted cell invasion and MMP-2/-9expression, JSH-23 was dissolved in dimethyl sulfoxide (DMSO) andadded to the culture media with final concentration of 10 mM (equalDMSO added to culture medium as solvent control groups).

2.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay

MTT experiment was performed to judge the effect of PFOA oncell proliferation viability (El-Rayes et al., 2006). Human breastcancer cells MDA-MB-231 were seeded in a 96-well plate withserum-free medium, 5000 cells/well, and 24 h later the cells weretreated with different doses of PFOA. At certain time points (72 h),the cells were incubated in 0.83 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in growth medium at37 �C for 4 h and lysed in 100 mL of dimethyl sulfoxide at roomtemperature for 15 min. The absorbance in each well wasmeasured at 490 nm by Synergy 2 Absorbance Microplate Reader(Biotek, USA). Cell growth inhibition rate was expressed as afraction of absorbance compared to control.

2.4. Cell invasion ability assay

After PFOA exposure, trans-well filter assays were conducted toinvestigate MDA-MB0–231 invasion ability by using 24-well ECM-coated trans-well inserts (Zhang et al., 2012). Briefly, the uppersurface of the filter (8.0 mm in pore size) was coated with 80 mgECM gel before being air-dried overnight at 37 �C. Approximately4 �104 cells were added to the upper chamber, cultured in serum-free DMEM culture medium and DMEM with 10% FBS was added tothe lower chamber. For drug inhibition experiments, JSH-23(10 mM) was added to the upper filters prior to invasion assays, andequal DMSO was added to culture medium of another group assolvent control. After 72 h incubation with different doses of PFOAin an environment of 5% CO2 at 37 �C, cells degraded the ECM andadhered to the opposite surface of the filter. The filters were thenfixed with 4% paraformaldehyde (dissolved in PBS) and stainedwith crystal violet before the cells on the upper surface wereremoved completely with a cotton swab. The cells, whichtranslocated from the upper to the lower side of the filter, wereobserved under light microscopy at a magnification of �100. Cellswere then lysed by cell lysis buffer and collected for the detectionof optical density (OD) at 570 nm. OD values of the cells treatedwith different concentrations of PFOA were normalized to those of

Fig. 1. PFOA stimulated MDA-MB-231 cells invasiveness. In order to exclude the influence on cell survival, we first performed a MTT assay. The data showed that high-dosePFOA treatments (>100 nM) can induce apoptosis of the MDA-MB-231 cells (P < 0.05), but no cell growth inhibition was observed after treatment with low concentrations(�100 nM) (P > 0.05) (A). Then the cells were treated with different concentrations of PFOA (0, 0.1, 1, 5, 10, 50 nM), and we found that low-doses exposure could stimulate theinvasion ability of the crystal violet stained cancer cells (B), which was subsequently confirmed by determining the OD value of the invaded cells (C). (Each experiment wasperformed at least triple times.)

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the control cells (no PFOA treatment). Breast cancer cells MDA-MB-231 invasive ability was defined as the mean OD volume of cells.

2.5. RNA extraction, reverse transcription PCR, and quantitative realtime PCR

We employed quantitative real time PCR (qRT-PCR) to detectthe mRNA expression levels of homo-mmp-2/-9, with homo-b-actin as housekeeping genes. After the cells were treated withPFOA for 72 h, total RNA was isolated from breast cancer cells withTrizol reagent according to the manufacturer's protocol (Invitro-gen, Carlsbad, CA). The purified total RNA (1 mg) was then reverselytranscribed using the First Strand cDNA Synthesis Kit (TaKaRa,Japan). The qRT-PCR primers of mmp-2 gene were: forward 50-GAGAAC CAA AGT CTG AAG AG-30 and reverse 50-GGA GTG AGA ATGCTG ATT AG-30. The primers of mmp-9 gene were: forward 50-TGCCCG GAC CAA GGA TAC AG-30 and reverse 50-TCA GGG CGA GGACCA TAG AG-30. PCR primers set of the internal control gene b-actinwere: forward 50-TGA CGT GGA CAT CCG CAA AG-30 and reverse 50-CTG GAA GGT GGA CAG CGA GG-30. Reactions were conducted in96-well plates with a final volume of 20 ml including 10 ml SYBRGreen PCR Master Mix (Invitrogen, Carlsbad, CA), plus 1 ml eachprimer (2 mM), 1 ml template DNA, and 7 ml ddH2O. Thermalcycling and fluorescence detection were conducted on a AppliedBiosystems ViiATM7 (Life Technologies, USA), using the followingprotocol: 95 �C for 5 min, followed by 40 cycles of 95 �C for 15 s,60 �C for 30 s and 72 �C for 30 s. Each reaction was run in triplicate.The levels of mmp-2/-9 gene expression were normalized tob-actin levels using the method of 2�DDct. As assessed by

electrophoresis of PCR products, no primer–dimer was observedfor both the target genes and b-actin, and the specificity of theproducts was also confirmed by melt curve analysis.

2.6. Luciferase assay

Luciferase reporter assay experiment was conducted to performthe effect of PFOA on the gene promoter activity of mmp-2/-9 .MDA-MB-231 cells were grown on a 24-well plate for 24 h to reachapproximately 70% confluence prior to transfection. The cells ineach well were co-transfected with 0.4 mg of mmp-2/-9 -promotorsite-derived luciferase reporter plasmid and 0.1 mg of b-galactosi-dase (b-gal) expression plasmid using Lipofectamine 2000(Invitrogen, China Limited, China) according to the manufacturer'sprotocol. After transfection for 6 h, cells were treated with differentdoses of PFOA, JSH-23 (10 mM) or both JSH-23 (10 mM) and PFOA(50 nM) (equal DMSO was added to culture medium of anothergroup as solvent control). 24 h later, cell lysates were prepared withReporter Lysis Buffer (Promega, Madison, WI) following themanufacturer's instructions, and luciferase activity was measuredwith Luciferase Assay Reagent 10-Pack (Promega). The b-galactivity of the cells was measured as follows: 1 mL of Mg solution(0.1 M MgCl2, 4.5 M beta-mercaptoethanol), 22 mL of 4 mg/mL O-nitrophenol-b-D-galactoside solution (O-nitrophenyl-beta-D-gal-actopyranoside) (in 0.1 M phosphate buffer (pH 7.5)), and 57 mL of0.1 M phosphate buffer (pH 7.5), were added into the obtained cellextract solution (20 mL) to reach a total volume of 100 mL. Ascontrol, the optical density (OD) volume of b-gal at 450 mm wasmeasured in parallel.

Fig. 2. PFOA promoted MMP-2/-9 expression and activity levels. qRT-PCR results demonstrated that PFOA treatment upregulated gene MMP-2/-9 expressions (A). Westernblotting showed that the protein levels of MMP-2/-9 were also increased after PFOA treatments at over 5 nM (B), which was further supported by luciferase reporter assay (C).Meanwhile, gelatin zymography analysis data demonstrated that MMP-2/-9 activation levels were increased after PFOA treatment (D). (Each experiment was performed atleast triple times.)

W. Zhang et al. / Toxicology Letters 229 (2014) 118–125 121

2.7. Western blotting

After the cells' protein componets were collected, a standardwestern blotting analysis was used to investigate the MMP-2/-9and NF-kB protein expression levels. Briefly, nuclear and cellularprotein extracts were prepared, 72 h after treated with differentdoses of PFOA, JSH-23 (10 mM) or both JSH-23 (10 mM) and PFOA(50 nM) (equal DMSO was added to culture medium of anothergroup as solvent control), by using KEYGEN Protein Extraction Kit(KEYGEN, Nanjing). For detecting the ratio of nuclei (p65):cytosol(p65), cytosol protein and nucleus protein were dissolved in equalvolume lysis buffer of each group cells. Protein lysates were boiledin SDS-sample buffer for 5 min and then subjected to 8% SDS-polyacrylamide gel electrophoresis and transferred to PVDF(polyvinylidene fluoride) membranes (Bio-Rad). Membranes werethen blocked for 2 h in 5% milk-Tris-Buffered Saline Tween-20(TBST) at room temperature, and incubated overnight at 4 �C eitherwith the monoclonal antibodies (Cell Signaling Technology, USA) ofanti-b-actin, anti-MMP-2, anti-MMP-9 or anti-NF-kB. Membraneswere washed four times in TBST and incubated with theappropriate horseradish peroxidase-conjugated secondary anti-bodies at room temperature for 2 h. Blots were visualized byenhanced chemiluminescence (Thermo, USA) and analyzed using ascanning densitometer with the molecular analysis softwareFluorChem M system (Protein Simple, USA).

2.8. Gelatin zymography

The enzyme activities of MMP-2 and MMP-9 were analyzed bygelatin zymography. Cells (4 �104) were seeded into a 24-wellplate and allowed to adhere for 6–8 h with the presence of serum.Subsequently, the culture medium was replaced by serum-freeDMEM medium (400 ml per well) with different doses of PFOA (0,0.1, 1, 5, 10, and 50 nM). After incubation for 72 h, the medium ineach well was gathered and centrifuged for 10 min at 2000 rpm toremove cell debris. The supernatant was collected and mixed withsample loading buffer containing no DTT (dithiothreitol) before 8%SDS-polyacrylamide gel (containing 1 mg/mL gelatin) electropho-resis. Then the gel was soaked in 2.5% Triton-X-100 for 30 mintwice to remove SDS, and transferred to bath buffer containing50 mM tris (pH 8.0), 5 mM CaCl2 and 2 mM ZnCl2 at 37 �C for 16 h.The gel was then stained with 0.1% Coomassie blue in 45%methanol and 10% acetic acid. The visualized bands indicating thepresence of a protein with gelatinolytic activity were analyzed bythe FluorChem M system (Protein Simple, USA).

2.9. Statistical analysis

The differences between the treated and the control cells theinvasion rates and protein expressions were by applying one wayANOVA (analysis of variance) method followed by post-hoc Tukey's

Fig. 3. PFOA activated NF-kB to translocate into nuclear from cytosolic. The cytosoland nuclear proteins were isolated from the PFOA-treated cells for western blottingof p65 (A). After PFOA treatment, the nucleus/cytosol ratio of NF-kB was elevatedsignificantly (B). (Each experiment was performed at least triple times.)

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analysis. A p-value of less than 0.05 was considered statisticallysignificant, each experiment was performed at least triple times.Computer-based calculations were conducted using SPSS version20.0 (SPSS Inc. Chicago, Illinois, USA).

3. Results

3.1. Effects of PFOA treatment on breast cancer cell invasion

In order to investigate the invasiveness of the breast cancer cells(MDA-MB-231) after different doses of PFOA treatment, weperformed matrigel invasion assays. First, we evaluated the effectof different doses of PFOA on cell survival ratios. High-dose PFOAtreatments (>100 nM) could inhibit the viability of MDA-MB-231cells (P < 0.05), but no cell viability inhibition was observed aftertreatment with low concentrations (�100 nM) (P > 0.05) (Fig. 1A).Furthermore, we found that low-doses exposure could stimulatethe invasion ability of the crystal violet stained cancer cells(Fig. 1B), which was subsequently confirmed by determining theOD value of the invaded cells (Fig. 1C). PFOA treatments at 5, 10, or50 nM significantly increased the number of the invaded cells(p < 0.05), while treatment at 0.1 or 1 nM caused no change on cellinvasion ability (p > 0.05). Compared with the control, cellinvasiveness was separately enhanced by 1.5, 2.17 and 3.14 foldafter PFOA exposure at 5,10 and 50 nM. PFOA stimulated the cancercells invasion in a dose-dependent manner (R2 = 0.8422).

3.2. Enhanced MMP-2/-9 expression and activation levels in PFOA-treated MBA-MB-231 cells

To confirm the effect of the PFOA treatment on MMP-2/-9expression, we investigated the gene promoter activity, mRNA andprotein expression levels of MMP-2/-9 in MDA-MB-231 breastcancer cells after 24 h treatment with different doses of PFOA. qRT-PCR results demonstrated that the PFOA treatment upregulatedgene mmp-2/-9 expressions (Fig. 2A). Low-dose of PFOA (<5 nM)showed no effect on mmp-2/-9 mRNA levels, however, higher-doseof PFOA (5–50 nM) increased MMP-2/-9 mRNA expressions(P < 0.05). Western blotting showed that the protein levels ofMMP-2/-9 were also increased after PFOA treatments at over 5 nM(Fig. 2B), which was further supported by luciferase reporter assay(Fig. 2C). Meanwhile, gelatin zymography analysis data demon-strated that MMP-2/-9 activation levels were increased after PFOAtreatment (Fig. 2D). Results of qRT-PCR, western blotting,luciferase reporter, and gelatin zymography results revealed thatPFOA exposure could evidently up-regulate both expression andactivation of MMP-2/-9 in MDA-MB-231 cells, which may besubsequently involved in the cell invasion promotion.

3.3. NF-kB activation in PFOA-treated cells

We further examined the effect of PFOA treatment on NF-kBactivity to explore the potential molecular mechanisms of MMP-2/-9 overexpression. The cytosol and nuclear proteins wereisolated from the PFOA-treated cells for western blotting of p65(Fig. 3A). The data showed that after PFOA treatment, ratio of thenucleus/cytosol level of NF-kB was significantly elevated from0.24 � 0.04 (the control) to 0.69 � 0.12 (50 nM PFOA) (Fig. 3B)(P < 0.05).

3.4. Mediation of NF-kB in MMP-2/-9 expression and cell invasion

Drug inhibition experiment was performed to confirm the roleof NF-kB activation in the MMP-2/-9 stimulation and cell invasiveability promotion. First we confirmed that after NF-kB inhibitorJSH-23 (10 mM) treatment, nucleus/cytosol ratio of NF-kB was

significantly decreased 12% compared to control group; we alsofound that after JSH-23 pre-incubation, nucleus/cytosol ratio ofNF-kB was only up-regulated 16% with PFOA exposure, while PFOAincreased 36% NF-kB nuclear translocation without JSH-23 pre-incubation, which indicated that NF-kB inhibitor JSH-23 partiallyinhibited PFOA-induced NF-kB nuclear translocation (Fig. 4A).Then we performed trans-well filter assay to certify the inhibitioneffect of JSH-23 on PFOA-induced MDA-MB-231 invasion. The datashowed that with JSH-23 pre-treatment, PFOA-promoted cancercell invasiveness was reduced significantly (Fig. 4B), whichsuggested that PFOA could activate cancer cell invasion throughinducing NF-kB activation. Meanwhile, JSH-23 could reverse thestimulation effect of PFOA on gene MMP-2/-9 promoter activities(Fig. 4C) and expression levels (Fig. 4D) by using luciferase reporterassay and qRT-PCR, which was consistent with western blottingdata (Fig. 4E). Furthermore, gelatin zymography demonstrated thatMMP-2/-9 activity was inhibited in the cells treated with both JSH-23 and PFOA (Fig. 4F), suggesting that NF-kB mediated MMP-2/-9overexpression and enzyme activation stimulated by PFOA.

4. Discussion

This study demonstrated that the environmental pollutantPFOA can increase invasiveness of breast cancer cells. Accumulat-ing evidence has shown that PFOA can act as a tumor initiator toinduce DNA damages and oxidative stress (Eriksen et al., 2010; Yaoand Zhong, 2005). However, little information is available aboutthe effect of PFOA on breast cancer cell invasion, although clinicalconcerns have focused on screening and developing drugs capableof inhibiting breast cancer metastasis (Wang et al., 2010; Yeh et al.,2010). Recent studies have indicated that cancer cell migration andinvasion progression can be induced by physicochemical factors

Fig. 4. Mediation of NF-kB in MMP-2/-9 expression and cell invasion. Drug inhibition experiment was performed to confirm the role of NF-kB activation in the MMP-2/-9stimulation and cell invasive ability promotion. After treatment with NF-kB inhibitor JSH-23 (10 mM), western blotting showed that ratio of the nucleus/cytosol level of NF-kBwas significantly decreased (A), and the cells exposed to PFOA (50 nM) appeared lower invasiveness (p < 0.05) (B). Meanwhile, luciferase reporter assay results demonstratedthat JSH-23 reversed the stimulation effect of PFOA on gene <!–Author has rejected a copyediting change here. "mmp" is retained.–!>mmp<!–Author has rejected acopyediting change here. "MMP" is deleted.–!>-2/-9 promoter activities (C). qRT-PCR showed that JSH-23 thwarted PFOA-induced <!–Author has rejected a copyeditingchange here. "mmp" is retained.–!>mmp<!–Author has rejected a copyediting change here. "MMP" is deleted.–!>-2/-9 expression (D), which was agreed with westernblotting data (E). Furthermore, gelatin zymography demonstrated that MMP-2/-9 activity was inhibited in the cells treated with both JSH-23 and PFOA (F). (Each experimentwas performed at least triple times.)

W. Zhang et al. / Toxicology Letters 229 (2014) 118–125 123

such as progesterone receptor (Fu et al., 2010), glutamate (Herneret al., 2011), extracellular calcium (Saidak et al., 2009), bacterialpeptidoglycan (Xie et al., 2010), microcystin-LR (Zhang et al., 2012)and so on, but there is little information reported on therelationship between environmental pollution and breast cancermetastasis.

Our results suggested that low-dose treatments (�100 nM)could not affect breast cancer cell MDA-MB-231 survival, but high-dose treatments (>100 nM) could suppress cell viability. In order toexclude the influence on cell survival, we chose the low-dosegroups (0–50 nM) to explore the effect and mechanism of PFOA onbreast cancer cells invasion ability. In the present study, invasiveability of breast cancer cell MDA-MB-231 was significantlyenhanced after PFOA exposure. MMP-2/-9, which can efficientlydegrade extracellular matrix, are well known to play critical rolesin breast cancer invasiveness. In order to elucidate the undergoingmolecular mechanisms, we detected the influence of PFOAtreatment on the promoter activity, mRNA expression, proteinexpression and enzyme activation levels of MMP-2/-9 by usingluciferase reporter assay, qRT-PCR, western blotting and gelatinzymography separately. results consistently showed that PFOAcould enhance MMP-2/-9 expression and enzyme activation levelsin the breast cancer cells.

MMPs over-expression is closely related to tumor metastasis,much effort has been devoted to screening and developing drugswhich can inhibit MMPs expression (Lee et al., 2008). However,few environmental pollutants have been known to be capable ofinducing MMPs expression. Recently, microcystin-LR has been welldocumented to induce MMP-2/-9 expression through activation ofNF-kB in human melanoma cells (Zhang et al., 2012). Apart fromchemicals, radiation can also up-regulate MMP-9 mRNA level,protein level and catalytic activity by activating NF-kB inhepatocellular carcinoma cells (Cheng et al., 2006). Corsini et al.have found that 100 mg/ml (approximately 0.24 mM) PFOA couldreduce LPS-induced MMP-9 via regulating NF-kB phosphorylationlevel, while in this study we found that environmental concentra-tion (nM level) of PFOA could induce NF-kB activation, whichmight be involved in the over-expressions of MMP-2/-9. NF-kBcomplex located in the cytoplasm in a latent form often transfers tothe nuclear when NF-kB was activated, so we detected the changesof NF-kB activity by assessing the ratio of nuclear (NF-kB):cytosol(NF-kB). Western blotting showed that PFOA treatment inducednuclear translocation of NF-kB, revealing that NF-kB activationmight mediate MMP-2/-9 overexpression induced by PFOA.

The proposed molecular mechanism was further confirmed bythe JSH-23 suppression assay, a well-established NF-kB inhibition

124 W. Zhang et al. / Toxicology Letters 229 (2014) 118–125

method for MDA-MB-231 cells. The increased MMP-2/-9 activityand invasiveness were inhibited by JSH-23 in the breast cancercells cultured with PFOA, suggesting that NF-kB plays a crucial rolein the MMP-2/-9 overexpression and invasiveness enhancement.NF-kB is an important transcription factor controlling cellapoptosis and survival (Karin and Lin, 2002), this study showedthat appropriate increase of NF-kB activity could promote MMPsexpression and breast cancer cell invasion, but did not affect cellviability, which is supported by the study of Zhang et al. (Zhanget al., 2012) and Cheng et al. (Cheng et al., 2006). It has beenidentified that thioredoxin can stimulate MMP-9 transcriptionthrough activating NF-kB in breast cancer MDA-MB-231 cells(Farina et al., 2011). NF-kB also could mediate MMP-2 expressionand activation through stimulating MT1-MMP expression (Shihet al., 2010) and activating pro-MMP-2 (Han et al., 2001). Takentogether, PFOA can activate NF-kB to induce MMP-2/-9 over-expression, resulting in enhanced breast cancer cell invasion. PFOAis widely used because of its stain-resistant and water-repellantproperties, and given its persistence, it is ubiquitous in manyindoor environments, including homes and workplaces (Fraseret al., 2012; Haug et al., 2011), and our findings pointed theimportance of assessing the induced public health risks in oursurrounding environments.

In conclusion, this study indicates that PFOA exposure canpromote breast cancer cell invasion by stimulating MMP-2/-9mRNA, protein expression and activation. PFOA is able to activateNF-kB by stimulating translocation into the nucleus. NF-kBactivation may be contributed to the MMP-2/-9 overexpressionand cancer cell invasiveness enhancement.

Conflict of interest

The authors declare that they have no competing interests.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgments

This study was financially supported by the Key project of theNational Natural Science Foundation of China (81330067), theNational Natural Science Foundation of China (81302304,81302306, and 81172501), Natural Science Foundation of JiangsuProvince (BK20130074).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.toxlet.2014.06.004.

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