Pien Tze Huang inhibits metastasis of human colorectal carcinoma cells via modulation of TGF-β1/ZEB/miR‑200 signaling network

Abstract

Tumor metastasis, a complex process involving the spread of malignant tumor cells from a primary tumor site to a distant organ, is a major cause of failure of cancer chemotherapy. Epithelial-mesenchymal transition (EMT) is a critical step for the initiation of cancer metastasis. The processes of EMT and metastasis are highly regulated by a double-negative feedback loop consisting of TGF-β1/ZEB pathway and miR‑200 family, which therefore has become a promising target for cancer chemotherapy. Pien Tze Huang (PZH), a well‑known traditional Chinese formula first prescribed in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of various types of human malignancy including colorectal cancer (CRC). Our published data proposed that PZH was able to induce apoptosis, inhibit cell proliferation and tumor angiogenesis, leading to the suppression of CRC growth in vitro and in vivo. To further elucidate the mode of action of PZH, in the present study we evaluated its effects on the metastatic capacities of human colorectal carcinoma HCT-8 cells and investigated the underlying molecular mechanisms. We found that PZH significantly inhibited the migration and invasion of HCT-8 cells in a dose-dependent manner. In addition, PZH treatment inhibited the expression of key mediators of TGF-β1 signaling, such as TGF-β1, Smad2/3 and Smad4. Moreover, PZH treatment suppressed the expression of ZEB1 and ZEB2, two critical target genes of TGF-β1 pathway, leading to a decrease in the expression of mesenchymal marker N-cadherin and an increased expression of epithelial marker E-cadherin. Furthermore, PZH treatment upregulated the expression of miR‑200a, miR‑200b and miR‑200c. Collectively, our findings in this study suggest that PZH can inhibit metastasis of colorectal cancer cells via modulating TGF-β1/ZEB/miR‑200 signaling network, which might be one of the mechanisms whereby PZH exerts its anticancer function.

Full text

INT ERNATIONAL JOUR NAL OF ONCOLOGY
Abstract. Tumor metastasis, a complex process involving the
spread of malignant tumor cells from a primary tumor site to
a distant organ, is a major cause of failure of cancer chemo-
therapy. Epithelial-mesenchymal transition (EMT) is a critical
step for the initiation of cancer metastasis. The processes
of EMT and metastasis are highly regulated by a double-
negative feedback loop consisting of TGF-β1/ZEB pathway
and miR-200 family, which therefore has become a promising
target for cancer chemotherapy. Pien Tze Huang (PZH), a
well‑known traditional Chinese formula rst prescribed in
the Ming Dynasty, has been demonstrated to be clinically
effective in the treatment of various types of human malig-
nancy including colorectal cancer (CRC). Our published data
proposed that PZH was able to induce apoptosis, inhibit cell
proliferation and tumor angiogenesis, leading to the suppres-
sion of CRC growth in vitro and in vivo. To further elucidate
the mode of action of PZH, in the present study we evaluated its
effects on the metastatic capacities of human colorectal carci-
noma HCT-8 cells and investigated the underlying molecular
mechanisms. We found that PZH signicantly inhibited the
migration and invasion of HCT-8 cells in a dose-dependent
manner. In addition, PZH treatment inhibited the expres-
sion of key mediators of TGF-β1 signaling, such as TGF-β1,
Smad2/3 and Smad4. Moreover, PZH treatment suppressed
the expression of ZEB1 and ZEB2, two critical target genes
of TGF-β1 pathway, leading to a decrease in the expression
of mesenchymal marker N-cadherin and an increased expres-
sion of epithelial marker E-cadherin. Furthermore, PZH
treatment upregulated the expression of miR-200a, miR-200b
and miR‑200c. Collectively, our ndings in this study suggest
that PZH can inhibit metastasis of colorectal cancer cells via
modulating TGF-β1/ZEB/miR-200 signaling network, which
might be one of the mechanisms whereby PZH exerts its anti-
cancer function.
Introduction
Colorectal cancer (CRC) is one of the most common cancers
and a leading cause of cancer-related deaths (1,2). To date,
the mainstay of anti-CRC treatment includes surgery, chemo-
therapy and radiotherapy. However, due to tumor recurrence
and metastasis the long-term survival and prognosis of patients
remains quite poor (3,4). Tumor metastasis is a complex process
involving the spread of malignant tumor cells from a primary
tumor site to a distant organ, which is a major cause of failure
of cancer treatment (5-7). Epithelial-mesenchymal transition
(EMT) is a critical step for the initiation of cancer metas-
tasis (8,9). The processes of EMT and metastasis are highly
regulated by multiple mechanisms, including TGF-β1/ZEB
pathways and miRNA 200 family (10-14).
TGF-β1 is the prototypic member of transforming growth
factor β superfamily. The activation of TGF-β signaling
pathway is initiated by the binding of ligands to a type II
receptor, resulting in the phosphorylation/activation of a type I
receptor. The activated type I receptor then phosphorylates
SMAD2/3 that in turn bind to SMAD4. The SMAD complex
translocates to the nucleus to regulate the expression of target
genes, including the ZEB (zinc nger E‑box‑binding homeobox)
transcription fac tor family (15,16). Upon activation, ZEB tran-
scription factors suppress epithelial marker gene expression
Pien Tze Huang inhibits metastasis of human colorectal carcinoma
cells via modulation of TGF-β1/ZEB/miR-200 signaling network
ALING SHEN1,2*, WEI LIN1,2*, YOUQIN CHEN3, LIYA LIU1,2, HONGWEI CHEN1,2, QUNCHUAN ZHUANG1,2,
JIUMAO LIN1,2, THOMAS J. SFERRA3 and JUN PENG1,2,4
1Academy of Integrative Medicine and 2Fujian Key Laboratory of Integrative Medicine on Geriatric,
Fujian University of Traditional Chinese Medicine, Minhou Shangjie, Fuzhou, Fujian 350122, P.R. China;
3Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine,
Cleveland, OH 44106, USA; 4Postdoctor Workstation, Zhangzhou Pien Tze Huang
Pharmaceutical Co., Ltd., Shangjie, Zhangzhou, Fujian 363000, P.R. China
Received October 8, 2014; Accepted November 10, 2014
DOI: 10.3892/ijo.2014.2772
Correspondence to: Dr Jun Peng, Academy of Integrative Medicine,
Fujian University of Traditional Chinese Medicine, 1 Huatuo Road,
Minhou Shangjie, Fuzhou, Fujian 350122, P.R. China
E-mail: pjunlab@hotmail.com
*Contributed equally
Abbreviations: CRC, colorectal cancer; PZH, Pien Tze Huang;
TCM, traditional Chinese medicine; TGF-β, transforming growth
fac tor-β; EMT, epithelial-to-mesenchymal transition; ZEB, zinc finger
E-box-binding homeobox
Key words: Pien Tze Huang, traditional Chinese medicine, cancer
metastasis, TGF-β1 pathway, miRNA

SHEN et al: PIEN TZE HUANG IN HIBITS CANCER METASTASIS
2
and upregulate mesenchymal gene expression, leading to the
processes of EMT and cancer metastasis (17,18). MicroRNAs
(miRNA) are a class of endogenous short non-coding RNAs
(19-24 nucleotides), which function primarily to negatively
regulate target gene expression by specically binding to the
3'-untranslational region (3'-UTR) of target mRNAs (19-21). It
has been shown that miRNAs function more likely as onco-
genes or tumor suppressors to modulate multiple oncogenic
cellular processes, such as cell proliferation, apoptosis and
metastasis (22-24). The miR-200 family members, including
miR-200a, miR-200b and miR-200c, have been proposed to
act as tumor suppressors that inhibit EMT by downregulating
the expression of ZEB1 and ZEB2 (11,14,25-27). However,
the expression of miR-200 family is negatively regulated by
TGF-β signaling, probably via TGF-β-induced DNA meth-
ylation of the miR-200 loci (12). Thus, TGF-β/ZEB/m iR-200
signaling network creates a double-negative feedback loop
that plays an essential role in the initiation of EMT and cancer
metastasis; which therefore becomes a promising target for
cancer chemotherapy (13,14).
Recently, traditional Chinese medicines (TCM) have
received great interest in the field of anticancer treatment
since they have fewer adverse effects as compared to modern
chemotherapeutics and have been used in China for thousands
of years as important alternative remedies for various diseases
including cancer (28,29). Pien Tze Huang (PZH) is a well-
known TCM formula that was rst prescribed >450 years ago
in the Ming Dynasty. The main ingredients of PZH include
Moschus, Calculus Bovis, Snake Gall and Radix Notoginseng.
These products together confer PZH proper ties of heat clearing,
detoxication, dissipation of hard mass, detumescence and
analgesia (30). Traditionally, PZH has been used to clinically
treat traumatic injuries and a variety of inammatory diseases,
particularly hepatitis (30-32). More importantly, PZH has also
been used in China and Southeast Asia for centuries as a folk
remedy for treatment of various types of human cancer. We
recently reported that PZH can inhibit colon cancer growth
through the promotion of cancer cell apoptosis, the inhibition
of cell proliferation and tumor angio genesis, which is prob-
ably mediated by modulation of multiple signaling pathways
(33-40). To further elucidate the mode of action of PZH, in
the present study we evaluated its effects on the metastatic
capacities of human colorectal carcinoma HCT-8 cells and
investigated the underlying molecular mechanisms.
Materials and methods
Materials and reagents. Roswell Park Memorial Institute
(RPMI)-1640 medium, fetal bovine serum (FBS), penicillin-
streptomycin, were obtained from Life Technologies Corp.
(Grand Island, NY, USA). N-cadherin and E-cadherin anti-
bodies were purchased from Abcam (HK) Ltd. (Hong Kong,
Ch ina). T GF-β1, SMAD2/3, SMAD4, ZEB1, ZEB2 and
β-actin antibodies, horseradish peroxidase (HRP)-conjugated
secondary antibodies were provided by Cell Signaling
Technology (Beverly, MA, USA). Transwell chambers were
obtained from Corning Life Sciences (Tewksbury, MA, USA).
BD BioCoat Matrigel Invasion Chamber was purchased from
BD Bioscience (San Jose, CA, USA). PrimeScript RT reagent
kit, RNAiso for Small RNA kit and SYBR Premix Ex Taq Ⅱ
kit were provided by Dalian Takara Biotechnology Co., Ltd.
(Dalian, Liaoning, China). All the other chemicals, unless
otherwise stated, were obtained from Sigma Chemicals
(St. Louis, MO, USA).
Preparations of PZH. PZH was obtained from, and authen-
ticated by the sole manufacturer Zhangzhou Pien Tze Huang
Pharmaceutical Co. Ltd., China (Chinese FDA approval
no. Z35020242). Stock solutions of PZH were prepared just
before use by dissolving the PZH powder in PBS (phosphate-
buffered saline) to a concentration of 20 mg/ml. The working
concentrations of PZH were made by diluting the stock solu-
tion in the culture medium.
Cell culture. Human colorectal carcinoma HCT-8 cells were
obtained from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing,
Jiangsu, China). Cells were grown in RPMI-1640 medium
containing 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml
streptomycin in a 37˚C humidied incubator with 5% CO2.
The cells were subcultured at 80‑90% conuency.
Evaluation of cell migration by wound-healing assay.
Migration of HCT-8 cells was examined by wound-healing
assay. Cells were seeded into 6-well plate at a density of
1x106 cells/well in 2 ml medium. After 24 h of incubation,
cells were scraped away vertically in each well by using a P100
pipette tip. Three randomly selected views along the scraped
line were photographed on each well using a phase-contrast
inverted microscope (Leica, Germany) at a magnification
of x100. Cells were then treated with indicated concentrations
of PZH for 24 h, and another set of images were taken using
the same method. A reduction in the scraped area indicates a
sign of migration.
Measurement of cell migration and invasion by transwell
as sa y. Migration assay was performed using transwell
cell culture chambers with 8‑µm pore lters (Corning Life
Sciences, USA). After treatment with various concentrations
of PZH for 24 h, HCT-8 cells were trypsinized and resus-
pended in serum-free RPMI-1640. A total of 5x104 cells in
200 µl of serum-free RPMI-1640 were plated in the upper
chambers. RPMI-1640 media containing 10% (v/v) FBS was
used in the lower chambers as a chemoattractant. Cells were
allowed to migrate for 12 h, and the non-migrated cells were
removed from the upper surface of transwell membranes by a
cotton swab. Membranes were then stained with crystal violet.
For quantification, the average number of migrating cells
per eld was assessed by counting 3 random elds under a
phase‑contrast microscope (Leica) at a magnication of x200.
For cell invasion assay, the procedure was the same as that
of above-described migration analysis, except that the upper
chambers were coated with Matrigel Matrix (BD Biosciences,
USA).
Western blot analysis. HCT-8 cells were seeded into 25 cm2
asks at a density of 1.5x106 cells/ask in 5 ml medium. After
incubation for 24 h, the cells were treated with the indicated
concentrations of PZH for 24 h. The treated cells were lysed
with mammalian cell lysis buffer containing protease and
phosphatase inhibitor cocktails. Total protein concentrations

INT ERNATIONAL JOUR NAL OF ONCOLOGY 3
were determined by BCA assay. Equal amounts of total
proteins were resolved in 12% SDS-PAGE gels and electrob-
lotted. The PVDF membranes were blocked with 5% skimmed
milk and probed with primary antibodies N-cadherin,
E-cadherin, TGF-β1, SMAD2/3, SMAD4, ZEB1, ZEB2 and
β‑actin overnight at 4˚C and subsequently with the appropriate
HRP-conjugated secondary antibody followed by enhanced
chemiluminescence detection.
Q-PCR analysis. Total small RNA from HCT-8 cells was
isolated with RNAiso for Small RNA kit. Total small RNA
(500 ng) was reverse-transcribed with SYBR PrimeScript
miRNA RT-PCR kit according to the manufacturer's instruc-
tions. The obtained cDNA was used to determine the miRNA
amount of miR-200a, miR-200b and miR-200c, U6 was used
as an internal control. The primers of miR-200a (DHM0178),
miR-200b (DHM0179), miR-200c (DHM0180) and U6
(D356-03) were obtained from Dalian Takara Biotechnology
Co., Ltd. Quantitative PCR was performed using SYBR Premix
Ex Taq II in an ABI 7500 Fast instrument. Q-PCR reactions
were carried out following the manufacturer's protocol. miR NA
expression values were determined as ∆Ct=Ct (sample)-Ct
(U6) and relative quantities between different samples were
determined as ∆∆Ct=∆Ct (sample 1)-∆Ct (sample 2), the values
were expressed as 2-∆∆Ct. All Q-PCR reactions were conducted
in triplicate.
Statistical analysis. The data are presented as the means of
three deter minations and was analyzed using the SPSS package
for Windows (Version 18.0). Statistical analysis of the data was
performed with Student's t-test and ANOVA. Differences with
P<0.05 were considered statistically signicant.
Results
PZH inhibits migration and invasion of HCT-8 cells. We
rst performed a wound‑healing assay to evaluate the effect
of PZH on the migration of HCT-8 cells. As shown in Fig. 1,
after post-wounding for 24 h, untreated HCT-8 cells migrated
into the clear area, whereas PZH treatment dose-dependently
inhibited HCT-8 cell migration. We further verified these
results using transwell assay; and the data showed that treat-
ment with 0.25-0.75 mg/ml of PZH for 24 h dose-dependently
reduced cell migratory rate of HCT-8 cells by 44.4-85.8%, as
compared to untreated cells (Fig. 2, P<0.05). We next deter-
mined the effect of PZH on the invasion capacity of HCT-8
cells using the transwell assay. As shown in Fig. 3, compared
with untreated cells (100%), the invasion rate of HCT-8 cells
following treatment with 0.25, 0.5 or 0.75 mg/ml of PZH was
46.0±8.4, 29.6±3.0 or 19.1±4.0%, respectively (P<0.05). Taken
together, these data suggest that PZH can inhibit metastasis of
human colorectal cancer cells.
PZH modulates the activation of TGF-β1 pathway and the
expression of EMT-regulatory genes in HCT-8 cells. To deter-
mine the PZH effect on the activation of TGF-β1 signaling, we
examined the protein expression of several key mediators of
this pathway using western blot analysis. As shown in Fig. 4,
the protein expression levels of TGF-β1, Smad2/3 and Smad4
were downregulated by PZH treatment in a dose-dependent
manner. Moreover, PZH treatment suppressed the expression
of TGF-β1 target genes ZEB1 and ZEB2, leading to the down-
regulation of expression of mesenchymal marker N-cadherin
as well as an increase in the expression of epithelial marker
E-cadherin (Fig. 4). Therefore, the inhibitory effect of PZH on
cancer cell metastasis might be mediated by the suppression of
TGF-β1 pathway and the process of EMT.
PZH upregulates the expression of miR-20 0a, miR-200b and
miR-200c in HCT-8 cells. To further explore the mechanism
of anti-metastasis activities of PZH, we determined the
expression of miR-200 family in HCT-8 cells using Q-PCR
assay. As shown in Fig. 5, PZH treatment signicantly and
dose-dependently increased the expression of miR-200a,
Figure 1. Effect of PZH on migration of HCT-8 cells by wound-healing assay. Following treatment with the indicated concentrations of PZH for 24 h, the
migration pattern of HCT‑8 cells was observed using phase‑contrast microscopy. Images were captured at a magnication of x100. Images are representative
of three independent experiments.

SHEN et al: PIEN TZE HUANG IN HIBITS CANCER METASTASIS
4
miR-200b and miR-200c, consistent with the observations that
PZH inhibited the TGF-β1 pathway and expression of ZEB
transcription factors (Fig. 4).
Discussion
Drug resistance and intrinsic cytotoxicity against normal
cells profoundly limit the long-term use of currently-used
chemotherapeutic regimens and thereby their therapeutic
effectiveness (41,42), emphasizing the need for the develop-
ment of novel antitumor drugs. Due to the relatively higher
safety and the long history of pharmacological applications,
traditional Chinese medicines (TCM) have attracted great
interest in the eld of cancer treatment (28,29). TCM formula
is a complex combination of many natural prod ucts, each of
which contains numerous chemical compounds. Therefore,
TCM formulas are considered to be multi-component and
multi-target agents exerting their therapeutic function in a
more holistic way; and discovering naturally-occurring agents
could be a promising approach of cancer treatment. Pien Tze
Huang (PZH) is a well-known TCM formula that has been used
in China and Southeast Asia for centuries as a folk remedy for
various types of cancer. We recently reported that PZH can
inhibit colon cancer growth through the promotion of cancer
cell apoptosis, the inhibition of cell proliferation and tumor
angio genesis, which is probably mediated by modulation of
multiple signaling pathways (33-40). These data demonstrate
that PZH possesses a broad range of anticancer activities due
to its ability to affect multiple intracellular targets, suggesting
that PZH could be a novel multi-target anticancer agent.
Tumor metastasis is a complex process involving the
spread of malignant tumor cells from a primary tumor site to
a distant organ, which is a major cause of failure of clinical
cancer chemotherapy and therefore has become an important
focus for anticancer therapies (5-7,13,14). To further elucidate
the mode of action of PZH, in the present study we evaluated
Figure 2. Effect of PZH on the migration of HCT-8 cells by transwell assay. HCT-8 cells were treated with indicated concentrations of PZH for 24 h. (A) The
migration of HCT-8 cells was determined using transwell cell culture chambers. Cells were stained with crystal violet; the photographs were taken at a
magnication of x200. (B) The average number of migrated cells was counted in 3 randomly selective elds. The data were normalized to the migration of
untreated HCT-8 cells (100%). Data are averages with SD (error bars) from three independent experiments. *P<0.05, versus untreated HCT-8 cells.
Figure 3. Effect of PZ H on the invasion of HCT-8 cells. HCT-8 cells were tre ated with indicated concentrations of PZH for 24 h. (A) The invasion of HCT-8 cells
was determined using transwell cell culture cha mbers with membranes coated with Matrigel matrix. Cells were stained with crystal violet; the photographs
were taken at a magnication of x200. (B) The average number of invaded cells was counted in 3 randomly selective elds. The data were normalized to the
invasion of untreated HCT-8 cells (100%). Data are averages with SD (error bars) from three independent experiments. *P<0.05, versus untreated HCT-8 cells.

INT ERNATIONAL JOUR NAL OF ONCOLOGY 5
its effects on cancer metastasis. Using wound healing and
transwell assays we found that PZH treatment signicantly
inhibited the migration and invasion of human colorectal carci-
noma HCT-8 cells in a dose-dependent manner, demonstrating
the inhibitory activity of PZH on the metastatic capacities
of colorectal cancer cells. Epithelial-mesenchymal transition
(EMT) is a biological process in which epithelial cells lose
their polarity and cell-cell adhesion, and acquire migratory
and invasive properties of mesenchymal cells (8,9,14,15). After
acquiring a mesenchymal phenotype through the process of
EMT, carcinoma cells invade adjacent tissues, break through
the basement membrane, and eventually enter the bloodstream
leading to cancer metastasis (8,9,14-17). Therefore, EMT is
an essential step for the initiation of cancer metastasis. Using
western blot analysis we found that PZH treatment reduced
the protein expression of mesenchymal marker N-cadherin
but increased that of epithelial marker E-cadherin, indicating
that the anti-metastasis activity of PZH was associated with its
inhibitory effect on EMT. The processes of EMT and metas-
tasis are highly regulated by multiple mechanisms, including
TGF-β1/SMAD pathways and miRNA 200 family (10-14).
The activation of TGF-β1 signaling enhances the expression
of ZEB transcription fac tors, which in turn modulates the
expression of EMT-regulatory genes resulting in the initiation
of EMT (15,16). Interestingly, the expression of ZEB transcrip-
tion factors can be downregulated by miR-200 family members
(11,14,25-27); but miR-200 family expression is negatively
regulated by TGF-β1 signaling (12), forming a double-negative
feedback loop to control the processes of EMT and metastasis
(13,14). Data from western blot and Q-PCR analyses indicated
that PZH suppressed the activation of TGF-β1 pathway and
the expression of ZEB1 and ZEB2, whereas the expression
of miR-200a, miR-200b and miR-200c was upregulated after
PZH treatment.
In conclusion, here we report that PZH can inhibit the
metastatic capacity of human colorectal carcinoma cells via
modulating TGF-β1/ZEB/miR-200 signaling network, which
might be one of the mechanisms whereby PZH exerts its anti-
cancer function.
Acknowledgements
This study was supported by the National Natural Science
Foundations of China (81373819 and 81202790) and the China
Postdoctoral Science Foundation (2013T60636).
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