Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability

Abstract

Background
Trans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line. Methods
The 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins. ResultsDPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin. Conclusion
Various concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.

Full text

Fong et al. Chin Med (2016) 11:45
DOI 10.1186/s13020-016-0116-7
RESEARCH
Inhibitory eect oftrans-ferulic acid
onproliferation andmigration ofhuman lung
cancer cells accompanied withincreased
endogenous reactive oxygen species
andβ-catenin instability
Yao Fong1†, Chia‑Chun Tang2†, Huei‑Ting Hu3, Hsin‑Yu Fang4, Bing‑Hung Chen3,10, Chang‑Yi Wu3,5,
Shyng‑Shiou Yuan6, Hui‑Min David Wang7, Yen‑Chun Chen3, Yen‑Ni Teng8 and Chien‑Chih Chiu3,5,6,9*
Abstract
Background: Trans‑ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans‑
FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the
cellular physiological effects of trans‑FA on the H1299 human lung cancer cell line.
Methods: The 2,2‑diphenyl‑1‑picrylhydrazyl assay was used to determine free radical scavenging capability. Assess‑
ment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ‑dichlorofluorescin diacetate
and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage‑independent growth assays
were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow
cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin
zymography was used to detect matrix metalloproteinase (MMP‑2 and MMP‑9) activity. Western blotting was used to
detect expression levels of various signaling pathway proteins.
Results: DPPH assay results indicated that trans‑FA exerted potent antioxidant effects. However, trans‑FA increased
intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans‑FA treatment inhib‑
ited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM).
Furthermore, trans‑FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and
attenuated MMP‑2 and MMP‑9 activity. Trans‑FA caused the phosphorylation of β‑catenin, resulting in proteasomal
degradation of β‑catenin. Conversely, trans‑FA treatment increased the expression of pro‑apoptotic factor Bax and
decreased the expression of pro‑survival factor survivin.
Conclusion: Various concentrations (0.06–0.6 mM) of trans‑FA exert both anti‑proliferation and anti‑migration effects
in the human lung cancer cell line H1299.
© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain D edication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Background
In 2013, the mortality rates for male and female lung
cancer patients in the United States were 28 and 26%,
respectively [1]. At present, chemotherapy is the primary
treatment for lung cancer [2–4], with both carboplatin
and cisplatin commonly used as chemotherapy drugs [5,
6]. However, the combination of cisplatin or vinblastine
with irradiation increases the level of unexpected toxicity
in the body [4]. Pemetrexed (Alimta®), a next-generation
antifolate drug, is used for treating malignant pleural
mesothelioma and non-small cell lung cancer (NSCLC)
Open Access
Chinese Medicine
*Correspondence: cchiu@kmu.edu.tw
†Yao Fong and Chia‑Chun Tang contributed equally to this work
3 Department of Biotechnology, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Full list of author information is available at the end of the article

Page 2 of 13
Fong et al. Chin Med (2016) 11:45
but can induce scleroderma-like induration of the lower
extremities [7]. It is therefore necessary to develop alter-
native treatment strategies for selective inhibition of lung
cancer cells growth.
e carcinogenic process may be driven by mutations,
leading to alterations in phenotypes, genetics, and epige-
netics. e induction of oxidative stress in various cancer
cells such as human pancreatic and colon adenocarci-
noma cancer cell lines [8] was shown to inhibit expression
of β-catenin and the matrix metalloproteinases MMP-2
and MMP-9 in colitis-associated colon carcinoma [9],
induce Bax expression in urothelial cell carcinoma [10],
induce apoptosis by blocking the AMPK-mTOR-survivin
pathway [11], and inhibit the anchorage-independent
growth (AIG) of transformed cells [12]. Chemopreven-
tive treatment is moderate or non-cytotoxic to normal
cells, but significantly inhibits cancer cell growth or
metastasis. Accumulating evidence shows that anti-oxi-
dative compounds isolated from plants exert potentially
chemopreventive effects. Among these compounds are
carotenoids, curcumin, and hesperidin [13, 14]. Recently,
anticancer compounds derived from plants, including
goniothalamin and feruloyl--arabinose, were shown to
inhibit the growth and metastasis of human lung cancer
cells [15, 16]. Additionally, moscatilin, isolated from the
orchid Dendrobrium loddigesii, inhibited metastasis of
both human breast and lung cancer cells [17, 18].
Ferulic acid (FA) is an aromatic compound, abundant
in plant cell walls [19, 20]. Both isomers of FA, cis-FA
and trans-FA, show a potent ability to remove reactive
oxygen species (ROS) and inhibit lipid peroxidation [20,
21]. Unlike cis-FA, trans-FA is abundant in plant cells
and easily isolated from various plants, thus significantly
reducing the cost of its preparation [22].
Trans-FA ameliorated ionizing radiation-induced
inflammation and glycerol-induced nephrotoxicity [23,
24] and modulated fluoride-induced oxidative hepato-
toxicity in male Wistar rats [25]. Water-soluble trans-FA
sugar esters protected normal rat erythrocytes against
peroxyl radical 2,2ʹ-azobis-2-amidinopropane dihydro-
chloride (AAPH)-induced oxidative damage [26] and
exhibited protective capacity against oxidative dam-
age caused by diabetes [27, 28]. Furthermore, trans-FA
exhibited antiproliferative effects in colon cancer cells
[29, 30], increased the radiosensitivity of cervical cancer
cells [31], and exerted protective effects against chemical-
induced DNA strand breaks [32, 33]. However, the effects
of trans-FA in lung cancer have not been reported to
date, and its biological mechanism remains unknown.
e study investigated the cellular and physiological
effects of trans-FA in human lung cancer cells.
Methods
Chemicals
Trans-FA (4-hydroxy-3-methoxycinnamic acid) was
purchased from Sigma-Aldrich Chemicals (#128708,
St. Louis, MO, USA). Trans-FA was freshly dissolved in
0.01% dimethyl sulfoxide (DMSO) and aliquoted before
use.
Cell culture
e human non-small cell lung cancer (NSCLC) cell
line H1299 and lung fibroblast cells HEL-299 were
obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA). Cells were maintained in
DMEM:F-12 medium (1:1 ratio) supplemented with 8%
fetal bovine serum, 2mM glutamine, 100units/mL peni-
cillin, and 100µg/mL streptomycin (Gibco BRL, Gaith-
ersburg, MD, USA) at 37°C in a humidified atmosphere
of 5% CO2 [4].
DPPH radical‑scavenging activity assay
e anti-oxidant activities of trans-FA were measured
based on the scavenging activity of 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) (#D9132, Sigma-Aldrich) free radical
[34, 35]. Briefly, vitamin C standards and various trans-
FA concentrations were freshly prepared and diluted in
methanol. Methanol (as a blank control; 10µM) or trans-
FA solution was added to 90µL DPPH solution to yield a
final trans-FA concentration of 0.15mg/mL in a 96-well
microplate. e mixture was incubated at 25°C and pro-
tected from light. After incubation, solution absorbance
was measured at 492nm using a Multiskan Ascent 354
microplate reader (ermo Fisher Scientific, Rockford,
IL, USA). DPPH radical scavenging activity was calcu-
lated as follows:
where A0 and A1 are the absorbances of the control and
trans-FA solutions, respectively. Each experiment was
repeated three times and found to be reproducible within
experimental error margins.
Cell viability andproliferation assay
Briefly, 5×104 cells were seeded into wells of a 12-well
plate and treated with phosphate-buffered saline (PBS)
(Sigma-Aldrich) as control or concentrations of trans-
FA (0.03–0.6mM) for 24 or 48h. After incubation, cell
viability and proliferation were analyzed by trypan blue
assay and an automated cell counter (Countess™) accord-
ing to the manufacturer’s instructions (Invitrogen, Carls-
bad, CA, USA).
DPPH radical scavenging activity (%)
=
(1
−A
0/
A
1)
×
100

Page 3 of 13
Fong et al. Chin Med (2016) 11:45
Colony formation assay
Fifty cells were seeded into wells of a 6-well plate and,
after 24h of incubation, were treated with different con-
centrations of trans-FA (0.03–0.6mM). After incubation
for 11days, cell colonies were glutaraldehyde-fixed and
stained with crystal violet (0.1% w/v) for 10min. Colony
diameter was determined using Image-Pro v3.0 software
(Media Cybernetics, Silver Spring, MD, USA).
Anchorage‑independent growth (AIG) assay
e assay procedure was performed as described in our
previous work, with minor modifications [36]. Briefly,
1×103 cells were mixed with 0.75% low melting agarose
(MDBio Inc, Taipei, Taiwan). e mixtures were placed on
a solidified layer of 1.5% agarose with medium in a 12-well
plate. After incubation for 13days, cells in the upper layer
were fixed with 1% glutaraldehyde and stained with 0.1%
w/v Giemsa (Merck, Darmstadt, Germany). Colony diam-
eter was determined using Image-Pro v3.0 software.
Cell cycle analysis
Cell cycle distribution was assessed using propidium
iodide (PI; Sigma-Aldrich) staining described previ-
ously [37]. Briefly, 1× 105 cells were treated with PBS
(as vehicle control) or various trans-FA concentrations
(0.03–0.6mM) for 48h. Next, cells were detachedusing
0.05% trypsin (Biological Industries, Kibbutz Beit Hae-
mek, Israel) for 5min, harvested, and washed with PBS.
Cells were then fixed with 75% ethanol overnight. After
centrifugation (Microfuge® 16, Beckman Coulter Life
Sciences, Taipei, Taiwan) at 664×g for 15min at 4°C, the
resulting supernatants were decanted. Cell pellets were
stained with 10µg/mL PI and 10µg/mL RNase A (Sigma-
Aldrich) in PBS buffer for 30min at 37°C in the dark.
e samples were assayed using a FACScan flow cytom-
eter (Becton–Dickinson, Mansfield, MA, USA) and the
results were analyzed using FlowJo v7.5.5 software (Tree
Star Inc., San Carlos, CA).
Assessment ofapoptosis
Apoptotic cell death was assessed by annexin V and PI dou-
ble staining (Pharmingen, San Diego, CA, USA) accord-
ing to our previous paper [15]. Briefly, 1×106 cells were
seeded into a 100-mm petri dish and treated with vehicle
or trans-FA at doses of 0–0.6mM for 48h. Next, cells were
inoculated with 10µg/mL of annexin V-fluorescein isothio-
cyanate (FITC) and 20µg/mL of PI and analyzed using a
FACScan flow cytometer FlowJo v7.5.5 software.
Detection ofendogenous ROS
Changes in endogenous ROS levels were assessed using
the fluorescent indicators 2′,7′-dichlorofluorescin
diacetate (DCFDA, Sigma-Aldrich) for hydrogen per-
oxide (H2O2) and dihydroethidium (DHE) (Invitrogen)
for superoxide anion (O2−). A total of 1× 105 H1299
cells were seeded into wells of a 6-well culture plate
and treated with various concentrations (from 0.03 to
0.15mM) of trans-FA for 24h. Next, cells were harvested
and stained with 100 nM DCFDA or 1 µM DHE for
30min at 37°C in PBS and then washed twice with PBS.
Fluorescence was measured by flow cytometry.
Western blot assay
Western blot assays were performed as described previ-
ously [38]. Briefly, cells were harvested and lysed, lysates
were centrifuged, and protein concentrations of cell pel-
lets were determined. Next, 40 mg quantities of pro-
tein lysate were resolved by 10% SDS–polyacrylamide
gel electrophoresis and electro-transferred to polyvi-
nylidene difluoride membranes. e membranes were
blocked with 5% nonfat milk and incubated with the fol-
lowing primary antibodies: r41/Ser45 phosphorylated
β-catenin (#2377-S, Epitomics, CA, USA), β-catenin (#sc-
7963, Santa Cruz Biotech., CA, USA), Bax (#ab32503,
Abcam Inc., Cambridge, MA, USA), survivin (#614701,
Biolegend, CA, USA), and β-actin (#AM1021b, Abgent,
San Diego, CA, USA), followed by appropriate second-
ary antibodies (anti-Mouse, #074-1806; anti-Rabbit,
#074-1506, KPL, Gaithersburg, MD, USA). e Western-
Bright™ ECL kit (Advansta, CA, USA) was used for signal
detection.
Wound‑healing assay
Quantities of 3×105 H1299 cells were seeded in wells of
a 12-well plate, treated with PBS (as vehicle control) or
trans-FA (0.03–0.6mM), and grown to 100% confluence.
Culture monolayers were scratched using a pipette tip
to create a clean 1-mm-wide wound area. After further
incubation for 16h, the wound gaps were photographed
and analyzed using TScratch software (CSE Lab, Zurich,
Switzerland) [39].
Transwell invasion assay
Invasion assays were performed as described in our
previous work, with minor modifications [40], using
8µm-pore Transwell® chambers (Greiner Bio-One, Fric-
kenhausen, Germany). Control and various concentra-
tions (from 0.03 to 0.6mM) of trans-FA treated cells were
cultured in triplicate at 5× 104 cells/well in the upper
inserts of 24-well Transwell® culture plates. Next, cells
were fixed for 5min and stained with 0.1% w/v Giemsa.
e cells which had invaded the lower inserts were
counted by arbitrarily selecting five fields from each well.
e experiments were repeated three times.

Page 4 of 13
Fong et al. Chin Med (2016) 11:45
Gelatin zymography
MMP-2 and -9 activity was assessed by gelatin zymog-
raphy as previously described [41], with minor modi-
fication. Briefly, 3×105 H1299 cells were seeded into
wells of 12-well plates and cultured with various con-
centrations of trans-FA for 24h, after which aliquots
of culture medium were harvested for gelatin zymog-
raphy analysis. Samples were prepared in standard
SDS-PAGE loading buffer containing 0.01% SDS with-
out β-mercaptoethanol and were not boiled before
use. Next, the samples were subjected to electropho-
resis (150V for 3h) on 10% SDS–polyacrylamide gels
containing 1% gelatin. After electrophoresis, the gels
were thoroughly washed with distilled water contain-
ing 2.5% Triton X-100 on a gyratory shaker for 30min
at room temperature. Gels were incubated in 100mL
reaction buffer (40 mM Tris–HCl, pH 8.0; 10 mM
CaCl2, and 0.02% NaN3) at 37°C overnight, followed
by staining with Coomassie brilliant blue R-250 (Bio
basic Inc., Markham, Ontario, Canada) and de-stain-
ing with methanol-acetic acid–water (50/75/875,
v/v/v). The gelatinase activities of MMP-2 and -9 were
determined by analyzing signal intensity using Gel Pro
v.4.0 software (Media Cybernetics, Silver Spring, MD,
USA).
Statistical analysis
All data are mean±SD from at least three experiments,
with three replicates per experiment. e significance of
the differences was analyzed using one-way analysis of
variance (ANOVA) and SigmaPlot v12.0 software (Systat
Software Inc., San Jose, CA, USA). P values less than 0.05
were considered statistically significant.
Results
Radical scavenging activity oftrans‑FA
e DPPH assay was used to assess the radical scaveng-
ing activity of trans-FA. Antioxidant activity was calcu-
lated by evaluating the capacity of trans-FA to scavenge
DPPH. As shown in Fig.1, trans-FA treatment produced
significant radical-scavenging activity compared with the
DMSO control. Vitamin C was serially diluted in metha-
nol (500–7.8µM) and used in triplicate as a positive con-
trol (Additional file1).
The short‑term eect oftrans‑FA onproliferation ofNSCLC
cells
H1299 cells were treated with PBS (as vehicle control)
or different concentrations of trans-FA for 24 or 48h
before gross morphological changes were examined by
light microscopy to determine the effect of trans-FA on
cell growth (Fig.2a). Cells exhibited no significant change
in morphology compared with the vehicle control. Next,
measurement of cell survival was performed by trypan
blue dye exclusion assay. Low doses (>0.15mM) of trans-
FA exerted no significant cytotoxic effect, but moderate
cytotoxicity was observed with 0.3 and 0.6mM treatment
for 48h (Fig.2b).
The long‑term eect oftrans‑FA oncolony formation
andAIG assay inNSCLC cells
As shown in Fig. 3a, trans-FA inhibited colony forma-
tion in H1299 cells after 11days of treatment. e calcu-
lated colony diameters for trans-FA concentrations of 0,
0.06, 0.15, 0.3 and 0.6mM were 100±0.00, 77.19±2.70,
64.19± 2.75, 56.75 ±4.23 and 4.59± 1.25 % (n= 3),
respectively. Figure3c shows that long-term treatment
with trans-FA inhibited the AIG capacity of H1299 cells.
Furthermore, the results of the colony formation showed
the selectively inhibitory effect of trans-FA on cellular
proliferation between lung cancer cells H1299 and lung
fibroblast cells HEL-299 (Additional file2). e calculated
colony diameters for trans-FA at 0, 0.03, 0.06, 0.15, 0.3
and 0.6mM were 100±3.10, 95.34±4.18, 85.64±1.08,
75.51±2.41, 70.41±1.71 and 61.36±2.70% (n=3),
respectively.
Tra ns ‑FA caused moderate G0/G1 accumulation
e effects of 48h trans-FA treatment on cell cycle pro-
gression in H1299 cells were examined. Trans-FA treat-
ments caused the arrest of the cell cycle at G0/G1 and a
decrease in the percentage of the G2/M phase (Fig.4a, b).
Tra ns ‑FA‑induced apoptosis inlung cancer cells
We investigated whether inhibition of proliferation by
trans-FA was achieved by apoptosis in H1299 cells.
Only the highest concentration (0.6 mM) of trans-FA
increased the proportion of Annexin V+/PI+ cells (from
1.81 to 5.4%) (Fig.5). Other concentrations of trans-FA
used in the study did not induce a significant increase in
apoptotic populations.
Tra ns ‑FA induced changes inintracellular ROS
e endogenous level of ROS can regulate a variety of
cellular physiological processes, including survival,
proliferation, angiogenesis, and signaling pathways
[42]. Flow cytometry-based detection with DCFDA and
DHE staining was used to detect endogenous H2O2 and
O2−, respectively. As shown in Fig.6, H2O2 concentra-
tions of 100±20, 317±28, 364±23 and 375±20%
(n=3) were observed in H1299 cells treated with dif-
ferent concentrations of trans-FA (0–0.15 mM) for
24 h. Furthermore, endogenous O2− concentrations
of 100±9, 100±2, 241±19 and 392±7% (n=3)
were observed with the same trans-FA concentrations
(Fig.6b).

Page 5 of 13
Fong et al. Chin Med (2016) 11:45
Regulation ofsurvival proteins bytrans‑FA
Western blotting was used to examine whether trans-FA
treatment affected the expression of β-catenin in H1299
cells. As shown in Fig. 7, trans-FA treatment increased
β-catenin phosphorylation at r41/Ser45 but did not affect
β-catenin protein levels. e anti-cancer effects of trans-
FA might act by selective inhibition of β-catenin, a tran-
scription factor associated with the growth and migration
of H1299 cells. Anti-survival Bax protein was increased
following trans-FA treatment in a dose-responsive man-
ner, although the protein level of survivin was decreased.
Tra ns ‑FA attenuated the motility oflung cancer cells
Wound-healing assays were performed to investigate
whether trans-FA affected migration of NSCLC cells.
trans-FA treatment moderately attenuated the migra-
tion of H1299 lung cancer cells (Fig.8). e area of the
denuded zone was used as an index of the migratory
ability of H1299 cells. e areas measured for trans-FA
concentrations of 0, 0.03, 0.06, 0.15, 0.3 and 0.6 mM
were 100 % ± 5.78, 96.86 % ± 2.69, 98.99 % ± 4.39,
93.95 % ± 6.77, 85.78 % ± 5.76 and 76.87 % ± 1.76
(n=3), respectively.
Fig. 1 DPPH radical‑scavenging capacity of trans‑FA. a Trans‑FA was tested in an antioxidant assay by measuring the DPPH radical‑scavenging activ‑
ity. The indicated concentrations of trans‑FA were incubated with DPPH respectively as described in the “Methods” section. b Quantificative analysis.
The radical‑scavenging capacity of trans‑FA at indicated concentrations was quantified as the percentage decrease in absorbance at 492 nm against
the DMSO control. *P < 0.05 and **P < 0.001, respectively
Fig. 2 Comparison of cell morphology and proliferation between of control and trans‑FA‑treated of NSCLC cells. a H1299 cells were treated with
PBS as vehicle control or the indicated doses of trans‑FA for 24 and 48 h, respectively. And then, morphology was examined and photographed with
a light microscope. (phase contrast, X200). b 5 × 104 H1299 cells were seeded onto a 12‑well plate, and cells were treated with indicated doses of
trans‑FA for 24 and 48 h, respectively. Cell viability was determined using the trypan blue exclusion assay described in “Methods” section. *P < 0.001
against vehicle control

Page 6 of 13
Fong et al. Chin Med (2016) 11:45
Tra ns ‑FA exerted an anti‑invasion eect
A Boyden chamber assay was used to evaluate inva-
sion ability. After treating H1299 cells with various con-
centrations (from 0.03 to 0.6mM) of trans-FA for 16h,
the percentages of invasive cells were 100 % ± 7.49,
95.79 % ± 3.42, 95.61 % ± 5.86, 88.39 % ± 7.01,
82.57%±5.87 and 68.57%±4.48 (n=3) (Fig.9).
Tra ns ‑FA reduced the activity ofMMP‑2 andMMP‑9
MMP-2 and MMP-9 are gelatinases which degrade
extracellular matrix and thus regulate the ability of cells
to migrate. Overexpression of MMP-2 and MMP-9 pro-
motes cancer progression and is highly correlated with
poor prognosis of cancer patients [43]. erefore, tar-
geting of MMP-2 and -9 represents a promising strat-
egy for cancer treatment [44]. e activity of MMP-2
and MMP-9 was determined using a gelatin zymogra-
phy assay (Fig.10a). Trans-FA treatment (0, 0.03, 0.06,
0.15, 0.3 and 0.6 mM) significantly reduced the activ-
ity of MMP-2 [100± 2.95, 90.59±1.96, 80.43 ± 4.46,
71.99± 2.9, 66.04±1.59 and 55.87 ±0.26% (n = 3)]
(Fig. 10b, c). e activity of MMP-9 [100 ± 4.44,
105.13±5.29, 94.04±0.99, 112.08±5.24, 78.04±1.86
and 53.32±2.38% (n=3)] was also reduced.
Discussion
e radical scavenger assay findings indicated the potent
anti-oxidant activity of trans-FA (Fig.1). Treatment with
trans-FA for 24 or 48h did not affect cellular morphol-
ogy and proliferation of lung cancer H1299 cells (Fig.2).
However, long-term treatment with trans-FA attenuated
colony formation and AIG, characteristics of advanced
cancer, in H1299 cells (Fig.3). Trans-FA induced moder-
ate cell proliferation at the lowest concentration tested
(0.03mM). ese results were consistent with previous
reports that trans-FA promoted proliferation of MCF7
and BT20 breast cancer cells and neural progenitor cells
[45, 46]. e colony formation assay also showed the dis-
crepant inhibitory effect of trans-FA on cellular prolifera-
tion of lung cancer cells H1299 and lung fibroblast cells
Fig. 3 The long‑term effect of trans‑FA on colony formation and anchorage‑independent growth assay of NSCLC. a The assessment of colony
formation, a 2D indicator of cell proliferation. Adherent H1299 cells were treated with indicated concentrations for 24 h. 5 × 10 cells treated with
trans‑FA were allowed to form colonies for 11 days. Afterward, colonies were fixed and stained using crystal violet. c Representative results showed
the formation of tumor spheres, an indicator of 3D for anchorage‑independent growth. H1299 cells were treated with indicated concentrations
of trans‑FA for 13 days using the soft agar assay. b, d Quantitative analysis of results from a and c showed the number and size of tumorspheres.
*P < 0.05 and **P < 0.001, respectively

Page 7 of 13
Fong et al. Chin Med (2016) 11:45
HEL-299 (Additional file2), suggesting the potential of
trans-FA for selectively inhibiting lung cancer. Further-
more, trans-FA significantly inhibited AIG capability in a
dose-responsive manner.
Trans-FA caused an accumulation of the G0/G1 popu-
lation and induced a moderate increase in the apop-
totic population at the highest concentration (0.6mM)
(Fig.4). G0/G1 cell cycle arrest is usually associated with
the upregulation of cell cycle regulatory protein p15INK4b
and p21WAF1/Cip1 [47]. A recent study showed that perillyl
alcohol, a natural compound purified from citrus fruits
and herbs, causes G1 arrest and inhibits proliferation of
human immortalized keratinocyte HaCaT cells through
inducing p15INK4b and p21WAF1/Cip1 [47]. Annexin V
staining confirmed that the anti-cancer effects of Trans-
FA were not mediated through apoptosis (Fig.5).
Excess endogenous ROS may inhibit cellular growth or
cause cell death [48–51]. e anti-cancer effects of trans-
FA might correlate with increased levels of ROS in H1299
cells (Fig.6). ROS content is higher in cancer cells than in
normal cells, and ROS are reported to be involved in can-
cer cell migration [42]. In this study, trans-FA treatment
caused the accumulation of both H2O2 and O2−. Trans-
FA (0.03mM) induced an increase in H2O2, but not O2−.
Changes in endogenous ROS levels were assessed using
the fluorescent indicators DCFDA for H2O2 and DHE for
O2− [52]. Superoxide dismutase (SOD) converts O2− into
H2O2, and is overexpressed in lung cancer compared with
Fig. 4 Effect of trans‑FA on cell‑cycle progression of H1299 cells. H1299 cells were treated with the indicated doses, 0.03, 0.06, 0.15, 0.3 and 0.6 mM
of trans‑FA for 48 h respectively. a An accumulation of G0/G1 population in trans‑FA‑treated H1299 cells and vehicle controls at 48 h. b The quantifi‑
cation analysis. Data are presented as mean ± SD (n = 3). Different letter notations indicate the statistical significance between control and trans‑FA
treatment groups (a no significance; a vs b and a vs c, statistically significant with P < 0.05 and 0.001, respectively)

Page 8 of 13
Fong et al. Chin Med (2016) 11:45
Fig. 5 Effect of trans‑FA on apoptosis of H1299 cells. H1299 cells were treated with indicated doses, 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans‑FA for
48 h respectively. Annexin V/PI double staining was performed to detect the apoptosis
Fig. 6 Trans‑FA up‑modulates the endogenous level of ROS in H1299 cells. 1 × 105 H1299 cells were seeded onto a 6‑well plate and treated with
indicated doses of trans‑FA (from 0.03 to 0.15 mM) 24 h respectively. Afterward, the induction of endogenous ROS was determined by a DCFDA or
b DHE staining combined with a flow cytometry analysis. **P < 0.001 against vehicle control

Page 9 of 13
Fong et al. Chin Med (2016) 11:45
normal and non-malignant lung tissues [53]. erefore,
a moderate increase in O2− might be rapidly converted
into H2O2 in lung cancer cells. However, the signifi-
cant increase in endogenous O2− induced by trans-FA
(>0.03mM) may cause saturation of SOD capacity, pre-
venting further conversion of O2− to H2O2. Accordingly,
increased levels of H2O2 might be the product of O2−
conversion by SOD in H1299 cells following low dose
(0.03mM) trans-FA treatment.
β-catenin is a transcription factor involved in cell
growth and cell migration pathways. Wnt/β-catenin
signaling is thus essential for the maintenance of neu-
ronal progenitor proliferation [54]. However, phos-
phorylated β-catenin is inactivated and undergoes
proteasomal degradation, causing the inhibition of cell
growth [55].
With respect to tumorigenesis, constitutive activation
or overexpression of β-catenin is frequently observed in
cancers, including rectal cancer [56], colon cancer [57],
breast cancer [58], prostate cancer [59], glioma [60],
and lung cancer [61]. Furthermore, overexpression of
Fig. 7 Effect of trans‑FA on survival protein of H1299 cells. H1299
cells were treated with the indicated doses, 0.03, 0.06, 0.15, 0.3 and
0.6 mM of trans‑FA for 48 h. Western blot assay was performed
Fig. 8 Effect of trans‑FA on the migration of H1299 cells. Trans‑FA inhibits cell migration of NSCLC tumor H1299 cells at the highest dose (0.6 mM).
a 3 × 105 cells were seeded in a 12‑well plate and cells were scraped to create a clean 1‑mm area within the confluent culture. Cells were treated
with the indicated doses of 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans‑FA for 16 h. Afterward, the wound gaps were photographed using an inverted
phase‑contrast microscopy. b The quantification analysis. *P < 0.05 for trans‑FA treatments against vehicle control
Fig. 9 Effect of trans‑FA on the invasion of H1299 cells. a Cells were treated with the indicated doses of 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans‑FA
for 16 h and stained with 0.1 % w/v Giemsa stain respectively. b The results of the quantificative analysis. *P < 0.05 and **P < 0.001 for trans‑FA treat‑
ments against vehicle control, respectively

Page 10 of 13
Fong et al. Chin Med (2016) 11:45
β-catenin enhances the expression of cyclin D1, a critical
factor for G1/S transition during cell cycle progression in
colon carcinoma cells [62]. S-adenosylmethionine and
its metabolite, methylthioadenosine, inhibited β-catenin
signaling by multiple mechanisms in colon cancer, and
thus might have the potential to prevent tumorigenesis
[63]. Furthermore, Wnt/β-catenin signaling was shown
to be a potent activator of ROS generation, resulting in
DNA damage and acceleration of cellular senescence
[64]. Furthermore, Wnt/β-catenin signaling potently
activated ROS generation in mesenchymal stem cells
[64–66].
To clarify the underlying mechanism of trans-FA-
induced anti-lung cancer activities, we examined
whether trans-FA could affect the expression of cell pro-
liferation-related transcription factor β-catenin using
western blotting (Fig. 7). Our results demonstrated
that trans-FA treatment promoted the phosphorylation
of β-catenin at residues r41 and Ser45 [55] and led to
the proteasomal degradation of cytoplasmic β-catenin,
causing the downregulation of β-catenin protein lev-
els. e Wnt pathway regulated MMP-2/-9 expression
by directly targeting the MMP promoter through T-cell
factor (TCF), a β-catenin interacting partner, therefore
promoting cellular migration [67]. In effector T cells,
endothelial cell-derived Wnt induced the expression of
MMP-2/-9 through activating the Frizzled receptors to
regulate the transmigration of T cells. In contrast, Wnt
signaling blockade reduced the migration of effector T
cells invitro [67].
In addition to β-catenin, we also examined the role
of pro-survival protein Bax, a key anti-survival factor,
can promote apoptosis by binding to and antagoniz-
ing pro-survival Bcl-2 proteins such as Bcl-2 or Bcl-xL
[68]. Conversely, survivin is a member of the inhibitor
of apoptosis (IAP) family and acts as an inhibitor of
Fig. 10 Effect of trans‑FA on activities of MMP‑2 and MMP‑9. H1299 cells were treated with indicated concentrations of trans‑FA for 24 h respec‑
tively. a The activities of MMP‑2 and MMP‑9 were determined by a gelatin zymography assay (n = 3). b, c The results of the quantificative analysis.
b *P < 0.05 and **P < 0.001 for trans‑FA treatments against vehicle respectively. c *P < 0.001 for trans‑FA treatments against vehicle control

Page 11 of 13
Fong et al. Chin Med (2016) 11:45
caspase activation, thereby negatively regulating apopto-
sis or programmed cell death [69]. Both the Bcl-2 family
and IAP proteins are critical regulators of cell prolifera-
tion and survival. In our study, the significant changes
in Bax and survivin expression occurred alongside the
anti-proliferation effects observed following trans-FA
treatment (Fig.7). As shown using colony formation and
AIG assays, trans-FA treatment might impair cell prolif-
eration of H1299 cells. Apart from in cells treated with
higher concentrations (0.3 and 0.6 mM) of trans-FA,
no significant increase in the population of apoptotic
cells was detected. Survivin is considered an apoptosis
inhibitor which promotes cellular proliferation, although
decreased expression of survivin may not always cause
apoptosis [69]. Ito etal. showed that both human hepa-
tocellular carcinoma (HCC) cell lines and patient tissues
expressed high levels of survivin mRNA, with detectable
levels not found in normal and non-tumor areas of liver
[70]. Survivin expression may be an indicator of cellular
proliferation but not apoptosis in HCC tissues [70]. e
degradation or expression Bax may represent a thresh-
old for inducing apoptosis [71]. ese results might
explain how trans-FA treatment caused an increase in
Bax protein expression but did not significantly induce
apoptosis in H1299 cells at most concentrations tested
(0.015–0.15mM). ese observations suggest that trans-
FA treatment attenuates cellular proliferation rather
than cellular survival. erefore, the results of the pro-
liferation assay imply that the anti-migratory effect of
trans-FA may also be mediated by regulating the balance
of pro-survival and anti-survival proteins in lung cancer
cells.
Extracellular matrix-degrading MMPs, especially
MMP-2 and MMP-9, are involved in the metastasis
of cancer cells [72]. Trans-FA treatment inhibited the
migration and invasion of lung cancer cells and con-
currently attenuated the activities of both MMP-2 and
MMP-9 (Figs. 8, 9, 10). ese observations suggest a
positive correlation between MMP activity and trans-FA-
induced anti-migration in lung cancer cells.
Based on these observations, we propose that the
anti-lung cancer effects of trans-FA might act through
the modulation of endogenous ROS and β-catenin sta-
bilization. Trans-FA induced the production of endog-
enous ROS and may cause β-catenin phosphorylation,
resulting in proteasomal degradation. In addition,
trans-FA regulated the balance between pro-survival
and pro-apoptosis signals and downregulated the
activities of metastasis-associated MMP-2 and MMP-9
(Fig.11).
Conclusion
Various concentrations (0.06–0.6mM) of trans-FA exert
both anti-proliferation and anti-migration effects in the
human lung cancer cell line H1299.
Abbreviations
AAPH: 2,2′‑azobis‑2‑amidinopropane dihydrochloride; AIG: anchorage‑inde‑
pendent growth; AMPK: AMP activated protein kinase; Bax: Bcl‑2‑associated X
protein; Bcl‑2: B‑cell lymphoma 2; Bcl‑xL: B‑cell lymphoma‑extra large; DCFDA:
2′,7′‑dichlorofluorescin diacetate; DHE: dihydroethidium; DMSO: dimethyl
sulphoxide; DPPH: 2,2‑diphenyl‑1‑picrylhydrazyl; ECL: enhanced chemilumi‑
nescence; FA: ferulic acid; NSCLC: non‑small cell lung cancer; IAP: inhibitor
Additional les
Additional le1. DPPH radical‑scavenging capacity of vitamin C as
a positive control. (A) Vitamin C as a positive control in DPPH assay. (B)
Quantificative analysis of (A). The radical‑scavenging capacity of Vitamin C
at indicated concentrations was quantified as the percentage decrease in
absorbance at 492 nm against the blank control. *P < 0.05 and **P < 0.001
for trans-FA treatments against vehicle respectively.
Additional le2. Discrepant proliferative effect of trans‑FA on long‑term
expansion of lung cancer cells and lung fibroblast. Lung fibroblast HEL‑
299 and NSCLC tumor cells H1299 were treated with indicated concentra‑
tions of trans‑FA respectively. Afterward, cells were fixed with glutaralde‑
hyde and stained with crystal violet.
Fig. 11 Schematic diagram of hypothesized mechanism of trans‑FA
effect of lung cancer cells. Trans‑FA induced ROS leading to degrada‑
tion of phosphorylated β‑catenin. Trans‑FA induced regulation of
pro‑survival proteins survivin and anti‑survival protein Bax caused the
anti‑proliferation of lung cancer H1299 cells. Trans‑FA also reduced
the activity of both MMP‑2 and MMP‑9, causing the down‑regulation
of migration of H1299 cells

Page 12 of 13
Fong et al. Chin Med (2016) 11:45
of apoptosis; MMP: matrix metallopeptidase; mTOR: mammalian target of
rapamycin; PBS: phosphate‑buffered saline; PI: propidium iodide; RNase A:
ribonuclease A; ROS: reactive oxygen species; SD: standard deviation; SDS:
sodium dodecyl sulfate; SOD: superoxide dismutase; TCF: T cell factor.
Authors’ contributions
YF, CCT, BHC and CCC designed the study. CC T, HTH, HYF, CYW and SSY per‑
formed the experiments. CYW, SSY, HMW and YNT analyzed and organized the
data. YF, YCC and CCC wrote the manuscript. All authors read and approved
the final manuscript.
Author details
1 Department of Thoracic Surgery, Chi‑Mei Medical Center, Tainan 710, Taiwan.
2 Division of Chest, Ten Chan General Hospital, Chung‑Li 320, Taiwan, ROC.
3 Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807,
Taiwan. 4 Department of Food Nutrition, Chung‑Hwa University of Medical
Technology, Tainan 701, Taiwan. 5 Department of Biological Sciences, National
Sun Yat‑sen University, Kaohsiung 804, Taiwan. 6 Translational Research Center,
Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan. 7 Gradu‑
ate Institute of Biomedical Engineering, National Chung Hsing University,
Taichung 402, Taiwan. 8 Department of Biological Sciences and Technology,
National University of Tainan, Tainan 700, Taiwan. 9 Research Center for Envi‑
ronment Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
10 The Institute of Biomedical Sciences, National Sun Yat‑Sen University,
Kaohsiung 804, Taiwan.
Acknowledgements
The study was financially supported by Grants NSC101‑2320‑B‑037‑046‑MY3,
NSC102‑2632‑B‑037‑001‑MY3 and MOST105‑2311‑B‑037‑001 from the
Ministry of Science and Technology (MOST), Taiwan; Grants 101‑CM‑KMU‑11,
102‑CM‑KMU‑09 and 104‑CM‑KMU‑006 from the ChiMei‑KMU Joint Research
Project, Grants NSYSU‑KMU104‑P031 from the NSYSU‑KMU Joint Research Pro‑
ject, and Grant MOHW103‑TD‑B‑111‑05 from the Ministry of Health and Wel‑
fare, Taiwan; by the grants Aim for the Top Universities Grant. KMU‑TP103A17,
KMU‑TP104A03, KMU‑TP105A03 and KMU‑M104008 from Kaohsiung Medical
University, Taiwan; Grant Ten Chan General Hospital, Chung‑Li and KMU Joint
Research Project (ST102004), Taiwan.
Competing interests
The authors declare that they have no competing interests.
Received: 18 March 2015 Accepted: 19 September 2016
References
1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin.
2013;63:11–30.
2. O’Rourke N, Roque IFM, Farre Bernado N, Macbeth F. Concurrent chemo‑
radiotherapy in non‑small cell lung cancer. Cochrane Database Syst Rev.
2010;16:CD002140.
3. Pirker R, Minar W. Chemotherapy of advanced non‑small cell lung cancer.
Front Radiat Ther Oncol. 2010;42:157–63.
4. Wagner TD, Yang GY. The role of chemotherapy and radiation in the treat‑
ment of locally advanced non‑small cell lung cancer (NSCLC). Curr Drug
Targets. 2010;11:67–73.
5. Atagi S, Kawahara M, Tamura T, Noda K, Watanabe K, Yokoyama A, et al.
Standard thoracic radiotherapy with or without concurrent daily low‑
dose carboplatin in elderly patients with locally advanced non‑small
cell lung cancer: a phase III trial of the Japan Clinical Oncology Group
(JCOG9812). Jap J Clin Oncol. 2005;35:195–201.
6. Guo S, Liang Y, Zhou Q. Complement and correction for meta‑analysis
of patients with extensive‑stage small cell lung cancer managed with
irinotecan/cisplatin versus etoposide/cisplatin as first‑line chemotherapy.
J Thorac Oncol. 2011;6:406–8.
7. Corbaux C, Marie J, Meraud JP, Lacroix S, Delhoume JY, Jouary T, et al.
Pemetrexed‑induced scleroderma‑like changes in the lower legs. Ann
Dermatol Venereol. 2015;142:115–20.
8. Sun Y, Chen J, Rigas B. Chemopreventive agents induce oxidative stress
in cancer cells leading to COX‑2 overexpression and COX‑2‑independent
cell death. Carcinogenesis. 2009;30:93–100.
9. Setia S, Nehru B, Sanyal SN. The PI3K/Akt pathway in colitis associated
colon cancer and its chemoprevention with celecoxib, a Cox‑2 selective
inhibitor. Biomed Pharmacother. 2014;68:721–7.
10. Madka V, Mohammed A, Li Q, Zhang Y, Patlolla JM, Biddick L, et al. Chem‑
oprevention of urothelial cell carcinoma growth and invasion by the dual
COX‑LOX inhibitor licofelone in UPII‑SV40T transgenic mice. Cancer Prev
Res (Phila). 2014;7:708–16.
11. Wang Y, Ma W, Zheng W. Deguelin, a novel anti‑tumorigenic agent
targeting apoptosis, cell cycle arrest and anti‑angiogenesis for cancer
chemoprevention. Mol Clin Oncol. 2013;1:215–9.
12. Zhu B, Liu GT, Wu RS, Strada SJ. Chemoprevention of bicyclol against
hepatic preneoplastic lesions. Cancer Biol Ther. 2006;5:1665–73.
13. Tanaka T, Shnimizu M, Moriwaki H. Cancer chemoprevention by carot‑
enoids. Molecules. 2012;17:3202–42.
14. Tanaka T, Makita H, Ohnishi M, Hirose Y, Wang A, Mori H, et al. Chemo‑
prevention of 4‑nitroquinoline 1‑oxide‑induced oral carcinogenesis by
dietary curcumin and hesperidin: comparison with the protective effect
of β‑carotene. Cancer Res. 1994;54:4653–9.
15. Chiu CC, Liu PL, Huang KJ, Wang HM, Chang KF, Chou CK, et al. Gonio‑
thalamin inhibits growth of human lung cancer cells through DNA
damage, apoptosis, and reduced migration ability. J Agric Food Chem.
2011;59:4288–93.
16. Fang HY, Wang HM, Chang KF, Hu HT, Hwang LJ, Fu TF, et al. Feruloyl‑
l‑arabinose attenuates migration, invasion and production of reactive
oxygen species in H1299 lung cancer cells. Food Chem Toxicol.
2013;58:459–66.
17. Pai HC, Chang LH, Peng CY, Chang YL, Chen CC, Shen CC, et al. Moscatilin
inhibits migration and metastasis of human breast cancer MDA‑MB‑231
cells through inhibition of Akt and Twist signaling pathway. J Mol Med
(Berl). 2013;91:347–56.
18. Kowitdamrong A, Chanvorachote P, Sritularak B, Pongrakhananon V.
Moscatilin inhibits lung cancer cell motility and invasion via sup‑
pression of endogenous reactive oxygen species. Biomed Res Int.
2013;2013:765894.
19. Buanafina MM, Langdon T, Hauck B, Dalton S, Timms‑Taravella E, Morris
P. Targeting expression of a fungal ferulic acid esterase to the apoplast,
endoplasmic reticulum or golgi can disrupt feruloylation of the growing
cell wall and increase the biodegradability of tall fescue (Festuca arundi-
nacea). Plant Biotechnol J. 2010;8:316–31.
20. Srinivasan M, Sudheer AR, Menon VP. Ferulic acid: therapeutic
potential through its antioxidant property. J Clin Biochem Nutr.
2007;40:92–100.
21. Pan GX, Spencer L, Leary GJ. Reactivity of ferulic acid and its derivatives
toward hydrogen peroxide and peracetic acid. J Agric Food Chem.
1999;47:3325–31.
22. Hartley RD, Jones EC. Phenolic components and degradability of cell
walls of grass and legume species. Phytochemistry. 1977;16:1531–4.
23. Das U, Manna K, Sinha M, Datta S, Das DK, Chakrabor ty A, et al. Role of
ferulic acid in the amelioration of ionizing radiation induced inflamma‑
tion: a murine model. PLoS One. 2014;9:e97599.
24. Manikandan R, Beulaja M, Thiagarajan R, Pandi M, Arulvasu C, Prabhu NM,
et al. Ameliorative effect of ferulic acid against renal injuries mediated by
nuclear factor‑κB during glycerol‑induced nephrotoxicity in Wistar rats.
Ren Fail. 2014;36:154–65.
25. Panneerselvam L, Subbiah K, Arumugam A, Senapathy JG. Ferulic acid
modulates fluoride‑induced oxidative hepatotoxicity in male Wistar rats.
Biol Trace Elem Res. 2013;151:85–91.
26. Yuan X, Wang J, Yao H. Antioxidant activity of feruloylated oligosaccha‑
rides from wheat bran. Food Chem. 2005;90:759–64.
27. Roy S, Metya SK, Sannigrahi S, Rahaman N, Ahmed F. Treatment with feru‑
lic acid to rats with streptozotocin‑induced diabetes: effects on oxidative
stress, pro‑inflammatory cytokines, and apoptosis in the pancreatic beta
cell. Endocrine. 2013;44:369–79.
28. Ramar M, Manikandan B, Raman T, Priyadarsini A, Palanisamy S, Velay‑
udam M, et al. Protective effect of ferulic acid and resveratrol against
alloxan‑induced diabetes in mice. Eur J Pharmacol. 2012;690:226–35.

Page 13 of 13
Fong et al. Chin Med (2016) 11:45
29. Janicke B, Hegardt C, Krogh M, Onning G, Akesson B, Cirenajwis HM, et al.
The antiproliferative effect of dietary fiber phenolic compounds ferulic
acid and p‑coumaric acid on the cell cycle of Caco‑2 cells. Nutr Cancer.
2011;63:611–22.
30. Jayaprakasam B, Vanisree M, Zhang Y, Dewitt DL, Nair MG. Impact
of alkyl esters of caffeic and ferulic acids on tumor cell proliferation,
cyclooxygenase enzyme, and lipid peroxidation. J Agric Food Chem.
2006;54:5375–81.
31. Karthikeyan S, Kanimozhi G, Prasad NR, Mahalakshmi R. Radiosensitizing
effect of ferulic acid on human cervical carcinoma cells in vitro. Toxicol
In Vitro. 2011;25:1366–75.
32. Stagos D, Kazantzoglou G, Magiatis P, Mitaku S, Anagnostopoulos K,
Kouretas D. Effects of plant phenolics and grape extracts from Greek
varieties of Vitis vinifera on mitomycin C and topoisomerase I‑induced
nicking of DNA. Int J Mol Med. 2005;15:1013–22.
33. Tanaka T, Kojima T, Kawamori T, Wang A, Suzui M, Okamoto K, et al. Inhibi‑
tion of 4‑nitroquinoline‑1‑oxide‑induced rat tongue carcinogenesis by
the naturally occurring plant phenolics caffeic, ellagic, chlorogenic and
ferulic acids. Carcinogenesis. 1993;14:1321–5.
34. Blois MS. Antioxidant determinations by the use of a stable free radical.
Nature. 1958;181:1199–200.
35. Barreto JC, Trevisan MT, Hull WE, Erben G, de Brito ES, Pfundstein B, et al.
Characterization and quantitation of polyphenolic compounds in bark,
kernel, leaves, and peel of mango (Mangifera indica L.). J Agric Food
Chem. 2008;56:5599–610.
36. Tseng CN, Hong YR, Chang HW, Yu TJ, Hung TW, Hou MF, et al. Brefeldin
A reduces anchorage‑independent survival, cancer stem cell potential
and migration of MDA‑MB‑231 human breast cancer cells. Molecules.
2014;19:17464–77.
37. Chiu CC, Li CH, Fuh TS, Chen WL, Huang CS, Chen LJ, et al. The suppressed
proliferation and premature senescence by ganciclovir in p53‑mutated
human non‑small‑lung cancer cells acquiring herpes simplex virus‑
thymidine kinase cDNA. Cancer Detect Prev. 2005;29:286–93.
38. Chiu CC, Chen JY, Lin KL, Huang CJ, Lee JC, Chen BH, et al. p38 MAPK and
NF‑κB pathways are involved in naphtho[1,2‑b] furan‑4,5‑dione induced
anti‑proliferation and apoptosis of human hepatoma cells. Cancer Lett.
2010;295:92–9.
39. Geback T, Schulz MM, Koumoutsakos P, Detmar M. TScratch: a novel and
simple software tool for automated analysis of monolayer wound healing
assays. Biotechniques. 2009;46:265–74.
40. Tu B, Ma TT, Peng XQ, Wang Q, Yang H, Huang XL. Targeting of COX‑2
expression by recombinant adenovirus shRNA attenuates the malig‑
nant biological behavior of breast cancer cells. Asian Pac J Cancer Prev.
2014;15:8829–36.
41. Chu SC, Yang SF, Liu SJ, Kuo WH, Chang YZ, Hsieh YS. In vitro and in vivo
antimetastatic effects of Terminalia catappa L. leaves on lung cancer cells.
Food Chem Toxicol. 2007;45:1194–201.
42. Wu WS. The signaling mechanism of ROS in tumor progression. Cancer
Metastasis Rev. 2006;25:695–705.
43. Liu J, Ping W, Zu Y, Sun W. Correlations of lysyl oxidase with MMP2/MMP9
expression and its prognostic value in non‑small cell lung cancer. Int J
Clin Exp Pathol. 2014;7:6040–7.
44. Jacob A, Prekeris R. The regulation of MMP targeting to invadopodia dur‑
ing cancer metastasis. Front Cell Dev Biol. 2015;3:4.
45. Chang CJ, Chiu JH, Tseng LM, Chang CH, Chien TM, Wu CW, et al. Modula‑
tion of HER2 expression by ferulic acid on human breast cancer MCF7
cells. Eur J Clin Invest. 2006;36:588–96.
46. Yabe T, Hirahara H, Harada N, Ito N, Nagai T, Sanagi T, et al. Ferulic acid
induces neural progenitor cell proliferation in vitro and in vivo. Neurosci‑
ence. 2010;165:515–24.
47. Koyama M, Sowa Y, Hitomi T, Iizumi Y, Watanabe M, Taniguchi T, et al.
Perillyl alcohol causes G1 arrest through p15(INK4b) and p21(WAF1/Cip1)
induction. Oncol Rep. 2013;29:779–84.
48. Ushio‑Fukai M, Nakamura Y. Reactive oxygen species and angiogenesis:
NADPH oxidase as target for cancer therapy. Cancer Lett. 2008;266:37–52.
49. Clerkin JS, Naughton R, Quiney C, Cotter TG. Mechanisms of ROS modu‑
lated cell survival during carcinogenesis. Cancer Lett. 2008;266:30–6.
50. Wu XJ, Hua X. Targeting ROS: selective killing of cancer cells by a
cruciferous vegetable derived pro‑oxidant compound. Cancer Biol Ther.
2007;6:646–7.
51. Geest CR, Buitenhuis M, Groot Koerkamp MJ, Holstege FC, Vellenga E,
Coffer PJ. Tight control of MEK‑ERK activation is essential in regulating
proliferation, survival, and cytokine production of CD34+ ‑derived neu‑
trophil progenitors. Blood. 2009;114:3402–12.
52. Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA
and flow‑cytometry. Methods Mol Biol. 2010;594:57–72.
53. Svensk AM, Soini Y, Paakko P, Hiravikoski P, Kinnula VL. Differential
expression of superoxide dismutases in lung cancer. Am J Clin Pathol.
2004;122:395–404.
54. Zechner D, Fujita Y, Hulsken J, Muller T, Walther I, Taketo MM, et al.
β‑Catenin signals regulate cell growth and the balance between progeni‑
tor cell expansion and differentiation in the nervous system. Dev Biol.
2003;258:406–18.
55. Kim SI, Park CS, Lee MS, Kwon MS, Jho EH, Song WK. Cyclin‑dependent
kinase 2 regulates the interaction of axin with β‑catenin. Biochem Bio‑
phys Res Commun. 2004;317:478–83.
56. Garcia‑Florez LJ, Gomez‑Alvarez G, Frunza AM, Barneo‑Serra L, Martinez‑
Alonso C, Fresno‑Forcelledo MF. Predictive markers of response to
neoadjuvant therapy in rectal cancer. J Surg Res. 2015;194:120–6.
57. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, et al.
Activation of β‑catenin‑Tcf signaling in colon cancer by mutations in
β‑catenin or APC. Science. 1997;275:1787–90.
58. Yook JI, Li XY, Ota I, Hu C, Kim HS, Kim NH, et al. A Wnt‑Axin2‑GSK3β
cascade regulates Snail1 activity in breast cancer cells. Nat Cell Biol.
2006;8:1398–406.
59. Verras M, Sun Z. Roles and regulation of Wnt signaling and β‑catenin in
prostate cancer. Cancer Lett. 2006;237:22–32.
60. Pu P, Zhang Z, Kang C, Jiang R, Jia Z, Wang G, et al. Downregulation of
Wnt2 and β‑catenin by siRNA suppresses malignant glioma cell growth.
Cancer Gene Ther. 2009;16:351–61.
61. Stewart DJ. Wnt signaling pathway in non‑small cell lung cancer. J Natl
Cancer Inst. 2014;106:djt356.
62. Tetsu O, McCormick F. β‑Catenin regulates expression of cyclin D1 in
colon carcinoma cells. Nature. 1999;398:422–6.
63. Li TW, Peng H, Yang H, Kurniawidjaja S, Panthaki P, Zheng Y, et al. S‑Adenosyl‑
methionine and methylthioadenosine inhibit β‑catenin signaling by multi‑
ple mechanisms in liver and colon cancer. Mol Pharmacol. 2015;87:77–86.
64. Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, et al. Augmented
Wnt signaling in a mammalian model of accelerated aging. Science.
2007;317:803–6.
65. Yoon JC, Ng A, Kim BH, Bianco A, Xavier RJ, Elledge SJ. Wnt signaling
regulates mitochondrial physiology and insulin sensitivity. Genes Dev.
2010;24:1507–18.
66. Zhang DY, Pan Y, Zhang C, Yan BX, Yu SS, Wu DL, et al. Wnt/β‑catenin sign‑
aling induces the aging of mesenchymal stem cells through promoting
the ROS production. Mol Cell Biochem. 2013;374:13–20.
67. Wu B, Crampton SP, Hughes CC. Wnt signaling induces matrix metal‑
loproteinase expression and regulates T cell transmigration. Immunity.
2007;26:227–39.
68. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl‑2 heterodimerizes in vivo with a
conserved homolog, bax, that accelerates programmed cell death. Cell.
1993;74:609–19.
69. Sah NK, Khan Z, Khan GJ, Bisen PS. Structural, functional and therapeutic
biology of survivin. Cancer Lett. 2006;244:164–71.
70. Ito T, Shiraki K, Sugimoto K, Yamanaka T, Fujikawa K, Ito M, et al. Survivin
promotes cell proliferation in human hepatocellular carcinoma. Hepatol‑
ogy. 2000;31:1080–5.
71. Bagci EZ, Vodovotz Y, Billiar TR, Ermentrout GB, Bahar I. Bistability in apop‑
tosis: roles of bax, bcl‑2, and mitochondrial permeability transition pores.
Biophys J. 2006;90:1546–59.
72. Noel A, Jost M, Maquoi E. Matrix metalloproteinases at cancer tumor‑host
interface. Semin Cell Dev Biol. 2008;19:52–60.