Arch Toxicol
DOI 10.1007/s00204-014-1301-z
Genotoxicity and Carcinogenicity
Bisphenol A modulates colorectal cancer protein profile
and promotes the metastasis via induction of epithelial
to mesenchymal transitions
Zhuo‑Jia Chen · Xiang‑Ling Yang · Hao Liu · Wei Wei ·
Kun‑Shui Zhang · Hong‑Bin Huang · John P. Giesy ·
Huan‑Liang Liu · Jun Du · Hong‑Sheng Wang Received: 16 January 2014 / Accepted: 17 June 2014
© Springer-Verlag Berlin Heidelberg 2014
Abstract More and more evidences indicate that endocrine disruptor chemicals such as bisphenol A (BPA) can
act as carcinogens and enhance susceptibility to tumorigenesis. Although the gut is in direct contact with orally
ingested BPA, effects of BPA on occurrence and development of colorectal cancer remain an unexplored endpoint.
Colorectal cancer SW480 cells treated with nanomolar
(10−8 M) or greater (10−5 M) concentrations of BPA were
compared with responses of a control group. Proteomic
study revealed that more than 56 proteins were modulated
following exposure to BPA, which are relevant to structure, motility and proliferation of cells, production of ATP,
oxidative stress, and protein metabolism. Further studies
revealed that BPA increased migration and invasion and
triggered transformations from epithelial to mesenchymal transitions (EMTs) of colorectal cancer cells, which
was characterized by acquiring mesenchymal spindle-like
Electronic supplementary material The online version of this
article (doi:10.1007/s00204-014-1301-z) contains supplementary
material, which is available to authorized users.
Z.-J. Chen (*) · H.-B. Huang Department of Pharmacy, State Key Laboratory of Oncology
in South China, Collaborative Innovation Center for Cancer
Medicine, Sun Yat-sen University Cancer Center,
Guangzhou 510060, China
e-mail: chenzhuojia86@163.com
X.-L. Yang · H.-L. Liu Guangdong Institute of Gastroenterology and the Sixth
Affiliated Hospital, Institute of Human Virology, Key Laboratory
of Tropical Disease Control (Ministry of Education), Sun Yat-sen
University, Guangzhou 510655, China
H. Liu Cancer Research Institute and Cancer Hospital, Guangzhou
Medical University, Guangzhou 510095, China
morphology and increasing the expression of N-cadherin
with a concomitant decrease of E-cadherin. Accordingly,
BPA treatment increased the expression of transcription
factor Snail. Furthermore, signal AKT/GSK-3β-mediated
stabilization of Snail is involved during BPA-induced EMT
of colon cancer cells. Our study first demonstrated that the
xenoestrogen BPA at nanomolar and greater concentrations
modulates the protein profiles and promotes the metastasis
of colorectal cancer cells via induction of EMT.
Keywords Colorectal cancer · BPA · EMT · Proteomic ·
Tumorigenesis · Migration
Abbreviations
BPABisphenol A
CRCColorectal cancer
DMSODimethyl sulfoxide
E2Estradiol
E-CadE-cadherin
EDCsEndocrine disruptor chemicals
W. Wei · J. Du (*) · H.-S. Wang (*) Department of Microbial and Biochemical Pharmacy, School
of Pharmaceutical Sciences, Sun Yat-sen University, No. 132
Waihuandong Road, University Town, Guangzhou 510006, China
e-mail: dujun@mail.sysu.edu.cn
H.‑S. Wang
e-mail: whongsh@mail.sysu.edu.cn; hongshengwang@foxmail.com
K.-S. Zhang Department of Pharmacy, Sun Yat‑sen Memorial Hospital, Sun
Yat-sen University, Guangzhou 510120, China
J. P. Giesy Department of Veterinary Biomedical Sciences, Toxicological
Center, University of Saskatchewan, Saskatoon, SK, Canada
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EMTEpithelial to mesenchymal transitions
ERREstrogen-related receptor
ERα/βEstrogen receptor α/β
FBSFetal bovine serum
FNFibronectin
GPERG-protein-coupled estrogen receptor
hsp27Heat-shock protein 27
N-CadN-cadherin
VimVimentin
ZO-1Zona occludin-1
Introduction
The xenoestrogen bisphenol A (BPA), a food contaminant
with endocrine disrupting activity, is widely used to manufacture consumer products including drinking water bottles
and reusable food containers (Brotons et al. 1995). BPA
can leach from the polymers into food and water under
normal conditions, furthermore, exposure to elevated temperatures (boiling, heating) greatly increases its rate of
migration (Le et al. 2008). Due to an increase in products
based on epoxy resins and polycarbonate plastics, exposure of human beings to BPA via food consumption and
environmental intakes has increased in recent years (Kang
et al. 2006). Numerous studies have suggested that human
exposure to BPA is widespread, which is evidenced by its
presence in urine, blood, fetal tissues, and amniotic fluid
(Geens et al. 2012; Vandenberg et al. 2007a).
Colorectal cancer (CRC), also called colon cancer or
large bowel cancer, is the third most common form of cancer and the second leading cause of death due to cancer in
the Western world (Siegel et al. 2013). Previous studies
indicated that more than 80 % of CRC cases and deaths
are attributable to diet and environmental factors (Jasperson et al. 2010). In recent years, several lines of clinical
and experimental evidences have reported that estrogens,
xenoestrogens, and their receptors are involved in development and regulation of CRC (Chen and Iverson 2012).
Classical estrogen receptor α/β (ERα/β), G-protein-coupled
estrogen receptor (GPER) and estrogen-related receptor (ERR) have been detected in various colon cancer cell
lines (Kennelly et al. 2008). Considering all these estrogen
receptors can mediate estrogenic signaling of endocrine
disruptor chemicals (EDCs), the roles of BPA on tumorigenesis and progression of CRC should be illustrated.
There is a global concern for adverse endocrine disruptive effect and disturbing of normal sex hormone balance caused by BPA. Recent studies revealed that chronic
exposure to reference doses of BPA can decrease sperm
production and fertility (Salian et al. 2011), stimulate
the development of mammary gland (Vandenberg et al.
2007b), increase body weight (Vom Saal et al. 2012), and
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promote the tumorigenesis and development. To date, these
studies examining effects of BPA on tumorigenesis have
focused on the reproductive tract and mammary glands.
Rats exposed prenatally to greater doses of BPA-induced
in situ development of carcinomas (Durando et al. 2007).
Exposure to BPA during nursing followed by exposure at
50 days of age to dimethylbenzantracene resulted in more
of tumors per rat and a shorter latency period compared
with animals not exposed to BPA (Jenkins et al. 2009).
Although gut is in direct contact with BPA consumed orally
(Braniste et al. 2010; Dekant and Voelkel 2008), there is
very limited data concerning the effects of BPA on progression and development of CRC.
Therefore, the present study was designed to investigate
effects of BPA on progression of colon cancer. Profiles of
proteins altered by exposure to either nanomolar or micromolar concentrations of BPA on SW480 cells were determined by proteomic analysis. According to the identified
proteins, expression of which were altered by exposure to
BPA on migration of colon cancer were further confirmed
and investigated. To our knowledge, this is the first study
to identify changes in expression of proteins and illustrate
effects of BPA on CRC metastasis.
Materials and methods
Reagents
All reagents used in 2-DE were bought from Bio-Rad (Hercules, CA, USA). All chemicals were of reagent grade or
better and purchased from Sigma Chemical Co. (St. Louis,
MO, USA) unless otherwise noted. BPA was dissolved
in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock
solution, and stored at −20 °C. Primary antibodies against
fascin, Krt8, TIF4A, p-ERK1/2 (Thr202/204), ERK1/2, Bcl2, E-cadherin (E-Cad), Zona occludin-1(ZO-1), N-cadherin
(N-Cad), Vimentin (Vim), Snail, Slug, ZEB, Twist, p-AKT
(Ser473), AKT, p-GSK-3β (Ser9), GSK-3β, p-p38-MAPK
(Thr180/Tyr182), p38-MAPK, and β-actin were purchased
from Cell Signaling Technology (Beverly, MA, USA). Primary antibody to fibronectin (FN) was obtained from Bioworld Technology, Inc (Minneapolis, MN, USA). Horseradish peroxidase-conjugated secondary antibody was from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). All
compounds were solubilized in DMSO. Medium containing 0.5 % DMSO was used as the control.
Cell line and cell culture
The human CRC cell lines SW480 (passage 8–15) and
LoVo (passage 15–18) were cultured in RPMI medium 1640
supplemented with 10 % fetal bovine serum (FBS). The
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HCT-116 cells (passage 12–15) were grown in DMEM supplemented with 10 % FBS. Cells were incubated at 37 °C in
a 5 % CO2 atmosphere. Both the plastic items used for the
experiments and the water used to prepare the reagents were
pretreated by enhanced sonochemical degradation to reduce
any potential background BPA (Sheng and Zhu 2011).
Analysis of cell proliferation and apoptosis
Cell proliferation and apoptosis were detected by use
of previously described procedures (Wang et al. 2012).
Briefly, SW480 cells were inoculated in 96-well plates for
24 h before exposure to BPA. Viability of cells after treated
with increasing concentrations of BPA for the indicated
times were evaluated by use of the CCK-8 kit (Dojindo
Molecular Technologies, Gaithers burg, MD, USA) according to the manufacturer’s instructions. For cell apoptosis,
both the suspension and adherent cells treated with BPA
for 48 h were collected into flow cytometry tubes and
centrifuged at 2,000 rpm for 5 min to obtain cell pellets.
Cells were washed twice with PBS, stained with Annexin
V-FITC for 15 min and propidium iodide for 5 min, and
then analyzed by flow cytometry using FL1 (Em: 525 nm)
and FL3 (Em: 670 nm).
time periods. Cells were washed three times with PBS,
fixed with 4 % paraformaldehyde for 20 min and permeabilized with 0.3 % Triton X-100 for 10 min. After blocking with goat serum for 2 h at room temperature, cells were
incubated with antibodies against E-Cad (1:100 dilutions)
at 4 °C overnight. Slides were washed three times with PBS
and incubated with Alexa Fluor 488-conjugated secondary
antibodies (1:1,000 dilutions) for 1 h at room temperature. Nuclei were stained with DAPI for 10 min. Samples
were examined with Confocal Laser Scanning Microscopy
(Zeiss) to analyze expression of E-Cad.
Statistical analysis
All values were reported as mean ± SD of three independent
experiments unless otherwise specified. Data were analyzed
by two-tailed unpaired Student’s t test between two groups
and by one-way ANOVA followed by Bonferroni test for
multiple comparison involved. Homogeneity of variance was
confirmed by use of Levine’s test. The statistical analyses
were performed using SPSS 17.0 for Windows. A p value of
<0.05 was considered to be statistically significant.
Results
Proteomic analysis of proteins in SW480 cells
Cytotoxic effects of BPA on SW480 cells
SW480 cells were exposed to vehicle (0.5 % DMSO), 10−8
or 10−5 M BPA and incubated for 48 h. Two-dimensional
gel electrophoresis (2-DE), protein visualization, image
analysis was performed essentially as described in the Supplementary Materials (SM). The detailed procedures for ingel tryptic digestion of proteins and MALDI–TOF–MS/MS
analysis were also described in the SM.
Wound healing and transwell migration/invasion assay
For the in vitro wound healing assay, confluent monolayers
of SW480 cells were scratched, and the migration distance
of the cells into the scratched area was measured in 10 randomly chosen fields. Migration and invasion assays were
performed in Boyden chambers according to the previous
study (Wang et al. 2013) and described in the SM.
Western blotting analysis
Western blotting was performed as previously described
(Jiang et al. 2013) and described in the SM.
To determine cytotoxicity of BPA, SW480 cells were
treated with concentrations of BPA ranging from 10−8
to 10−4 M for 48 or 72 h, and then cell viability was
assessed by CCK-8 kit assay. Our results revealed that
concentrations of BPA >10−5 M significantly inhibited
proliferation of SW480 cells (Fig. 1a). There was no
statistically significant effect on apoptotic/necrotic populations at both 10−8 and 10−5 M BPA as compared to
vehicle treated cells of both cell lines after 48 h (Fig. 1b).
Concentrations of BPA in blood, fetuses, urine, saliva,
and various tissues are in the range of 0.5–10 ng/mL
or ng/g, which is equivalent to 2 × 10−9–4.4 × 10−8
M (Vandenberg et al. 2010). Micromolar BPA has been
reported to stimulate human cancer cell migration (Derouiche et al. 2013) and proangiogenic of human primary
endothelial cells (Andersson and Brittebo 2012). Therefore, 10−8 and 10−5 M were chosen to represent the oral
equivalent and high concentrations of BPA for further
proteomic study (Ge et al. 2014).
Immunofluorescence
The analysis and validation of BPA‑induced proteome
alterations in SW480 cells
SW480 cells were cultured on chamber slides, serum
starved for 12 h, then exposed to BPA for the indicated
The differentially expressed proteins in SW480 cells
treated with BPA were analyzed by MALDI–TOF–MS/
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Fig. 1 Effects of BPA on
proliferation and apoptosis
of SW480 cells. a Cells were
treated with various concentrations (10−8–10−4 M) of BPA for
48 or 72 h. Viability of cells was
assessed by CCK8 kit assay.
b Cells were exposed to BPA
for 48 h. Collected cells were
stained with annexin V-FITC
and PI, and then analyzed
by flow cytometry. Data are
presented as mean ± SD of
three independent experiments.
*p < 0.05 compared with
control
MS. Representative two-dimensional gel images of control,
10−8 and 10−5 M BPA-treated cells are shown in Fig. S1.
The 2-DE maps were compared with PDQuest software to
identify protein spots that varied among treatments. After
exposure to BPA, significantly (p < 0.05) differentially
expressed protein spots observed in all replicate gels were
scored. The fold difference is represented by the ratio of the
intensity value of the BPA-treated group to the value of the
control group (Table S1). Totally, 68 differently expressed
proteins were found as indicated by the spots marked with
arrows (Fig. S1). All these spots were excised from gels for
protein identification. And then 56 differentially expressed
proteins were identified by MALDI–TOF–MS/MS analysis
(Table S1).
The Western blotting analysis was performed to further verify the up- or down-regulation of identified proteins (Fig. S2). Validation confirmed up-regulation of
Fascin (spot 13) and TIF4A (spot 29) by BPA in SW480
(p53 wt, APC wt, and MSI−), LoVo (p53 mutant, APC wt,
and MSI+) and HCT-116 (p53 mutant, APC mutant, and
MSI−) cells. Furthermore, down-regulation of Krt8 (spot
64) was also confirmed in SW480 cells treated with BPA.
The results not only validated the results of proteomic
13
study but also indicated that protein profiles changed by
BPA on SW480 cells were representative for other colorectal cancer cells.
Functional categories of identified proteins
The PANTHER classification system was used to classify
the 56 identified proteins. Those identified proteins were
classified into seven categories according to their functions during biological processes (Fig. 2a). Our results
indicated that cell structure and motility-related proteins,
cell proliferation and apoptosis-related proteins, energy
metabolism, and oxidative stress proteins accounted for
the major proportions among the detected proteins. Protein–protein interaction network among the identified
proteins was predicted based on the STRING system.
Notably, five major clusters of interacting proteins were
determined by STRING (Fig. 2b), including cell structure and motility, cell proliferation and cell cycle, energy
metabolism, oxidative stress, protein folding and metabolism. It suggested that BPA significantly changed expression of proteins related to tumorigenesis and metastasis in
colorectal cancer cells.
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Fig. 2 Functional classification
and distribution of all identified
proteins. a Seven protein groups
were categorized based on the
putative biological functions
of identified proteins and the
percentages of each protein
group were indicated. b The
protein–protein interaction network of the identified proteins.
The network containing 56
identified proteins was mapped
using STRING system based on
evidence with different types
BPA promotes the metastasis of colorectal cancer cells
The proteomic study revealed that proteins of cell motility
and structure were markedly changed after treatment with
oral equivalent or greater concentrations of BPA. Therefore, metastasis of SW480 cell was further investigated
by use of wound healing and transwell migration/invasion
assays. Treatment with either 10−8 or 10−5 M BPA for 48
or 72 h significantly increased wound closure as compared
to the control group (p < 0.05) (Fig. 3a, b). Furthermore,
exposure to BPA resulted in a significantly more migration
(Fig. 3c) and invasion (Fig. 3d) cells. Compared with the
untreated cells, the number of migrated and invaded cells
was approximately 45 % (migration) and 72 % (invasion)
greater after treatment with 10−8 M BPA for 72 h, and about
88 % (migration) and 125 % (invasion) greater after treatment with 10−5 M BPA for 72 h. Collectively, our results
indicated that both the oral equivalent and greater concentrations of BPA promotes metastasis of SW480 cells.
BPA triggers epithelial to mesenchymal transition (EMT)
of SW480 cells
Previous studies indicated that increased migration and
invasion abilities of tumor cells are consistent with events
at EMT, during which, the epithelial makers such as E-cad
and ZO-1 are down-regulated, whereas mesenchymal markers such as N-cad and FN are up-regulated (Wang et al.
2013). The EMT of SW480 cells was observed after stimulation with 10−8 and 10−5 M BPA for 96 h. Cells resulted in
a significant change in morphology, from cobblestone morphology to mesenchymal spindle-like and fusiform features
(Fig. 4a). Western blotting analysis revealed that this morphological change was associated with down-regulation of
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Fig. 3 BPA-triggered migration and invasion of SW480 cells. a Representative images of the monolayer wound healing assay utilizing
the SW480 cell line immediately after scratching 48 and 72 h. Cells
were treated with 0.5 % DMSO (control), 10−8 and 10−5 M BPA, b
quantitative analysis of wound healing assay for SW480 cells treated
with BPA; SW480 cells were allowed to migrate (c) and invasive
(d) transwell chambers for 48 and 72 h in the presence or absence
of BPA (10−8 or 10−5 M). Migrated and invasion cells were fixed,
stained, and photographed. The % number of migrated and invasion
cells. Data represent the average of three independent experiments.
*p < 0.05 compared with control, **p < 0.01 compared with control
expression of epithelial characteristics E-Cad, ZO-1 and
the up-regulation of expression of mesenchymal characteristics FN, N-cad and Vim (Fig. 4b). Furthermore, BPA
treatment for 96 h significantly decreased the expression
of E-Cad while increased the expression of Vim in HCT116 cells (Fig. 4c). These results confirmed that BPA can
induce EMT of colorectal cancer cells.
Since transcription factors Snail, ZEB1, Twist, and Slug
play essential roles in regulating EMT (Cano et al. 2007),
their expressions were investigated to determine whether
they were involved in BPA-induced EMT of colorectal cancer cells. Compared to untreated cells, 10−8 M BPA significantly increased protein levels of Snail and Twist, but not
Slug or ZEB, while 10−5 M BPA significantly increased protein levels of Snail, Twist, and ZEB, but not Slug (Fig. 4c).
We further performed knockdown assays to investigate
the role of Snail in BPA-induced EMT. SW480 cells were
transfected with nontargeting control siRNA or siSnail for
24 h, and then treated with 10−5 M BPA for another 96 h.
The expression of Vim, E-Cad, and Snail were detected by
Western blotting. Results showed that silencing of Snail by
siRNA significantly attenuated BPA-induced up-regulation
of Vim and down-regulation of E-Cad (Fig. 4d). Collectively,
these observations demonstrated that Snail acts as a key regulator in BPA-induced EMT of SW480 cells.
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Akt/GSK‑3β mediates BPA‑induced EMT of colorectal
cancer cells
Recent studies revealed that the occurrence of EMT
requires AKT/GSK-3β-mediated stabilization of Snail in
colorectal cancer cells (Wang et al. 2013). Therefore, phosphorylation and protein levels of Akt, GSK-3β, ERK1/2,
and p38-MAPK were detected by Western blotting in
SW480 cells exposed to BPA for 96 h. Phosphorylation of
Akt and GSK-3β were significantly increased after stimulated by BPA, particularly in cells exposed to 10−5 M BPA
for 96 h. However, there was lesser phosphorylation of
p-ERK1/2 and no significant change in phosphorylation
of p38-MAPK in SW480 cells treated with BPA (Fig. 5a).
These results suggested that AKT/GSK-3β may be involved
in BPA-induced EMT of CRC.
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Fig. 4 BPA treatment induced
EMT of SW480 cells. a SW480
cells were treated with 10−8
or 10−5 M BPA for 96 h. Cell
morphological changes associated with EMT are shown in
the phase-contrast image. b
The expression of epithelial
makers (E-cad and ZO-1) and
mesenchymal markers (N-cad,
Vim and FN) in SW480 cells
treated with BPA for 96 h. c
The expression of EMT-related
transcription factors Snail, Slug,
ZEB, and Twist in SW480 cells
treated with BPA for 96 h. d
SW480 cells transfected with siSnail or si-NC were stimulated
with or without 10−5 M BPA for
96 h, and the expression of Vim,
E-Cad, and Snail were detected
by Western blotting. β-Actin
servers as the loading control
for Western blotting analysis
To verify whether AKT/GSK-3β is involved in BPAinduced EMT of colorectal cancer cells, SW480 and HCT116 cells were treated with the inhibitor of PI3 K/Akt,
LY294002 (20 μM), prior to exposure to BPA (10−5 M),
expression of p-AKT, p-GSK-3β, Snail, and markers of
EMT were determined by Western blotting. The results
showed that levels of p-AKT, p-GSK-3β, Snail, and mesenchymal marker Vim were increased while E-Cad was
decreased after exposure to BPA for 96 h. However, these
effects were reversed upon treating with LY294002 alone
or in combination with BPA in both SW480 (Fig. 5b) and
HCT-116 (Fig. 5c) cells. Furthermore, the inhibitor of
PI3 K/Akt abolished the BPA-induced EMT-like morphological changes and down-regulation of E-Cad in SW480
cells (Fig. 5d). Collectively, these observations suggested
that colorectal cancer cells had undergone an EMT after
treated by BPA and that AKT/GSK-3β-mediated stabilization of Snail mediated this process.
Discussion
Proteomic modifications of SW480 cells in response to oral
and high concentrations of BPA
The results of the proteomic analysis revealed that
treatment with BPA significantly modulates expression of proteins involved in various functional activities
such as cytoskeletal dynamic flexibility, cell cycle and
proliferation, ATP production, antioxidant mechanisms and
protein metabolism. Because of the limitations of in identifying proteins, proteomics data must be validated by use
of independent methods. Due to the large number of “hits”
observed in this study, validation of all proteomic results
could not be accomplished. Therefore, three proteins were
selected from different functional protein networks, Fascin, Krt8, and TIF4A for Western blotting analysis. The
results confirmed the significant greater expression of Fascin and TIF4A and lesser amount of Krt8 in SW480 cells
after exposure to BPA (Fig. S2). Furthermore, the results
also showed that BPA can increase the protein expression
of Fascin and TIF4A in both LoVo (p53 mutant, APC wt,
and MSI+) and HCT-116 (p53 mutant, APC mutant, and
MSI−) cells, suggesting that proteomic observations are
representative in various colorectal cancer cells. Further
studies showed that BPA can modulate the proliferation
and promote metastasis of SW480 cells. Thus, together
Western blotting and functional studies confirmed validity
of the proteomic results.
The five most represented functional activities for the
modulated proteins are cell structure and motility (Krt 8
and fascin), cell cycle and proliferation (translation initiation factor), protein folding and metabolism (heat-shock
cognate 71 kDa protein and elongation factor 1), oxidative
stress (complex I and protein DJ-1), and energy metabolism (glucose-6-phosphate dehydrogenase and glutamate
dehydrogenase 1) (Table S1). These functions are known
to be essential in steroid signaling which involves fast
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Fig. 5 Akt/GSK-3β-mediated BPA-induced EMT of colorectal cancer cells. a SW480 cells were treated with 10−8 or 10−5 M BPA for
96 h, and the phosphorylation and protein levels of Akt, GSK-3β,
ERK1/2, and p38-MAPK were detected by Western blotting. SW480
cells (b) and HCT-116 cells (c) were pretreated with or without
LY294002 (20 μM) for 1 h, followed by stimulation with or without BPA (10−5 M) for 96 h. The expression of Snail, E-Cad and the
activation of AKT and GSK-3β were examined by Western blotting. d
SW480 cells were pretreated with or without LY294002 (20 μM) for
1 h, followed by stimulation with or without BPA (10−5 M) for 96 h.
Changes in morphology and expression of E-Cad were analyzed by a
phase-contrast microscopy and immunofluorescence staining, respectively. Nuclei were visualized with DAPI staining
nongenomic activities (including the transport and metabolism of signaling molecules) and genomic mechanisms
mediated by receptors of environmental estrogens. Considering the functions of these proteins in colorectal cancer,
the directions in which their expression modulated by BPA
were confirmed by the following results that BPA can promote the metastasis of colorectal cancer cells.
presented here revealed, for the first time, that BPA can
promote the metastasis of colorectal cancer cells.
EMT is considered to be the first step of invasion of adjacent tissues by tumors and metastasis. It occurs at the invasive front of colon carcinoma concomitant with a selective
loss of basement membrane (Sheehan et al. 2008). Results of
the present study revealed, for the first time, that oral equivalent or greater concentrations of BPA can induce EMT in
SW480 cells, which resulted in down-regulation of epithelial
characteristics, such as E-cad, ZO-1, up-regulation of mesenchymal characteristics FN, N-cad and Vim, and increased
expression of transcription factors Snail and Twist (Fig. 4).
Up-regulation of the mesenchymal biomarker Vim has also
been found at in mammary glands of rat offspring exposed
in utero to BPA at day 50 (Betancourt et al. 2010). Estradiol
(E2) has been reported to enhance the EMT of lung adenocarcinoma through ERβ and regulate the expression of E-cad
in breast cancer via ERα (Ye et al. 2010). These studies suggested that as an xenoestrogen, BPA can promote the metastasis of colorectal cancer via trigger the EMT of cancer cells.
Furthermore, our study found that Snail mediated the
BPA, particularly for 10−5 M BPA, induced EMT of colorectal cancer cells. Snail, a zinc finger transcription factor
first identified in Drosophila, can bind to the E-box site
BPA triggers metastasis and EMT of colorectal cancer
via Akt/GSK‑3β/Snail
Proteomic study revealed that many proteins related to
cell structure and motility were significantly modulated by
BPA. Exposure to both oral equivalent and greater concentrations of BPA can significantly promote migration and
invasion ability of SW480 cells (Fig. 3). There is very limited published data about effects of EDCs on the metastasis of colorectal cancer. It has been reported that BPA can
induce migration of breast cancer—associated fibroblasts
(CAFs) and conditioned medium from BPA-treated CAFs
can induce migration of SKBR3 cells (Pupo et al. 2012).
In the cell migration and invasion assay, exposure to BPA
or E2 resulted in greater motility or invasiveness in neuroblastoma SK–N–SH cells (Zhu et al. 2010). Our results
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in the promoter of E-cad and trigger the EMT of many
types of cancer (Kudo-Saito et al. 2009). Snail can directly
inhibit transcription of the epithelial markers such as Krt-8
(Christiansen and Rajasekaran 2006), which is confirmed
by the results of proteomic study (Krt 50.9 and 84.4 %
of control, respectively). Furthermore, silencing of Snail
by siRNA attenuated BPA-induced EMT of SW480 cells,
which was not observed in control siRNA-transfected cells
(Fig. 4). These results demonstrated that Snail is essential
for BPA-induced EMT in CRC.
In the present study, our results found that BPA can up
regulate the expression of Snail via Akt/GSK-3β signal
pathway. Phosphorylation of Akt has been reported to be
associated with a loss of cell adhesion, decrease in cell–
matrix adhesion, loss of apico-basolateral cell polarization and induction of cell motility (Bellacosa et al. 2005).
Phosphorylation of Akt was increased and phenotypic
changes associated with EMT has been suggested in mammary glands of 50-day-old rats exposed prenatally to BPA
(Betancourt et al. 2010). The results of previous study
revealed that stabilization of Snail mediated by AKT/GSK3β mediates EMT of colorectal cancer induced by TNFα
(Wang et al. 2013). In the present study, BPA-induced
EMT of both SW480 and HCT-116 cells, down-regulation
of E-Cad, up-regulation of p-AKT, p-GSK-3β, and Snail,
was blocked by treatment with the PI3K inhibitor, suggesting that PI3K/AKT signaling pathway plays an essential
role for BPA-induced EMT of CRC cells.
The functional proteomic study also identified other functional signaling molecules and pathways that may be also
involved in the promotion effects of metastasis of SW480
cells. Annexin A1 and A2 are well-studied receptors for plasminogen, as it converts plasminogen to plasmin after binding. Accumulating evidences suggested that Annexin A1
and A2 and their receptor axis can significantly promote the
tumor metastasis (Lokman et al. 2011). Results of the present study revealed that 10−8 and 10−5 M BPA resulted in
significantly greater expression of proteins Annexin A1 and
A2 (Table S1). This is the first evidence that Annexin can be
up-regulated by BPA. Expression of the actin-bundling protein fascin was greater in cells exposed to BPA than that of
control, which can result in actin reorganization and synaptic remodeling and therefore promoting the colorectal cancer
invasion and migration (Vignjevic et al. 2007).
BPA promotes tumorigenesis and progression of colorectal
cancer
The proteomic study revealed that levels of proteins related
to cell cycle and proliferation were significantly modulated
by BPA. Our previous studies revealed that micromolar BPA
inhibited proliferation of Sertoli cells while nanomolar BPA
stimulated proliferation of cells (Ge et al. 2014). The present
study found that micromolar BPA inhibited cells proliferation while nanomolar BPA had limited effects on SW480
cells. This might be due to nanomolar and micromolar BPA
had opposite effects on proteins related to cell proliferation
of SW480 cells. For example, the small heat-shock protein
27 (hsp27) was increased by 10−8 M BPA (182 %) while
decreased by 10−5 M BPA (93.2 %). Hsp27 has been documented to inhibit both the Fas- and mitochondria-mediated
apoptosis pathways and over expressed in tumors (Mehlen
et al. 1996). It suggested that effects of oral equivalent or
greater concentrations of BPA might be different on proliferation of CRC cells, which was confirmed by cytotoxic tests.
Other proteins related to cell proliferations were also significantly altered by treatment with BPA. Ran (Ras-related
nuclear protein) is a small GTP-binding protein belonging to
the Ras super family (Di Fiore et al. 2004). Over-expression
of Ran caused by 10−5 M BPA might result in disruption of
transport of crucial cell-cycle regulators or effectors and thus
lesser proliferation of cells (Clarke and Zhang 2008). 14–3–3
is a family of highly conserved proteins that are crucial in
regulating multiple cellular processes, including maintenance
of cell-cycle checkpoints and repair of DNA, the onset of cell
differentiation and senescence, and the coordination of cell
adhesion and motility (Betancourt et al. 2011). Prenatal BPA
exposure increased expression of 14–3–3 eta and increased
susceptibility for mammary cancer (Betancourt et al. 2011).
Up-regulation of 14–3–3 epsilon has also been found in rat
offspring on postnatal day 50 following prenatal BPA exposure (Betancourt et al. 2010). In the present study, both oral
equivalent and greater concentrations of BPA significantly
elevated expression of 14–3–3, suggesting that BPA can promote the tumorigenesis of colorectal cancer. Collectively, the
effects of BPA on proliferation of CRC cells and progression
of colorectal cancer should be further investigated.
BPA alters energy metabolism and increases antioxidant
capacity
Many proteins altered by exposure to BPA belong to the
“Metabolism” group (Table S1). Regulation of cellular energetic pathways by BPA was evidenced by greater
expression of enzymes of the glycolysis (glucose-6-phosphate dehydrogenase and glutamate dehydrogenase 1)
and TCA cycle (aldehyde dehydrogenase family 1 member A3, malate dehydrogenase, and fumarate hydratase).
On the other hand, proteins within the electron transport
chain and complex I were increased in expression (complex I, ATP synthase subunit alpha, Human Mitochondrial Acetoacetyl-Coa Thiolase cytochrome b-c1 complex
subunit). The result is consistent with increased glycolytic
metabolic activity and suggests that energy consumption
of colorectal cancer cells was covered from sources of
glycolysis (Deberardinis et al. 2008). Metabolic activity
13
regulates the production of free radicals. Enhanced oxidative phosphorylation, and thus increased ATP synthesis via
the respiratory chain, requires more oxygen and generates
more free radicals, which can damage cells (Vander Heiden
et al. 2009). In the present study, BPA, particularly 10−8
M BPA, also increased the protein levels of peroxiredoxin
(PRDX-3), which naturalized the majority of the mitochondrial peroxides (Cox et al. 2010). It indicated that BPA is
not only an oxidant hormone, but also induces synthesis
of mitochondrial antioxidant proteins (Barros and Gustafsson 2011). Confirming our protein expression data, BPA
did induce generation of ROS in liver of rat and resulted in
greater expression of peroxiredoxin in mammary glands of
offspring in the rat (Betancourt et al. 2010).
BPA regulates protein folding, metabolism,
and modification
Heat-shock proteins (HSPs) play an important role in
physiological processes, such as division, apoptosis, and
differentiation of cells by interacting with multiple key
components of signaling pathways that regulate growth
and development. HSP70 has been previously identified as
a potential target of BPA (Papaconstantinou et al. 2001).
The proteomic study reported upon here also revealed that
BPA significantly down-regulated HSP70 in SW480 cells.
Results of previous studies indicated that HSP70 is a good
marker for proliferation of colorectal cancer cells (Wang
2011), it was consistent with the fact that 10−5 M BPA
inhibited proliferation of SW480 cells and down-regulated
expression of HSP70. Greater expression of HSP27 protein has previously been reported in colon cancer cells and
associated with poor prognosis (Thanner et al. 2005). The
fact that exposure to the oral equivalent concentration of
BPA resulted in significantly greater expression of HSP27
protein might indicate that low dose of BPA can also promote tumorigenesis and development of colorectal cancer.
Acknowledgments This research was supported by the National
Natural Science Foundation of China (Grant Nos. 31101071 and
81302317), the Fundamental Research Funds for the Central Universities (Sun Yat-sen University) (No. 12ykpy09), the Opening Project
of Guangdong Provincial Key Laboratory of New Drug Design and
Evaluation (No. 2011A060901014-007), the National Basic Research
Program of China (973 Program, No. 2011CB9358003), the Science
and Technology Planning Project of Guangdong Province, China (No.
2012B031500005), and the Seed Collaborative Research Fund from
the State Key Laboratory in Marine Pollution (SCRF0003).
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