Journal of Ethnopharmacology 155 (2014) 847851

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Ethnopharmacological communication

Korean Scutellaria baicalensis Georgi methanol extracts inhibits

metastasis via the Forkhead Box M1 activity in hepatocellular
carcinoma cells
Hyeon Soo Park a, Kwang Il Park b, Gyeong Eun Hong a, Arulkumar Nagappan a,
Ho Jeong Lee a, Eun Hee Kim c, Won Sup Lee d, Sung Chul Shin e, On Nuri Seo e,
Chung Kil Won a, Jae Hyeon Cho a, GonSup Kim a,n
a
Research Institute of Life Science, College of Veterinary Medicine (BK21 plus project), Gyeongsang National University, Gazwa, Jinju 660-701,
Republic of Korea
b
Department of Biological Science, Center for Colon Cancer Research, University of South Carolina, Columbia, SC 29208, USA
c
Department of Nursing Science, International University of Korea, Jinju 660-759, Republic of Korea
d
Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Gyeongnam Regional Cancer Center,
Gyeongsang National University Hospital, Jinju 660-702, Republic of Korea
e
Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history:
Received 11 November 2013
Received in revised form
28 May 2014
Accepted 28 May 2014
Available online 6 June 2014

Ethnopharmacological relevance: Scutellaria baicalensis Georgi, commonly known as skullcaps, and it has
been widely used as traditional therapeutic herb in several eastern Asia including Korea, China and Japan
because of its remarkable anti-inammatory and anti-cancer effects. Our study focuses on the antimetastatic effects of Scutellaria baicalensis Georgi in hepatocellular carcinoma (HCC).
Materials and methods: Methanol extract of Scutellaria baicalensis Georgi was examined for identication
of its composition by HPLC-MS/MS. The extract was evaluated for the anti-metastasis activity using
HepG2 hepatocellular carcinoma cells via immunoblotting and RT-PCR. For mechanical study, specic
Forkhead Box M1 (FOXM1) vector was transfected to HepG2 cells.
Results: Scutellaria baicalensis Georgi potentially inhibited proliferation of HepG2 cells dose dependently.
Scutellaria baicalensis Georgi decreased metastasis through the regulation of matrix metalloproteinase 2
(MMP-2) and FOXM1 activities at the transcription and translation levels.
Conclusions: The results of the present study suggest that Scutellaria baicalensis Georgi could be a potent
chemotherapeutic agent against HCC. Its clinical use guarantee for further study and individual
avonoids from Scutellaria baicalensis Georgi should also be investigated.
& 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Korean Scutellaria Baicalensis Georgi
Flavonoids
Human hepatocellular carcinoma
Anti-metastasis
Forkhead Box M1 (FOXM1)

1. Introduction
Despite of the encouraging progress in medical practice,
hepatocellular carcinoma (HCC) is still the third most critical cause
of cancer-related death, with approximately 500,000 people dying
every year and has not been decreased over the past few decades
(Sanyal et al., 2010; Villanueva and Llovet, 2011). During tumor
growth, HCC cells undergo drastic changes in extracellular matrix
to phenotype changes and metastasize to circumjacent organs. For

Corresponding author. Tel.: 82 55 772 2346; fax: 82 55 772 2349.

E-mail address: gonskim@gnu.ac.kr (G. Kim).

http://dx.doi.org/10.1016/j.jep.2014.05.053
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

these reasons, strategies of anti-hepatic carcinoma usually focused

on the novel therapeutic strategies.
Scutellaria baicalensis Georgi, commonly known as skullcaps,
has been widely used as medicinal herb in several eastern Asia
including Korea, China and Japan because of its anti-microbial,
anti-inammatory and anti-cancer effects (Ye et al., 2002;
Kumagai et al., 2007). Flavonoids are abundant constituents in
Scutellaria baicalensis Georgi and more than 60 structures have
been identied (Li et al., 2004). The major active component of
Scutellaria baicalensis Georgi is baicalin which exhibits ultraviolet
protective effect (Zhou et al., 2011) and anti-cancer effects (Ye et
al., 2009; Du et al., 2010; Chen et al., 2012). However, contents of
major active components are different depending on climate,
country and soil conditions. Thus, study about the Scutellaria

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H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847851

baicalensis Georgi which is grown in different countries is necessary to gure out the exact ethno pharmacological effects.
Human Forkhead Box M1 (FOXM1) is a family of forkhead box
transcription factor which consist of more than 50 transcription
factors (Myatt and Lam, 2007). FOXM1 is proliferation related
factor which is up regulated in human solid tumor and it is
overexpressed in hepatocellular carcinoma (Okabe et al., 2001).
Interestingly, there is growing evidence that increased expression
of FOXM1 correlates with metastasis in various malignant tumors
(Chandran et al., 2007; Dai et al., 2007). Previous studies showed
that inhibition of FOXM1 transcription factor lead to decrease
metastasis and invasion in breast and pancreatic cancer cells
(Wang et al., 2007; Ahmad et al., 2010). Metastasis is a fundamental property of malignant tumors and complex and multiple
processes involving the overexpression of matrix metalloproteinases (MMPs). Thus, effective control of FOXM1 can be an
excellent strategy in HCC treatment.

2.5. Gelatin zymography

2. Materials & methods

2.7. Plasmid construction and cell transfection

2.1. Sample preparation and high-performance liquid

chromatography- tandem mass spectrometry (HPLC-MS/MS)

To further investigate the mechanism of the G2/M cell cycle

arrest, we overexpressed FOXM1 in the HepG2 cells. To make
overexpression in HepG2 cells, FOXM1 DNA were isolated by PCR
from single-stranded cDNA harvested from HepG2 cells using
following specic primers with restriction enzymes (dashed
underline: EcoR I and Hind III): 50 -GATCAAGCTTATGAAAACTAGCCCCCGTCGGC-30 and 50 -GATCGAATTCCTACTGTAGCTCAGGAATAAACT-30 . PCR products were digested with EcoR I and
Hind III, cloned into the corresponding restriction sites of
pcDNA3.1 (Invitrogen, USA), and veried by DNA sequencing.
HepG2 cells were transiently transfected with 10 mg of FOXM1expressing constructs or empty vector (negative control) using
Lipfectamine LTX Reagent (Invitrogen) according to the manufacturer's instruction. Transfected cells were cultured for 24 h in
serum-free DMEM at 37 1C in 5% CO2, and treated with various
concentrations of avonoids (0, 50, 100, 200 and 400 g/mL) for
48 h until western blot analysis.

Korean Scutellaria baicalensis Georgi were purchased from the

Animal Bio Resources Bank (Jinju, Korea). The voucher specimen
(#00100B) was deposited at the Animal Bio Resources Bank,
Gyeongsang National University. The sample was prepared according to the method of Seo et al. (2013).
2.2. Cell culture and treatment
HepG2 human HCC cells were obtained from the Korean Cell
Line Bank (Seoul, Korea). The cells were maintained in DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin (p/s)
in a 5% CO2 atmosphere at 37 1C.

HepG2 cells (2  105 cells/well) were seeded in wells of a 6-well

plate and were treated with various concentrations of avonoids
for 48 h at 37 1C. After that, conditioned media were collected and
gelatin zymography was performed to conrm matrix metalloproteinase-2 (MMP-2) activity according to the methods of
Newcomb et al. (2005) with minor adaptions.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from HepG2 cells using TRIzol reagent
(GeneALL Biotechnology, Seoul, Korea) according to the manufacturer's instructions. PCR primers used to amplify the Homo
sapiens FOXM1, MMP-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were purchased from Bioneer (Seoul,
Korea). The oligonucleotids used and RT-PCR conditions were the
same as previously described (Park et al., 2012).

2.3. Cell proliferation assay

2.8. Western blot analysis
HepG2 cells or Chang liver cells (1  104 cells/well) were plated
onto 12-well plates and treated with avonoids at concentrations
of 50, 100, 200 and 400 g/mL or vehicle alone for 48 h. Cell
viability was estimated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cell viability was expressed as a
percentage of proliferation versus controls.
2.4. Wound healing assay
Cell migration was measured by a wound healing assay. HepG2
cells were cultured on 12-well plates (1  106 cells/well) for 34 h.
After conuent growth, the cells were scratched with a yellow tip
to obtain a constant straight line devoid of cells. Suspended cells
and media were aspirated and phenol-free medium containing
various concentrations of avonoids (0, 50, 100, 200 and 400 mg/
mL) were added. A negative control containing only cells and 10 L
DMSO was used as a reference for each sample. Three randomly
selected spaces along the scratched line were photographed using
an inverted microscope. The area of the wound was quantied by
Image J software using the polygon selection mode. The migration
of cells towards the wounds was expressed as a percentage of
wound closure: % of wound closure[(At 0 h  At h)/At 0 h] 
100% (Yue et al., 2010), where At 0 h is the area of the wound
measured immediately after scratching, and At h is the area of
the wound measured for 48 h after scratching.

Total cell lysates were prepared using lysis buffer (1 M TrisHCl

[pH 8.0], 5 M NaCl, 1% NaN3, 10% SDS, 10% NP-40 and 0.5%
C24H39NaO4). Cell lysates (40 mg of protein) were separated by
10% polyacrylamide gel and then electrophoretically transferred
onto a polyvinylidene (PVDF) membrane. After blocking in Tris
buffered saline-Tween containing 5% skimmed milk powder for
30 min, each membrane was incubated or 14 h at 4 1C with
primary antibodies against each target protein. After washing four
times, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies, and protein levels
were detected using an enhanced chemiluminescence reagent
(Anigen, Seoul, Korea).
2.9. Statistical analysis
Data are analyses as the mean 7standard deviation (SD) of a
minimum of three independent experiments. A Student's t-test
was performed using Instats software (Graphpad, San Diego, CA).
P values o0.05 were considered statistically signicant.
3. Results and discussion
Recent studies have been shown that Scutellaria baicalensis root
extract or major components inhibit cancer cell metastasis through

H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847851

various signal pathways {131 Dong, P. 2011; 478 Zhang, Z. 2014}.

However, there are only few studies related with anti- FOXM1
activities and avonoids. Especially, this is rst report on avonoids
which are originated from Korean Scutellaria baicalensis Georgi
inhibited FOXM1 protein expression. The main purpose of this work
is to reveal the effects and underlying molecular mechanism of
avonoids isolated from Korean Scutellaria baicalensis Georgi treatment on human HCC HepG2 cell lines. The results showed that 50
400 mg/mL of avonoids from Scutellaria baicalensis Georgi signicantly inhibited metastasis of HepG2 cells. As shown in Fig. 1A, the

849

avonoids decreased proliferation rate to 44.4% at 400 g/mL after

48 h incubation. Moreover, avonoids did not decreased the Chang
liver cells viability lesser than 80%. The anti-metastatic effects of
avonoids on HepG2 cells were evaluated using gelatin-zymography
and wound healing assay. After treatment with various concentrations of avonoids for 48 h, MMP-2 activity was remarkably down
regulated at dose dependent manner (Fig. 1B). As shown in Fig. 1C,
avonoids inhibited wound closure in dose dependent manner,
specically, wound closure were 44.173.0%, 21.570.8% and
13.972.8% at 100, 200 and 400 g/mL, respectively. Most hepatic

Fig. 1. Effects of avonoids on cell viability and cell migration in HepG2 cells. (A) Cell proliferation was determined by a standard MTT assay. (B) HepG2 cells were treated
with indicated concentrations of avonoids for 48 h and then subjected to gelatin zymography. (C) Conuent cultured HepG2 cells were wounded with a micropipette tip
and incubated at 37 1C for 48 h. Cell migration score was calculated as wound closure percentage. The data represent the mean 7 SD of three replicates independent
experiments. The asterisk (*) indicates a signicant difference from the control group (*p o 0.05).

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H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847851

Fig. 2. FOXM1 is essential for the MMP-2 regulation. (A) The total mRNA was isolated, and the mRNA level of FOXM1 and MMP-2 were performed by RT-PCR.
(B) Representative expression for FOXM1 protein. (C) Western blotting showed that transient transfection with FOXM1 could promote MMP-2 expression and avonoids
treatment revealed the down-regulation of FOXM1 and MMP-2 in HepG2 cells. The data represent the mean 7SD of three replicates independent experiments. The asterisk
(*) indicates a signicant difference from the control group (*p o0.05).

cancer is diagnosed at late advanced tumor stage and because of

metastasis to near other organs patients abandon surgical removal
(Katyal et al., 2000; Kummar and Sha, 2003). For these reason,
inhibition of metastasis can be excellent strategy in HCC treatment.
Previous in vitro studies on FOXM1 implicated its involvement in the
early stage of metastasis. FOXM1 stimulate invasion and angiogenesis
of pancreatic cancer cells via induction of MMP-2 and -9 (Wang et al.,
2007). Interestingly, our results also showed that avonoid inhibits
MMP-2 expression and cell migration. To examine the effect of
avonoids on FOXM1 and MMP-2 gene expression, HepG2 cells were
pretreated with various concentrations of avonoids for 48 h. The
level of FOXM1 and MMP-2 gene expression was decreased signicantly following avonoids treatment in dose dependent fashion
(Fig. 2A). We then examined FOXM1 expression pattern of HepG2
cells. Clearly, FOXM1 protein was much lower in the avonoids
treated groups compared to the control (Fig. 2B). Extracellular
protease like matrix metalloprotease-2 is important marker to the
acquisition of the metastatic ability. Hence, we examined the pattern
of MMP-2 to conrm the effect of FOXM1 cell metastasis. Transient
over-expression of FOXM1 increased MMP-2 level by 1.3 fold. However, avonoids treatment decreased MMP-2 level by 0.86 fold
(Fig. 2C). These data suggest that FOXM1 regulates the cell metastasis
through increasing the level of MMP-2. These ndings corroborate
with crucial results that avonoids decreased metastasis through
inhibition of FOXM1 and these results correlated with MMP-2
inhibition. Further investigations will be needed to understand the
molecular mechanism of avonoids in interrelation with FoxM1 and
MMP-2 inhibition. The concentration of major components of
Scutellaria baicalensis root extract such as wogonin, baicalin and
norwogonin might be different depending on various climatic

conditions which differ from country to country. Thus, comparative

experiment of Scutellaria baicalensis root extract from different
countries is required to study its effect on cancer cell line. In
conclusion, our study suggests that avonoids identied from Korean
Scutellaria baicalensis Georgi could inhibit HCC cell metastasis, and
may provide alternative offers in the treatment for HCC. Lastly, we
expect our study will benet the verication of anti-cancer effects of
Scutellaria baicalensis Georgi for Ethnopharmacological evidence.

Acknowledgments
This work was supported by a grant from the National Research
Foundation (NRF) of Korea funded by the Ministry of Science, ICT &
Future Planning (Nos.2012M3A9B8019303 and 2012R1A2A2A0604
5015) and National R&D Program for Cancer Control, Ministry for
Health, Welfare and Family Affairs, Republic of Korea. (No. 0820050).

References
Ahmad, A., Wang, Z., Kong, D., Ali, S., Li, Y., Banerjee, S., Ali, R., Sarkar, F.H., 2010.
FoxM1 down-regulation leads to inhibition of proliferation, migration and
invasion of breast cancer cells through the modulation of extra-cellular matrix
degrading factors. Breast Cancer Research and Treatment 122, 337346.
Chandran, U.R., Ma, C., Dhir, R., Bisceglia, M., Lyons-Weiler, M., Liang, W.,
Michalopoulos, G., Becich, M., Monzon, F.A., 2007. Gene expression proles of
prostate cancer reveal involvement of multiple molecular pathways in the
metastatic process. BMC Cancer 7, 64.
Chen, W.C., Kuo, T.H., Tzeng, Y.S., Tsai, Y.C., 2012. Baicalin induces apoptosis in
SW620 human colorectal carcinoma cells in vitro and suppresses tumor growth
in vivo. Molecules, 17; , pp. 38443857.

H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847851

Dai, B., Kang, S.H., Gong, W., Liu, M., Aldape, K.D., Sawaya, R., Huang, S., 2007.
Aberrant FoxM1B expression increases matrix metalloproteinase-2 transcription and enhances the invasion of glioma cells. Oncogene 26, 62126219.
Du, G., Han, G., Zhang, S., Lin, H., Wu, X., Wang, M., Ji, L., Lu, L., Yu, L., Liang, W., 2010.
Baicalin suppresses lung carcinoma and lung metastasis by SOD mimic and HIF1alpha inhibition. European Journal of Pharmacology 630, 121130.
Katyal, S., Oliver 3rd, J.H., Peterson, M.S., Ferris, J.V., Carr, B.S., Baron, R.L., 2000.
Extrahepatic metastases of hepatocellular carcinoma. Radiology 216, 698703.
Kumagai, T., Muller, C.I., Desmond, J.C., Imai, Y., Heber, D., Koefer, H.P., 2007.
Scutellaria baicalensis, a herbal medicine: anti-proliferative and apoptotic
activity against acute lymphocytic leukemia, lymphoma and myeloma cell
lines. Leukemia Research 31, 523530.
Kummar, S., Sha, N.Q., 2003. Metastatic hepatocellular carcinoma. Clinical Oncology (Royal College of Radiologists (Great Britain) 15, 288294.
Li, H.B., Jiang, Y., Chen, F., 2004. Separation methods used for Scutellaria baicalensis
active components. Journal of Chromatography B: Analytical Technologies in
the Biomedical and Life Sciences 812, 277290.
Myatt, S.S., Lam, E.W., 2007. The emerging roles of forkhead box (Fox) proteins in
cancer. Nature Reviews Cancer 7, 847859.
Newcomb, E.W., Ali, M.A., Schnee, T., Lan, L., Lukyanov, Y., Fowkes, M., Miller, D.C.,
Zagzag, D., 2005. Flavopiridol downregulates hypoxia-mediated hypoxia-inducible factor-1alpha expression in human glioma cells by a proteasomeindependent pathway: implications for in vivo therapy. Neuro-Oncology 7,
225235.
Okabe, H., Satoh, S., Kato, T., Kitahara, O., Yanagawa, R., Yamaoka, Y., Tsunoda, T.,
Furukawa, Y., Nakamura, Y., 2001. Genome-wide analysis of gene expression in
human hepatocellular carcinomas using cDNA microarray: identication of
genes involved in viral carcinogenesis and tumor progression. Cancer Research
61, 21292137.

851

Park, K.I., Kang, S.R., Park, H.S., Lee do, H., Nagappan, A., Kim, J.A., Shin, S.C., Kim, E.
H., Lee, W.S., Chung, H.J., An, S.J., Kim, G.S., 2012. Regulation of proinammatory
mediators via NF-kappaB and p38 MAPK-dependent mechanisms in RAW 264.7
macrophages by polyphenol components isolated from Korea Lonicera japonica
THUNB. Evidence-Based Complementary and Alternative Medicine: eCAM.
2012, 828521.
Sanyal, A.J., Yoon, S.K., Lencioni, R., 2010. The etiology of hepatocellular carcinoma
and consequences for treatment. The Oncologist 15 (Suppl. 4), S14S22.
Seo, O.N., Kim, G.S., Kin, Y.H., Park, S., Jeong, W.S., Lee, S.J., Jin, J.S., Shin, S.C., 2013.
Determination of polyphenol components of Korean Scutellaria baicalensis
Georgi using liquid chromatographytandem mass spectrometry: Contribution
to overall antioxidant activity. Journal of Functional Foods 5, 17411750.
Villanueva, A., Llovet, J.M., 2011. Targeted therapies for hepatocellular carcinoma.
Gastroenterology 140, 14101426.
Wang, Z., Banerjee, S., Kong, D., Li, Y., Sarkar, F.H., 2007. Down-regulation of
Forkhead Box M1 transcription factor leads to the inhibition of invasion and
angiogenesis of pancreatic cancer cells. Cancer Research 67, 82938300.
Ye, F., Che, Y., McMillen, E., Gorski, J., Brodman, D., Saw, D., Jiang, B., Zhang, D.Y.,
2009. The effect of Scutellaria baicalensis on the signaling network in hepatocellular carcinoma cells. Nutrition and Cancer 61, 530537.
Ye, F., Xui, L., Yi, J., Zhang, W., Zhang, D.Y., 2002. Anticancer activity of Scutellaria
baicalensis and its potential mechanism. Journal of Alternative and Complementary Medicine 8, 567572.
Yue, P.Y., Leung, E.P., Mak, N.K., Wong, R.N., 2010. A simplied method for
quantifying cell migration/wound healing in 96-well plates. Journal of Biomolecular Screening 15, 427433.
Zhou, B.R., Liu, W.L., Luo, D., 2011. Protective effect of baicalin against multiple
ultraviolet B exposure-mediated injuries in C57BL/6 mouse skin. Archives of
Pharmacal Research 34, 261268.