Perfluorooctanoic Acid Stimulates Breast Cancer

Home , Perfluorooctanoic acid

Toxicology Letters 229 (2014) 118–125

Contents lists available at ScienceDirect

Toxicology Letters

journal homepage: www.elsevier.com/locate/toxlet

Perﬂuorooctanoic acid stimulates breast cancer cells invasion and up-

regulates matrix metalloproteinase-2/-9 expression mediated by

activating NF-kB

b, 1 c, 1 a d a

Weidong Zhang , Fengliang Wang , Pengfei Xu , Chen Miao , Xin Zeng ,

a c c c c c c

Xianwei Cui , Cheng Lu , Hui Xie , Hong Yin , Fei Chen , Jingjing Ma , Sheng Gao , a,

Ziyi Fu *

Nanjing Maternal and Child Health Medical Institute, Afﬁliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing 210004, China

Department of Ophthalmology, The First Afﬁliated Hospital, Nanjing Medical University, Nanjing 210029, China

Department of Breast Surgery, Afﬁliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing 210004, China

Department 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, which

deserves more environmental health concerns.

A R T I C L E I N F O A B S T R A C T

Article history: Perﬂuorooctanoic acid (PFOA) is widely used because of its stain-resistant and water-repellant

Received 6 November 2013

properties. This study aimed to explore the molecular mechanisms undergoing the stimulation effects of

Received in revised form 25 May 2014

PFOA on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Trans-well ﬁlter assay

Accepted 1 June 2014

showed that PFOA exposure (5 nM) evidently enhanced the invasion ability of the breast cancer cells

Available online 21 June 2014

MDA-MB-231. Luciferase reporter assay, quantitative real-time PCR, western blotting and gelatin

zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the

Keywords:

cells after PFOA treatment (P < 0.05 each). Western blotting revealed that PFOA could activate nuclear

Perﬂuorooctanoic acid

factor kappaB (NF-kB) by accelerating NF-kB translocation into the nucleus. Furthermore, addition of NF-

Breast cancer

Invasion B inhibitor in culture medium could suppress the breast cancer cells invasiveness enhancement and

MMP-2/-9 overexpression. This study indicates that PFOA can stimulate breast cancer cells invasion and

up-regulate matrix metalloproteinase-2/-9 expression mediated by activating NF-kB, which deserves

more environmental health concerns.

1. Introduction

* Corresponding author. Nanjing Maternal and Child Health Medical Institute,

Afﬁliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University,

Perﬂuorooctanoic acid (PFOA), a man-made chemical, is

123 Mochou Rd., Nanjing 210004, China. Tel.: +86 25 52226159; fax: +86 25

52226159. frequently used in industrial and consumer products, correspond-

E-mail address: [email protected] (Z. Fu). ing to its stain-resistant and water-repellant characteristics. So far,

Weidong Zhang and Fengliang Wang have contributed equally to this work. it has been detected in everything surrounding us, such as non-

http://dx.doi.org/10.1016/j.toxlet.2014.06.004

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

stick cookware, cosmetics, upholstery, bio-accumulate in food 2. Materials and methods

chains (Bartell et al., 2010; Fraser et al., 2012), even in drinking

water (Shin et al., 2011). As known, the concentration of PFOA 2.1. Reagents and antibodies

existed in drinking water varied from picogram per liter to

microgram per liter (Rayne and Forest, 2009), meanwhile, The PFOA (purity 95%, by HPLC) was purchased from Sigma

regression analysis indicated that PFOA contamination increasing (USA). MDA-MB-231 human breast cancer cell line was obtained

on an average of 7–11% per year during past decades (Holmstrom from the American Type Culture Collection. Cell culture medium

et al., 2005). More importantly, several reports demonstrated that DMEM was purchased from Gibco (Invitrogen China Limited,

PFOA contamination of human serum attained on an average of China). All antibodies used in this study were provided by Cell

28.2 ng/ml (approximately 68.1 nM) (Bartell et al., 2010). Because Signaling Technology (USA). Other reagents were provided by

of its widespread occurrence, it has attracted much concentration Sigma–Aldrich Inc. (USA) unless otherwise mentioned.

since 2001. Recent epidemiological studies have linked the toxicity

of PFOA to tumorigenesis, such as pancreatic cancer, liver cancer, 2.2. Cell culture and PFOA exposure

testicular cancer (Lau et al., 2007; Vieira et al., 2013), and probably

breast cancer (Bonefeld-Jorgensen et al., 2011). However, informa- Human breast cancer cells MDA-MB-231 were maintained in

tion about the relationship between PFOA exposure levels and DMEM medium containing 100 U/ml streptomycin and 100 U/ml

tumor metastasis is omitted, although tumor metastasis is an penicillin supplemented with 10% fetal bovine serum (FBS). Cells

important section during carcinoma progression, which might were culturedina humidiﬁedatmosphere of5%carbondioxide(CO2)

cause the majority of cancer treatment failure and poor prognosis at 37 C. PFOA was dissolved in sterile water to make a 2 mM stock

(Chambers et al., 2002; Siegel et al., 2013). solution and added to the media to reach different doses (0, 0.1, 1, 5,

Cancer metastasis undergoes a series of steps including vessel 10, and 50 nM). In order to explore the role of NF-kB in the molecular

formation, cell attachment, invasion and cell proliferation pathway mediating the promoted cell invasion and MMP-2/-9

(Chambers et al., 2002). Cancer cells must migrate through and expression, JSH-23 was dissolved in dimethyl sulfoxide (DMSO) and

digest surrounding tissue barriers to escape from the primary site added to the culture media with ﬁnal concentration of 10 mM (equal

and form the distant organ colonization, so the degradation of DMSO added to culture medium as solvent control groups).

basement membranes and extracellular matrix (ECM) is a crucial

step for tumor metastasis (Geiger and Peeper, 2009). This 2.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

procedure requires different cellular proteolytic enzymes, in- (MTT) assay

cluding matrix metalloproteinases (MMPs) family, which is one of

the important families of proteinases responsible for the ECM MTT experiment was performed to judge the effect of PFOA on

destruction and rebuilding. The MMPs are a family of 24 human cell proliferation viability (El-Rayes et al., 2006). Human breast

zinc-binding endopeptidases which can degrade virtually all cancer cells MDA-MB-231 were seeded in a 96-well plate with

extracellular matrix (ECM) components (Overall and Dean, 2006). serum-free medium, 5000 cells/well, and 24 h later the cells were

These MMPs are synthesized in most cells and immediately treated with different doses of PFOA. At certain time points (72 h),

secreted into the ECM (Visse and Nagase, 2003). They play an the cells were incubated in 0.83 mg/mL 3-(4,5-dimethylthiazol-2-

important role in various cellular processes including embryo- yl)-2,5-diphenyltetrazolium bromide (MTT) in growth medium at

genesis, inﬂammation, wound healing, arthritis, cardiovascular 37 C for 4 h and lysed in 100 mL of dimethyl sulfoxide at room

diseases and cancer invasion (Egeblad and Werb, 2002). In those temperature for 15 min. The absorbance in each well was

MMPs, MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelati- measured at 490 nm by Synergy 2 Absorbance Microplate Reader

nase), which can efﬁciently degrade native collagen types I/IV, (Biotek, USA). Cell growth inhibition rate was expressed as a

ﬁbronectin, entactin, and elastin, are well known to play critical fraction of absorbance compared to control.

roles in cancer progress, such as invasion and distant metastasis

of breast cancer as well (Deryugina and Quigley, 2006). 2.4. Cell invasion ability assay

Meanwhile, MMP-2/-9 overexpression is closely related to poor

prognosis in patients. After PFOA exposure, trans-well ﬁlter assays were conducted to

MMP-2/-9 expression could be regulated by nuclear factor investigate MDA-MB0–231 invasion ability by using 24-well ECM-

kappaB (NF-kB) (Adya et al., 2008). NF-kB is an important coated trans-well inserts (Zhang et al., 2012). Brieﬂy, the upper

transcription factor that controls several cellular responses surface of the ﬁlter (8.0 mm in pore size) was coated with 80 mg

including invasion, inﬂammation (Karin and Greten, 2005), ECM gel before being air-dried overnight at 37 C. Approximately

apoptosis, and survival (Karin and Lin, 2002). Once the activated 4 10 cells were added to the upper chamber, cultured in serum-

NF-kB complex translocates from cytoplasm to nuclei and bind to free DMEM culture medium and DMEM with 10% FBS was added to

target genes, it could initiate the genes transcription (Karin and the lower chamber. For drug inhibition experiments, JSH-23

Ben-Neriah, 2000). Corsini et al. found that PFOA could regulate (10 mM) was added to the upper ﬁlters prior to invasion assays, and

LPS-induced MMP-9 release (Corsini et al., 2011), and previous equal DMSO was added to culture medium of another group as

studies have indicated that NF-kB activation contributes to the solvent control. After 72 h incubation with different doses of PFOA

enhanced invasiveness of carcinoma cells by regulating MMP-9 in an environment of 5% CO2 at 37 C, cells degraded the ECM and

and MMP-2 expression (Adya et al., 2008; Cheng et al., 2006), adhered to the opposite surface of the ﬁlter. The ﬁlters were then

while the effects of environmental pollutants on cancer cell ﬁxed with 4% paraformaldehyde (dissolved in PBS) and stained

invasion via this pathway are still unknown. In this study, we have with crystal violet before the cells on the upper surface were

demonstrated that low concentration of PFOA contributed to removed completely with a cotton swab. The cells, which

enhancing MDA-MB-231 breast cancer cells invasiveness, so we translocated from the upper to the lower side of the ﬁlter, were

further explored the role of NF-kB in the underlying molecular observed under light microscopy at a magniﬁcation of 100. Cells

mechanism mediating the overexpression of MMP-2/-9 and were then lysed by cell lysis buffer and collected for the detection

enhancement of invasiveness of the breast cancer cells exposed to of optical density (OD) at 570 nm. OD values of the cells treated

PFOA. with different concentrations of PFOA were normalized to those of

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

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-dose

PFOA 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 the

invasion 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 was

performed at least triple times.)

the control cells (no PFOA treatment). Breast cancer cells MDA-MB- electrophoresis of PCR products, no primer–dimer was observed

231 invasive ability was deﬁned as the mean OD volume of cells. for both the target genes and b-actin, and the speciﬁcity of the

products was also conﬁrmed by melt curve analysis.

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

time PCR 2.6. Luciferase assay

We employed quantitative real time PCR (qRT-PCR) to detect Luciferase reporter assay experiment was conducted to perform

the mRNA expression levels of homo-mmp-2/-9, with homo- the effect of PFOA on the gene promoter activity of mmp-2/-9 .

b-actin as housekeeping genes. After the cells were treated with MDA-MB-231 cells were grown on a 24-well plate for 24 h to reach

PFOA for 72 h, total RNA was isolated from breast cancer cells with approximately 70% conﬂuence prior to transfection. The cells in

Trizol reagent according to the manufacturer's protocol (Invitro- each well were co-transfected with 0.4 mg of mmp-2/-9 -promotor

gen, Carlsbad, CA). The puriﬁed total RNA (1 mg) was then reversely site-derived luciferase reporter plasmid and 0.1 mg of b-galactosi-

transcribed using the First Strand cDNA Synthesis Kit (TaKaRa, dase (b-gal) expression plasmid using Lipofectamine 2000

Japan). The qRT-PCR primers of mmp-2 gene were: forward 5 -GAG (Invitrogen, China Limited, China) according to the manufacturer's

0 0

AAC CAA AGT CTG AAG AG-3 and reverse 5 -GGA GTG AGA ATG protocol. After transfection for 6 h, cells were treated with different

0 0

CTG ATT AG-3 . The primers of mmp-9 gene were: forward 5 -TGC doses of PFOA, JSH-23 (10 mM) or both JSH-23 (10 mM) and PFOA

0 0

CCG GAC CAA GGA TAC AG-3 and reverse 5 -TCA GGG CGA GGA (50 nM) (equal DMSO was added to culture medium of another

CCA TAG AG-3 . PCR primers set of the internal control gene b-actin group as solvent control). 24 h later, cell lysates were prepared with

0 0 0

were: forward 5 -TGA CGT GGA CAT CCG CAA AG-3 and reverse 5 - Reporter Lysis Buffer (Promega, Madison, WI) following the

CTG GAA GGT GGA CAG CGA GG-3 . Reactions were conducted in manufacturer's instructions, and luciferase activity was measured

96-well plates with a ﬁnal volume of 20 ml including 10 ml SYBR with Luciferase Assay Reagent 10-Pack (Promega). The b-gal

Green PCR Master Mix (Invitrogen, Carlsbad, CA), plus 1 ml each activity of the cells was measured as follows: 1 mL of Mg solution

primer (2 mM), 1 ml template DNA, and 7 ml ddH2O. Thermal (0.1 M MgCl2, 4.5 M beta-mercaptoethanol), 22 mL of 4 mg/mL O-

cycling and ﬂuorescence detection were conducted on a Applied nitrophenol-b-D-galactoside solution (O-nitrophenyl-beta-D-gal-

Biosystems ViiA 7 (Life Technologies, USA), using the following actopyranoside) (in 0.1 M phosphate buffer (pH 7.5)), and 57 mL of

protocol: 95 C for 5 min, followed by 40 cycles of 95 C for 15 s, 0.1 M phosphate buffer (pH 7.5), were added into the obtained cell

60 C for 30 s and 72 C for 30 s. Each reaction was run in triplicate. extract solution (20 mL) to reach a total volume of 100 mL. As

The levels of mmp-2/-9 gene expression were normalized to control, the optical density (OD) volume of b-gal at 450 mm was

DDct

b-actin levels using the method of 2 . As assessed by measured in parallel.

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

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). Western

blotting 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 at

least triple times.)

2.7. Western blotting 2.8. Gelatin zymography

After the cells' protein componets were collected, a standard The enzyme activities of MMP-2 and MMP-9 were analyzed by

western blotting analysis was used to investigate the MMP-2/-9 gelatin zymography. Cells (4 10 ) were seeded into a 24-well

and NF-kB protein expression levels. Brieﬂy, nuclear and cellular plate and allowed to adhere for 6–8 h with the presence of serum.

protein extracts were prepared, 72 h after treated with different Subsequently, the culture medium was replaced by serum-free

doses of PFOA, JSH-23 (10 mM) or both JSH-23 (10 mM) and PFOA DMEM medium (400 ml per well) with different doses of PFOA (0,

(50 nM) (equal DMSO was added to culture medium of another 0.1, 1, 5, 10, and 50 nM). After incubation for 72 h, the medium in

group as solvent control), by using KEYGEN Protein Extraction Kit each well was gathered and centrifuged for 10 min at 2000 rpm to

(KEYGEN, Nanjing). For detecting the ratio of nuclei (p65):cytosol remove cell debris. The supernatant was collected and mixed with

(p65), cytosol protein and nucleus protein were dissolved in equal sample loading buffer containing no DTT (dithiothreitol) before 8%

volume lysis buffer of each group cells. Protein lysates were boiled SDS-polyacrylamide gel (containing 1 mg/mL gelatin) electropho-

in SDS-sample buffer for 5 min and then subjected to 8% SDS- resis. Then the gel was soaked in 2.5% Triton-X-100 for 30 min

polyacrylamide gel electrophoresis and transferred to PVDF twice to remove SDS, and transferred to bath buffer containing

(polyvinylidene ﬂuoride) membranes (Bio-Rad). Membranes were 50 mM tris (pH 8.0), 5 mM CaCl2 and 2 mM ZnCl2 at 37 C for 16 h.

then blocked for 2 h in 5% milk-Tris-Buffered Saline Tween-20 The gel was then stained with 0.1% Coomassie blue in 45%

(TBST) at room temperature, and incubated overnight at 4 C either methanol and 10% acetic acid. The visualized bands indicating the

with the monoclonal antibodies (Cell Signaling Technology, USA) of presence of a protein with gelatinolytic activity were analyzed by

anti-b-actin, anti-MMP-2, anti-MMP-9 or anti-NF-kB. Membranes the FluorChem M system (Protein Simple, USA).

were washed four times in TBST and incubated with the

appropriate horseradish peroxidase-conjugated secondary anti- 2.9. Statistical analysis

bodies at room temperature for 2 h. Blots were visualized by

enhanced chemiluminescence (Thermo, USA) and analyzed using a The differences between the treated and the control cells the

scanning densitometer with the molecular analysis software invasion rates and protein expressions were by applying one way

FluorChem M system (Protein Simple, USA). ANOVA (analysis of variance) method followed by post-hoc Tukey's

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

analysis. A p-value of less than 0.05 was considered statistically signiﬁcantly decreased 12% compared to control group; we also

signiﬁcant, each experiment was performed at least triple times. found that after JSH-23 pre-incubation, nucleus/cytosol ratio of

Computer-based calculations were conducted using SPSS version NF-kB was only up-regulated 16% with PFOA exposure, while PFOA

20.0 (SPSS Inc. Chicago, Illinois, USA). increased 36% NF-kB nuclear translocation without JSH-23 pre-

incubation, which indicated that NF-kB inhibitor JSH-23 partially

3. Results inhibited PFOA-induced NF-kB nuclear translocation (Fig. 4A).

Then we performed trans-well ﬁlter assay to certify the inhibition

3.1. Effects of PFOA treatment on breast cancer cell invasion effect of JSH-23 on PFOA-induced MDA-MB-231 invasion. The data

showed that with JSH-23 pre-treatment, PFOA-promoted cancer

In order to investigate the invasiveness of the breast cancer cells cell invasiveness was reduced signiﬁcantly (Fig. 4B), which

(MDA-MB-231) after different doses of PFOA treatment, we suggested that PFOA could activate cancer cell invasion through

performed matrigel invasion assays. First, we evaluated the effect inducing NF-kB activation. Meanwhile, JSH-23 could reverse the

of different doses of PFOA on cell survival ratios. High-dose PFOA stimulation effect of PFOA on gene MMP-2/-9 promoter activities

treatments (>100 nM) could inhibit the viability of MDA-MB-231 (Fig. 4C) and expression levels (Fig. 4D) by using luciferase reporter

cells (P < 0.05), but no cell viability inhibition was observed after assay and qRT-PCR, which was consistent with western blotting

treatment with low concentrations (100 nM) (P > 0.05) (Fig. 1A). data (Fig. 4E). Furthermore, gelatin zymography demonstrated that

Furthermore, we found that low-doses exposure could stimulate MMP-2/-9 activity was inhibited in the cells treated with both JSH-

the invasion ability of the crystal violet stained cancer cells 23 and PFOA (Fig. 4F), suggesting that NF-kB mediated MMP-2/-9

(Fig. 1B), which was subsequently conﬁrmed by determining the overexpression and enzyme activation stimulated by PFOA.

OD value of the invaded cells (Fig. 1C). PFOA treatments at 5, 10, or

50 nM signiﬁcantly increased the number of the invaded cells 4. Discussion

(p < 0.05), while treatment at 0.1 or 1 nM caused no change on cell

invasion ability (p > 0.05). Compared with the control, cell This study demonstrated that the environmental pollutant

invasiveness was separately enhanced by 1.5, 2.17 and 3.14 fold PFOA can increase invasiveness of breast cancer cells. Accumulat-

after PFOA exposure at 5,10 and 50 nM. PFOA stimulated the cancer ing evidence has shown that PFOA can act as a tumor initiator to

cells invasion in a dose-dependent manner (R = 0.8422). induce DNA damages and oxidative stress (Eriksen et al., 2010; Yao

and Zhong, 2005). However, little information is available about

3.2. Enhanced MMP-2/-9 expression and activation levels in PFOA- the effect of PFOA on breast cancer cell invasion, although clinical

treated MBA-MB-231 cells concerns have focused on screening and developing drugs capable

of inhibiting breast cancer metastasis (Wang et al., 2010; Yeh et al.,

To conﬁrm the effect of the PFOA treatment on MMP-2/-9 2010). Recent studies have indicated that cancer cell migration and

expression, we investigated the gene promoter activity, mRNA and invasion progression can be induced by physicochemical factors

protein expression levels of MMP-2/-9 in MDA-MB-231 breast

cancer cells after 24 h treatment with different doses of PFOA. qRT-

PCR results demonstrated that the PFOA treatment upregulated

gene mmp-2/-9 expressions (Fig. 2A). Low-dose of PFOA (<5 nM)

showed no effect on mmp-2/-9 mRNA levels, however, higher-dose

of PFOA (5–50 nM) increased MMP-2/-9 mRNA expressions

(P < 0.05). Western blotting showed that the protein levels of

MMP-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 PFOA

treatment (Fig. 2D). Results of qRT-PCR, western blotting,

luciferase reporter, and gelatin zymography results revealed that

PFOA exposure could evidently up-regulate both expression and

activation of MMP-2/-9 in MDA-MB-231 cells, which may be

subsequently 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-kB

activity to explore the potential molecular mechanisms of MMP-

2/-9 overexpression. The cytosol and nuclear proteins were

isolated from the PFOA-treated cells for western blotting of p65

(Fig. 3A). The data showed that after PFOA treatment, ratio of the

nucleus/cytosol level of NF-kB was signiﬁcantly elevated from

0.24 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 role

Fig. 3. PFOA activated NF-kB to translocate into nuclear from cytosolic. The cytosol

of NF-kB activation in the MMP-2/-9 stimulation and cell invasive

and nuclear proteins were isolated from the PFOA-treated cells for western blotting

ability promotion. First we conﬁrmed that after NF-kB inhibitor

of p65 (A). After PFOA treatment, the nucleus/cytosol ratio of NF-kB was elevated

JSH-23 (10 mM) treatment, nucleus/cytosol ratio of NF-kB was signiﬁcantly (B). (Each experiment was performed at least triple times.)

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

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/-9

stimulation 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-kB

was signiﬁcantly decreased (A), and the cells exposed to PFOA (50 nM) appeared lower invasiveness (p < 0.05) (B). Meanwhile, luciferase reporter assay results demonstrated

that JSH-23 reversed the stimulation effect of PFOA on gene mmp

copyediting change here. "MMP" is deleted.–!>-2/-9 promoter activities (C). qRT-PCR showed that JSH-23 thwarted PFOA-induced

change here. "mmp" is retained.–!>mmp-2/-9 expression (D), which was agreed with western

blotting 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 experiment

was performed at least triple times.)

such as progesterone receptor (Fu et al., 2010), glutamate (Herner MMPs over-expression is closely related to tumor metastasis,

et al., 2011), extracellular calcium (Saidak et al., 2009), bacterial much effort has been devoted to screening and developing drugs

peptidoglycan (Xie et al., 2010), microcystin-LR (Zhang et al., 2012) which can inhibit MMPs expression (Lee et al., 2008). However,

and so on, but there is little information reported on the few environmental pollutants have been known to be capable of

relationship between environmental pollution and breast cancer inducing MMPs expression. Recently, microcystin-LR has been well

metastasis. documented to induce MMP-2/-9 expression through activation of

Our results suggested that low-dose treatments (100 nM) NF-kB in human melanoma cells (Zhang et al., 2012). Apart from

could not affect breast cancer cell MDA-MB-231 survival, but high- chemicals, radiation can also up-regulate MMP-9 mRNA level,

dose treatments (>100 nM) could suppress cell viability. In order to protein level and catalytic activity by activating NF-kB in

exclude the inﬂuence on cell survival, we chose the low-dose hepatocellular carcinoma cells (Cheng et al., 2006). Corsini et al.

groups (0–50 nM) to explore the effect and mechanism of PFOA on have found that 100 mg/ml (approximately 0.24 mM) PFOA could

breast cancer cells invasion ability. In the present study, invasive reduce LPS-induced MMP-9 via regulating NF-kB phosphorylation

ability of breast cancer cell MDA-MB-231 was signiﬁcantly level, while in this study we found that environmental concentra-

enhanced after PFOA exposure. MMP-2/-9, which can efﬁciently tion (nM level) of PFOA could induce NF-kB activation, which

degrade extracellular matrix, are well known to play critical roles might be involved in the over-expressions of MMP-2/-9. NF-kB

in breast cancer invasiveness. In order to elucidate the undergoing complex located in the cytoplasm in a latent form often transfers to

molecular mechanisms, we detected the inﬂuence of PFOA the nuclear when NF-kB was activated, so we detected the changes

treatment on the promoter activity, mRNA expression, protein of NF-kB activity by assessing the ratio of nuclear (NF-kB):cytosol

expression and enzyme activation levels of MMP-2/-9 by using (NF-kB). Western blotting showed that PFOA treatment induced

luciferase reporter assay, qRT-PCR, western blotting and gelatin nuclear translocation of NF-kB, revealing that NF-kB activation

zymography separately. results consistently showed that PFOA might mediate MMP-2/-9 overexpression induced by PFOA.

could enhance MMP-2/-9 expression and enzyme activation levels The proposed molecular mechanism was further conﬁrmed by

in the breast cancer cells. the JSH-23 suppression assay, a well-established NF-kB inhibition

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

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(Farina et al., 2011). NF-kB also could mediate MMP-2 expression Farina, A.R., Cappabianca, L., DeSantis, G., Di Ianni, N., Ruggeri, P., Ragone, M.,

Merolle, S., Tonissen, K.F., Gulino, A., Mackay, A.R., 2011. Thioredoxin stimulates

and activation through stimulating MT1-MMP expression (Shih

MMP-9 expression, de-regulates the MMP-9/TIMP-1 equilibrium and promotes

et al., 2010) and activating pro-MMP-2 (Han et al., 2001). Taken

MMP-9 dependent invasion in human MDA-MB-231 breast cancer cells. FEBS

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Fraser, A.J., Webster, T.F., Watkins, D.J., Nelson, J.W., Stapleton, H.M., Calafat, A.M.,

expression, resulting in enhanced breast cancer cell invasion. PFOA

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Fu, X.D., Goglia, L., Sanchez, A.M., Flamini, M., Giretti, M.S., Tosi, V., Genazzani, A.R.,

indoor environments, including homes and workplaces (Fraser

Simoncini, T., 2010. Progesterone receptor enhances breast cancer cell motility

et al., 2012; Haug et al., 2011), and our ﬁndings pointed the

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In conclusion, this study indicates that PFOA exposure can

Han, Y.P., Tuan, T.L., Wu, H., Hughes, M., Garner, W.L., 2001. TNF-alpha stimulates

promote breast cancer cell invasion by stimulating MMP-2/-9 activation of pro-MMP2 in human skin through NF-(kappa)B mediated

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mRNA, protein expression and activation. PFOA is able to activate induction of MT1-MMP. J. Cell Sci. 114, 131 139.

Haug, L.S., Huber, S., Schlabach, M., Becher, G., Thomsen, C., 2011. Investigation on

NF-kB by stimulating translocation into the nucleus. NF-kB

per- and polyﬂuorinated compounds in paired samples of house dust and

activation may be contributed to the MMP-2/-9 overexpression

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Conﬂict of interest

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Holmstrom, K.E., Jarnberg, U., Bignert, A., 2005. Temporal trends of PFOS and PFOA

in guillemot eggs from the Baltic Sea 1968–2003. Environ. Sci. Technol. 39, 80–

The authors declare that they have no competing interests.

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cancer development and progression. Nat. Rev. Immunol. 5, 749–759.

Karin, M., Lin, A., 2002. NF-kappaB at the crossroads of life and death. Nat. Immunol.

The Transparency document associated with this article can be

3, 221–227.

found in the online version. Lau, C., Anitole, K., Hodes, C., Lai, D., Hutchens, Pfahles-, Seed, A., J, 2007.

Perﬂuoroalkyl acids: a review of monitoring and toxicological ﬁndings. Toxicol.

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Acknowledgments Lee, K.W., Kang, N.J., Oak, M.H., Hwang, M.K., Kim, J.H., Kerth, Schini-, Lee, V.B., H.J,

2008. Cocoa procyanidins inhibit expression and activation of MMP-2 in

vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP

This study was ﬁnancially supported by the Key project of the

activities. Cardiovasc. Res. 79, 34–41.

National Natural Science Foundation of China (81330067), the Overall, C.M., Dean, R.A., 2006. Degradomics: systems biology of the protease web.

Pleiotropic roles of MMPs in cancer. Cancer Metastasis Rev. 25, 69–75.

National Natural Science Foundation of China (81302304,

Rayne, S., Forest, K., 2009. Perﬂuoroalkyl sulfonic and carboxylic acids: a critical

81302306, and 81172501), Natural Science Foundation of Jiangsu

review of physicochemical properties, levels and patterns in waters and

Province (BK20130074). wastewaters, and treatment methods. J. Environ. Sci. Health A Tox. Hazard.

Subst. Environ. Eng. 44, 1145–1199.

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Appendix A. Supplementary data

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Shih, Y.W., Chien, S.T., Chen, P.S., Lee, J.H., Wu, S.H., Yin, L.T., 2010. Alpha-mangostin

Supplementary data associated with this article can be found, in

suppresses phorbol 12-myristate 13-acetate-induced MMP-2/MMP-9 expres-

the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.06.004.

sions via alphavbeta3 integrin/FAK/ERK and NF-kappaB signaling pathway in

human lung adenocarcinoma A549 cells. Cell Biochem. Biophys. 58, 31–44.

Shin, H.M., Vieira, V.M., Ryan, P.B., Steenland, K., Bartell, S.M., 2011. Retrospective

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