ESC reverses epithelial mesenchymal transition induced by transforming growth factor-β via inhibition of Smad signal pathway in HepG2 liver cancer cells
Liu et al. Cancer Cell Int
ESC reverses epithelial mesenchymal transition induced by transforming growth factor-β via inhibition of Smad signal pathway in HepG2 liver cancer cells
Xiao‑Ni Liu 1
Shuang Wang 1
Qing Yang 0
YaJ‑ie Wang 0
DeX‑i Chen 1
XiaoX‑in Zhu 0
0 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences , No 16 Nan Xiao Jie, Dong Zhi Men Nei, Dong Cheng Qu, Beijing 100700 , China
1 Beijing Institute of Hepatology and Beijing YouAn Hospital, Capital Medical University , No 8 Xi Tou Tiao, You An Men Wai, Feng Tai Qu, Beijing 100069 , China
Background: Epithelial mesenchymal transition (EMT) mediated by TGF‑ β pays an important role in malignant tumor acquired abilities of migration and invasion. Our previous study showed that the extract of Stellera chamaejasme L. (ESC) was against proliferation of a variety of tumor cells, but there were no studies in the effects of ESC on EMT in tumor cells. In this study, TGF‑ β was adopted to induce EMT in HepG2 cells and the influence of ESC on EMT was observed. Methods: MTT assay was used to observe the cell viability. Wound healing assay and transwell assay were used to observe the migration and invasion activities. Western blot and immunofluorescence methods were used to observe the expression of proteins. Results: We found that HepG2 cells induced by TGF‑ β showed mesenchymal morphology, down‑ regulation of epithelial marker E‑ cadherin and up‑ regulation of mesenchymal marker Vimentin, indicating that TGF‑ β could mediate epithelial mesenchymal induction in HepG2 cells. ESC could reverse the mesenchymal morphology and regulate expressions of marker proteins in HepG2 induced by TGF‑ β and significantly inhibit TGF‑ β induced HepG2 cell migration and invasion. We further found that ESC could also significantly depress Smad2 phosphorylation and nuclear translocation, and ESC had coordination with SB432542, a specific inhibitor of TβRI kinases. Conclusions: These results suggested that the ESC could reverse epithelial mesenchymal transition induced by TGF‑ β via inhibition Smad2 signaling pathway.
Hepatocellular carcinoma; Stellera chamaejasme L; Transforming growth factor; Metastasis; Epithelial mesenchymal transition; Smad signaling pathway
In recent years, it is found that epithelial mesenchymal
transition (EMT) is an important biological process for
malignant tumor cells to obtain migratory and invasive
ability and a key initiative step of invasion and
metastasis in tumors. EMT is characterized by up-regulation of
mesenchymal markers (such as Vimentin)
down-regulation of epithelial markers (such as E-cadherin) [
and loss of cell–cell adhesion, which enables tumor
cells to dissociate and migrate from the primary tumor
]. Because EMT is closely related to the proliferation,
metastasis and prognosis of malignant tumor, it has
become an important hot spot for pharmacological
studies on tumors [
]. Transforming growth factor (TGF-β)
is one of the most important signal molecular that can
initiate the EMT process [
]. During TGF-β-mediated
EMT, TGF-β initiates responses by contacting two types
of transmembrane serine/threonine kinases called
receptors type I and type II, promoting activation of the type I
by the type II kinase. The activated type I receptor then
propagates the signal to the nucleus by phosphorylating
Smad2 and Smad3. Once phosphorylated, Smad2 and
Smad3 associate with the shared partner Smad4 and the
complexes accumulate in the nucleus where they regulate
the expression of TGF-β target genes through
cooperative interactions with transcriptional partners, which is
process of the classical Smad-dependent signaling
pathway that TGF-β induced [
]. Many studies showed that
a variety of Stellera chamaejasme L. extracts or
monomers had anti-tumor activities and could induce
apoptosis of tumor cells [
]. Our previous studies also
got the same results [
], but there were no
experiments on its anti-metastasis effects. In this study, we
further observed the effects of ESC on TGF-β-mediated
EMT and classical Smad-dependent signaling pathway in
HepG2 liver cancer cells.
Cell line and cell culture
HepG2 liver cancer cell lines were preserved in Beijing
Institute of Hepatology. HepG2 cells were cultured in
DMEM medium (Gibco, Grand Island, NY, USA)
supplemented with 10 % fetal bovine serum (China Hangzhou
Sijiqing Biological Technology Co., Ltd) and maintained
at 37 °C in a humidified incubator with 5 % CO2.
Reagents and antibodies
Process of ESC (Extract of Stellera Chamaejasme L.)
and determination of part components of ESC was
provided in another paper [
Trypsin-ethylene-diaminetetraacetic acid (EDTA) and DMEM medium were
purchased from Gibco (Grand Island, NY,
bromide (MTT), dimethyl sulfoxide (DMSO) and SB431542
were provided by Sigma Chemical Co. (St. Louis, MO,
USA); TGF-β was from R&D Systems (Miniieapolis, MN,
USA); E-cadherin, Vimentin and β-actin primary
monoclonal antibody were purchased by Abcam Ltd
(Cambridgem MA, USA); Matrigel was from BD Biosciences
(Los Angeles, CA, USA); Crystal violet was from Beijing
Solarbio Science and Technology Co., Ltd; Smad and
p-Smad primary monoclonal antibody were from Cell
Signal Technology, Inc (Beverly, MA, USA).
Cells in the logarithmic growth phase were plated in 96-well
plates in a seeding density of 5000 cells/well and incubated
in a 37 °C incubator with 5 % CO2 overnight. After cells
were treated with ESC (final concentration was respectively,
100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0 μg/mL) for 24, 48, 72 h,
the culture medium in each well was abandoned, incubating
with 0.5 g/L MTT 100 μL for 4 h. Then each well was added
with 150 μL DMSO and vibrated for 10 min, and
absorbance of each well was detected with microplate reader
(ELX800 type, BIO-TEX Instruments, INC, Winooski, VT,
USA) at the 490 nm wavelength. The inhibition rate (IR) was
calculated as follows: IR (%) = (1 − ODtreatment/ODcontrol) ×
100 %. Then half-maximal inhibitory concentration (IC50)
was determined by logistic method.
Cells (2 × 104) were seeded in each well of 24-well plate
and incubated at 37 °C with 5 % CO2 overnight. Cells
were pretreated with indicated concentration of ESC for
the appropriated time and then TGF-β was added to each
well with the final concentration of 5 ng/mL. Cells were
incubated at 37 °C with 5 % CO2 for 24 h. Representive
photographs were taken using an inverted microscope
Wound healing assay
Cells (5 × 105) were plated in a 6-well plate (three lines were
drawn at the external bottom of each well) and incubated in
a 37 °C incubator with 5 % CO2 overnight to form a
confluent monolayer. The monolayers were scratched vertically
to the lines by a plastic tip and washed by PBS to remove
cell debris. Indicated concentrations of ESC and 5 ng/mL
TGF-β were then added to each well, and the plates were
incubated at 37 °C incubator with 5 % CO2 for 24 h.
Photographs of wound closure were taken at the intersection
point of lines and scratches at the point time of 0 and 24 h
by the inverted microscope and the distance of wound
closure was measured by Photoshop 8.0 software.
Relative closure rate (%)
= 1 − Distance of treatment group at 0 h − Distance of treatment group at 24 h
Distance of control group at 0 h − Distance of control group at 24 h
In vitro invasion assay
The matrigel invasion experiment was analyzed in
24-well transwell plates. 25 μL matrigel matrix was
resolved at 4 °C overnight and coated on the transwell
insert membrane (Corning Incorporated). After the
inserts were incubated at 37 °C for 30 min, 2 × 104 cells
in 100 μL of DMEM medium with 1 % BSA and different
concentrations of ESC were added to the top chamber
and 500 μL of 10 % serum-containing DMEM and 5 ng/
mL TGF-β were added in the bottom chamber. The cells
were then incubated at 37 °C with 5 % CO2 for 24 h. After
incubation, the medium was removed, and non-invading
cells were scrubbed by a wet cotton swab. The invading
cells were washed by PBS for three times and fixed by 4 %
paraformaldehyde for 15 min. Fixed cells were washed
three times by PBS and stained by 0.1 % crystal violet
in PBS for 10 min. Excess stain was washed by distilled
water for three times. The invading cells were counted in
five random fields using an inverted microscope.
In vitro migration assay
The methods of migration assay were same to the
invasion assay, but the transwell insert membranes were not
coated by matrigel.
Western blot analysis
Cells were seeded in 100 mm tissue culture dishes at the
density of 2 × 106 cells per dish and incubated for
overnight. Cells were then treated with various agents as
indicated in figure legends, then washed with ice-cold PBS
and harvested in 400 μL of cell lysis buffer. The protein
concentrations of lysates were determined using the
bicinchonininc acid method. Cell lysates (40 μg per lane) were
separated using 10 % SDS-PAGE and transferred
electrophoretically to polyvinylidenedifluoride membrane.
Membranes were blocked with tris-buffered saline/0.1 % tween
20 containing 5 % bovine serum albumin and then
incubated overnight at 4 °C with primary antibodies (1:1000).
Membranes were washed three times with TBST and
incubated for 1 h at room temperature with the
appropriate secondary antibody conjugated to goat anti-rabbit
horseradish peroxidase (1:2000). Membranes were then
washed and immunoreactive band were developed with
ECL and visualized by autoradiography. Protein loading
was normalized using β-actin antibody. Gray-scale analysis
of protein bands was performed using image software.
Cells were seeded into 24-well plates and treated as
described above. Cells were fixed with 4 % formaldehyde for
30 min, washed with PBS, blocked with 5 % BSA for 30 min
at room temperature, and then stained with anti-human
primary antibody (1:100) at 4 °C overnight. Cells were
incubated with anti-rabbit-FITC secondary antibody (1:500)
for 2 h at 4 °C, and then washed with PBS. Cells were then
incubated for 10 min at room temperature with DAPI to
stain nuclei, washed twice with PBS, and observed using an
inverted fluorescence microscope (Olympus, Japan).
All the data were expressed as the mean ± SD. The
results were subjected to the one way ANOVA test using
SPSS software (17.0 version).
TGF‑β induced EMT in HepG2 cells
First, the optimal concentrations of TGF-β to initiate the
EMT in HepG2 cells were determined. Changes of cell
morphology were observed after treatment with various
concentrations (0.1–10 ng/mL) of TGF-β for 24 h. HepG2
cells were subjected to morphological changes under
exposure to various concentrations of TGF-β. HepG2
cells showed a classical cobblestone epithelial morphology
in the absence of TGF-β, but after stimulation with
various concentrations of TGF-β for 24 h, the cells showed
a fibroblast-like morphology and cell–cell adhesion also
reduced (Fig. 1a). The expressions of epithelial phenotype
marker, E-cadherin, and mesenchymal phenotype marker,
Vimentin, were also determined after treatment with
various concentration of TGF-β for 24 h in HepG2 cells.
TGF-β decreased the E-cadherin and induced the
Vimentin expression in HepG2 cells (Fig. 1b, c). These results
suggested that TGF-β could induce EMT in HepG2 cells.
The cytotoxic effect of ESC on HepG2 cells
The dosage of ESC for EMT experiments must be
determined to avoid the influence of anti-proliferation of ESC.
Cytotoxicity of ESC was evaluated in HepG2 cells using
MTT assay. Doses of ESC up to 12.5 μg/mL (0, 1.56, 3.13,
6.25) exhibited no significant inhibition on HepG2 cells,
whereas higher doses of ESC (>12.5 μg/mL) significantly
inhibited HepG2 cell viability in HepG2 cells (Fig. 2).
IC50 of ESC on HepG2 cells for 24, 48, 72 h were
respectively, 85.99, 59.75 and 54.57 μg/mL. Therefore, the doses
less than 12.5 μg/mL were chosen for following EMT
ESC reversed cell scattering induced by TGF‑β in HepG2 cells
The TGF-β induced EMT was characterized by cell
scattering (Fig. 1). To make out whether ESC could
influence TGF-β-induced cell scattering, HepG2 cells were
stimulated with TGF-β (5 ng/mL) before treated with
ESC. In order to eliminate the interference of
proliferation inhibition of ESC on its block-scattering, we chose
low toxicity concentrations (<12.5 μg/mL) of ESC for the
further experiments. HepG2 cells were pretreated with
various concentrations of ESC (1–5 μg/mL) for 2 h before
stimulated with TGF-β for 24 h. The results showed that
5 and 1 μg/mL ESC blocked this scattering significantly
(Fig. 3A). We pretreated HepG2 cells with 5 μg/mL ESC
for varying periods from 2 to 6 h before TGF-β addition
and time-dependent reversing effect of ESC was showed
(Fig. 3B). These results showed that ESC could inhibit the
cell scattering induced by TGF-β in HepG2 cells.
ESC reversed EMT induced by TGF‑β in HepG2 cells by down‑regulation of Vimentin and up‑regulation of E‑cadherin
Pretreated with ESC (0.2–5 μg/mL) for 2 h in HepG2
cells significantly inhibited up-regulation of Vimentin
and down-regulation of E-cadherin induced by 5 ng/mL
TGF-β for 24 h (Fig. 4a, b). HepG2 cells were pretreated
with 5 μg/mL ESC for different hours (1–4 h) prior to
5 ng/mL TGF-β stimulation, the time-dependent effects
of ESC were showed (Fig. 4c). These results suggested
that ESC reversed marker proteins changes of EMT
induced by TGF-β in HepG2 cells.
ESC inhibited the TGF‑β‑induced cell migration and invasion
We also further examined whether ESC affected
TGFβ-induced cell migration and invasion. Our results
showed that TGF-β significantly induced cell
migration and ESC (0.2–5 μg/mL) could notably block this
migration (Fig. 5a). The same results were observed
with transwell assay and the results were not showed.
A modified invasion assay with transwell method was
also performed to further determine whether ESC
blocked TGF-β-induced invasion. ESC (0.2–5 μg/mL)
obviously decreased the number of TGF-β induced
invasive cells (Fig. 5b). These results suggested that
ESC inhibited the TGF-β-induced cell migration and
invasion in HepG2.
ESC inhibited TGF‑β‑initiated Smad signaling pathway
Since TGF-β could induce the Smad2 phosphorylation, we
tried to figure out whether ESC could inhibit this initiation.
We found that 5 ng/mL of TGF-β induced Smad2
phosphorylation within 0.5–6 h, and the level of Smad2
phosphorylation reached a maximum between 30 and 60 min after
treatment but total Smad2 expression was not be affected
during the whole stimulation period. Pretreatment with
5 μg/mL ESC for 0.5–6 h significantly inhibited the
expression of phosphorylation of Smad2 induced by TGF-β, but
no influence on the whole smad2 expression (Fig. 6a). Our
results found that 5 ng/mL TGF-β could induce Smad2
nucleus translocation and pretreatment of ESC (5 μg/mL)
for 2 h could reverse this nucleus translocation in hepG2
cells (Fig. 6b). These results suggested that ESC could
inhibit TGF-β-induced Smad2 phosphorylation and Smad2
Nuclear import. The western blot analysis revealed that
TGF-β induced smad2 phosphorylation, down-regulated
the E-cadherin and up-regulated the Vimentin. SB431542
(25 μM) could reverse these effects of TGF-β in HepG2
cells. The ESC (5 μg/mL) also could inhibit Smad2
phosphorylation, up-regulate E-cadherin and down-regulate
Vimentin induced by TGF-β. The reversing effects of
copretreatment with ESC (5 μg/mL) and SB432542 (25 μM) on
phosphorylation of smad2 and Vimentin were more potent
than pretreatment with SB431542 or ESC alone (Fig. 6c).
Cell migration and invasion were significantly inhibited by
treatment with SB431542 and ESC, copretreatment with
ESC and SB432542 showed more potent inhibition on cell
migration and invasion induced by TGF-β than treatment
with SB431542 or ESC alone (Fig. 6d). These results
suggested that ESC could reversing EMT induced by TGF-β
and this reversing effect might be related to its inhibition of
Smad signaling pathway.
Hepatocellular carcinoma is an extreme malignant tumor,
and early metastasis is one of the important reasons for
its poor prognosis. EMT is an important step in the
invasion and metastasis of many cancers, and TGF-β induces
progression of cancer through EMT [
researches confirmed that high expression or activation
of TGF-β in metastatic tumors [
]. So, as a classic
model of EMT induced by TGF-β, it was widely used
and we made this model in HepG2 cells to mimic the
metastasis in vitro. Our experiment results showed that
HepG2 cells were sensitive to the different concentration
of TGF-β (0.1–10 ng/mL) and TGF-β not only changed
the morphology but also the molecular markers of the
HepG2 cells. HepG2 cells induced by TGF-β appeared
the fibroblast-like morphology, with down-regulation
of the E-cadherin and up-regulation of Vimentin, which
was consistent with other reports [
]. These results
suggested that TGF-β could induce EMT in HepG2 cells.
ESC, the extract of Stellera Chamaejasme L., inhibited
proliferation and induced apoptosis in various tumor cells
by activation apoptotic death receptor signaling pathway in
our previous researches [
]. In this study, we found that
EMT induced by TGF-β in HepG2 cells could be reversed
by ESC (doses less than 12.5 μg/mL were used to avoid the
influence of anti-proliferation of ESC). The results showed
ESC (1–10 μg/mL) could reverse the cell scattering induced
by TGF-β and this effect appeared time-dependent style. To
further clarify whether ESC inhibition of TGF-β-induced
scattering in HepG2 cells resulted from dysregulation of
EMT related proteins, influence of ESC on E-cadherin and
Vimentin expression was observed. E-cadherin (epithelial
marker protein) is a well-studied member of the cadherin
family. In epithelial cells, E-cadherin-containing
cell-tocell junctions are often adjacent to actin-containing
filaments of the cytoskeleton . Loss of E-cadherin function
or expression has been implicated in cancer progression
and metastasis. E-cadherin downregulation decreases the
strength of cellular adhesion within a tissue, resulting in
an increase in cellular motility [
]. Vimentin is a type III
intermediate filament (IF) protein that is expressed in
mesenchymal cells. Vimentin is the major cytoskeletal
component of mesenchymal cells. Because of this, Vimentin is
often used as a marker of mesenchymally-derived cells or
cells undergoing an epithelial mesenchymal transition
during both normal development and metastatic progression
]. We examined the expressions of these two marker
proteins in HepG2 cells with immunofluorescence and
western blot assay. ESC (0.2–5 μg/mL) could reverse the
(see figure on next page)
Fig. 4 ESC reversed EMT induced by TGF‑β in HepG2 cells by down‑regulation of Vimentin and up ‑regulation of E‑ cadherin. a Expressions of
Vimentin and E‑ cadherin in HepG2 cells pretreated with different concentrations (1–5 μg/mL) of ESC prior 2 h to TGF‑β (5 ng/mL) incubation for
24 h with immune‑fluorescence assay. The magnification is ×400. b Western blot results of Vimentin and E‑ cadherin in HepG2 cells pretreated with
different concentrations (0.2–5 μg/mL) of ESC prior 2 h to TGF‑β (5 ng/mL) incubation for 24 h. c Western blot results of Vimentin and E‑ cadherin in
HepG2 cells pretreated with 5 μg/mL ESC for different hours (1–4 h) prior to 5 ng/mL TGF‑β stimulation. Western blot data presented were repre ‑
sentative of those obtained in at least 3 separate experiments. The value of the control cells was set to 1
changes of two marker proteins in hepG2 cells induced by
TGF-β which dedicated that ESC could decrease cellular
motility and metastasis. The results of wound closure and
transwell assay further confirmed this inhibition effect of
ESC in migration and invasion induced by TGF-β. These
data suggested that ESC could restrain the EMT mediated
How does the ESC reverse TGF-β-induced EMT in
hepG2 cells? Smad-dependent signaling was
classical pathway mediated by TGF-β. TGF-β superfamily
ligands bind to a type II receptor, which recruits and
phosphorylates a type I receptor. The type I receptor
then phosphorylates receptor-regulated Smad2/3 which
can bind the Smad4. P-Smads/Smad4 complexes
accumulate in the nucleus where they act as transcription
factors and participate in the regulation of EMT target
gene expression [
]. Under resting state, Smad2 is
unphosphorylated and retain in the cytoplasma. Upon
activation of TGF-β, Smad2 is phosphorylated and
undergoes dimerization with Smad3, thus permitting its
translocation into nucleus . Our results showed that
ESC could inhibit the up-regulation of p-Smad2 but did
not influence the expression of Smad2. The subcellular
location results showed that ESC could inhibit the Smad2
nuclear translocation. These results suggested that ESC
really could reverse TGF-β induced EMT by regulating
Smad signal pathway.
In Smad signal pathway, which was the key
regulating point of ESC in reversing EMT induced by TGF-β in
hepG2 cells? We use a specific inhibitor of TβRI kinases,
] to inhibit TGF-β-induced
phosphorylation of Smad2 [
]. We found that blocking the function
of TβRI kinases with SB432542 resulted in inhibition of
Smad2 phosphorylation, up-regulation of E-cadherin,
down-regulation of Vimentin, with the decreasing
activities of migration and invasion in hepG2 cells induced by
TGF-β. ESC not only had same effects to SB432542, but
also enhanced these effects of SB432542. These results
(see figure on next page)
Fig. 6 ESC inhibited TGF‑β‑induced Smad signaling pathway. a Pretreatment with 5 μg/mL ESC for 2 h significantly suppressed the expression of
phosphorylation of Smad2 induced by TGF‑β for 0.5–6 h, but no influence on expression of Smad2. b Pretreatment of 5 μg/mL ESC for 2 h could
inhibit the nucleus translocation induced by TGF‑β in hepG2 cells. The magnification is 200 times. c 5 μg/mL ESC and 25 μM SB432542 significantly
reversed EMT proteins induced by TGF‑β, inhibiting expression of phosphorylation of Smad2 and Vimentin, enhancing expression of E‑ cadherin and
the coordination of ESC and SB432542 was showed. d 5 μg/mL ESC and 25 μM SB432542 could inhibited cell migration and invasion induced by
TGF‑β and also showed coordination effects
suggested that ESC might be a potential inhibitor of TβRI
kinases which might be the key regulating point of ESC
in reversing EMT induced by TGF-β in hepG2 cells.
In summary, our observations showed that ESC could
reverse the EMT mediated by TGF-β in hepG2 liver cells
via inhibition of Smad signaling pathway. ESC may have
good prospects in the prevention and treatment of
hepatocellular carcinoma metastasis.
XL was the main experimental investigator and had drafted the manuscript.
SW carried out the cell culture. QY participated the data analysis. YW helped to
complete the molecular experiments. DC helped to draft the manuscript. XZ
supervised the study and the manuscript. All authors read and approved the
This work was supported by National Major Scientific and Technological
Special Project for “Significant New Drugs Development” during the Twelfth
Five‑ year Plan Period (No. 2013ZX09301307001004); Foundation of Beijing
Institute of Hepatology (No. BJIH‑01504).
The authors declare that they have no competing interests.
The authors received no financial support for the research, authorship, and/or
publication of this article.
1. Thiery JP , Acloque H , Huang YJR , Nieto MA . Epithelial‑mesenchymal transitions in development and disease . Cell . 2009 ; 5 : 871 - 90 .
2. Scanlon CS , Van Tubergen EA , Inglehart RC , D'Silva NJ . Biomarkers of epithelial‑mesenchymal transition in squamous cell carcinoma . J Dent Res . 2013 ; 2 : 114 - 21 .
3. Liu X , Xie L , Wang Y , Chen D , Zhu X . Advance of pharmacological studies on reversing epithelial‑mesenchymal transition of tumors . Chin Pharmacol Bull . 2013 ; 11 : 1481 - 5 .
4. Kalluri R , Weinberg RA . The basics of epithelial‑mesenchymal transition . J Clin Investig . 2009 ; 6 : 1420 - 8 .
5. Battaglia S , Benzoubir N , Nobilet S , Charneau P , Samuel D , Zignego AL , Atfi A , Bréchot C , Bourgeade MF . Liver cancer‑ derived Hepatitis C virus core proteins shift TGF‑Beta responses from tumor suppression to epithelial‑mesenchymal transition . PLoS One . 2009 ; 2 : e4355 .
6. Nakao A , Imamura T , Souchelnytskyi S , Kawabata M , Ishisaki A , Oeda E , Tamaki K , Hanai J , Heldin CH , Miyazono K , Dijke P. TGF‑beta receptor ‑ mediated signalling through Smad2, Smad3 and Smad4 . EMBO J . 1997 ; 17 : 5353 - 62 .
7. Li J , Zhang JJ , Pang XX , ZhengChen XL , Gan LS . Biflavanones with antiproliferative activity against eight human solid tumor cell lines from Stellera chamaejasme . Fitoterapia . 2014 ; 93 : 163 - 7 .
8. Yang D , Wang P , Ren X ( 2015 ) Apoptosis induced by chamaejasmine in human osteosarcoma cells through p53 pathway . Tumour Biol (Epub ahead of print).
9. Yang QX , Cheng MC , Wang L , Kan XX , Zhu XX , Xiao HB . Antitumor components screening of Stellera chamaejasme L. under the case of discrete distribution of active data . Yao Xue Xue Bao . 2014 ; 6 : 927 - 31 .
10. Zhang C , Zhou SS , Feng LY , Zhang DY , Lin NM , Zhang LH , Pan JP , Wang JB , Li J. In vitro anti‑ cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L . Acta Pharmacol Sin . 2013 ; 2 : 262 - 70 .
11. Liu X , Zhu X. Stellera Chamaejasme L. extract induces apoptosis of human lung cancer cells via activation of the death receptor‑ dependent pathway . Exp Ther Med . 2012 ; 4 : 605 - 10 .
12. Liu X , Yang Q , Zhang G , Li Y , Chen Y , Weng X , Wang Y , Wang Y , Zhu X . Anti‑tumor pharmacological evaluation of extracts from Stellera chamae - jasme L. based on hollow fiber assay . BMC Complement Altern Med . 2014 ; 14 : 116 .
13. Miyazono K. Transforming growth factor‑b signaling in epithelial‑mesenchymal transition and progression of cancer . Proc Jpn Acad Ser B Phys Biol Sci . 2009 ; 8 : 314 - 23 .
14. Chaffer CL , Weinberg RA. A perspective on cancer cell metastasis . Science . 2011 ; 6024 : 1559 - 64 .
15. Longo V , Brunetti O , D'Oronzo S , Ostuni C , Gatti P , Silvestris F . Bone metastases in hepatocellular carcinoma: an emerging issue . Cancer Metastasis . 2014 ; 1 : 333 - 42 .
16. Li W , Kang Y. A new Lnc in metastasis: long noncoding RNA mediates the prometastatic functions of TGF‑beta . Cancer Cell . 2014 ; 5 : 557 - 9 .
17. Ru NY , Wu J , Chen ZN , Bian H. HAb18G/CD147 is involved in TGF‑β‑ induced epithelial‑mesenchymal transition and hepatocellular carcinoma invasion . Cell Biol Int . 2015 ; 1 : 44 - 51 .
18. Fleming TP , Papenbrock T , Fesenko I , Hausen P , Sheth B . Assembly of tight junctions during early vertebrate development . Semin Cell Dev Biol . 2000 ; 4 : 291 - 9 .
19. Weinberg R. The biology of cancer . Garland Science; 2006 . pp. 864 .
20. Eriksson JE , Dechat T , Grin B , Helfand B , Mendez M , Pallari HM , Goldman RD . Introducing intermediate filaments: from discovery to disease . J Clin Invest . 2009 ; 17 : 1763 - 71 .
21. Ogrodnik M , Salmonowicz H , Brown R , Turkowska J , Średniawa W , Pattabiraman S , Amen T , Abraham AC , Eichler N , Lyakhovetsky R , Kaganovich D . Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of Vimentin . Proc Natl Acad Sci USA . 2014 ; 22 : 8049 - 54 .
22. Ko H , So Y , Jeon H , Jeong MH , Choi HK , Ryu SH , Lee SW , Yoon HG , Choi KC . TGF‑β1‑induced epithelial‑mesenchymal transition and acetylation of Smad2 and Smad3 are negatively regulated by EGCG in human A549 lung cancer cells . Cancer Lett . 2013 ; 1 : 205 - 13 .
23. Zi Z , Chapnick DA , Liu X . Dynamics of TGF‑β/Smad signaling . FEBS Lett . 2012 ; 14 : 1921 - 8 .
24. Liu LC , Tsao TC , Hsu SR , Wang HC , Tsai TC , Kao JY , Way TD . EGCG inhibits transforming growth factor‑β‑mediated epithelial‑to ‑mesenchymal transition via the inhibition of Smad2 and Erk1/2 signaling pathways in nonsmall cell lung cancer cells . J Agric Food Chem . 2012 ; 39 : 9863 - 73 .
25. Pang K , Ryan JF , Baxevanis AD , Martindale MQ . Evolution of the TGF‑β signaling pathway and its potential role in the ctenophore , Mnemiopsisleidyi. PLoS One . 2011 ; 9 : e24152 .