Ethnopharmacological communication

Korean Scutellaria baicalensis Georgi methanol extracts inhibitsmetastasis via the Forkhead Box M1 activity in hepatocellularcarcinoma 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 Koreab Department of Biological Science, Center for Colon Cancer Research, University of South Carolina, Columbia, SC 29208, USAc Department of Nursing Science, International University of Korea, Jinju 660-759, Republic of Koread 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 Koreae Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea

a r t i c l e i n f o

Article history:Received 11 November 2013Received in revised form28 May 2014Accepted 28 May 2014Available online 6 June 2014

Keywords:Korean Scutellaria Baicalensis GeorgiFlavonoidsHuman hepatocellular carcinomaAnti-metastasisForkhead Box M1 (FOXM1)

a b s t r a c t

Ethnopharmacological relevance: Scutellaria baicalensis Georgi, commonly known as skullcaps, and it hasbeen widely used as traditional therapeutic herb in several eastern Asia including Korea, China and Japanbecause of its remarkable anti-inflammatory and anti-cancer effects. Our study focuses on the anti-metastatic effects of Scutellaria baicalensis Georgi in hepatocellular carcinoma (HCC).Materials and methods: Methanol extract of Scutellaria baicalensis Georgi was examined for identificationof its composition by HPLC-MS/MS. The extract was evaluated for the anti-metastasis activity usingHepG2 hepatocellular carcinoma cells via immunoblotting and RT-PCR. For mechanical study, specificForkhead 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 potentchemotherapeutic agent against HCC. Its clinical use guarantee for further study and individualflavonoids from Scutellaria baicalensis Georgi should also be investigated.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

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

these reasons, strategies of anti-hepatic carcinoma usually focusedon the novel therapeutic strategies.

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

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.05.0530378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

n Corresponding author. Tel.: þ82 55 772 2346; fax: þ82 55 772 2349.E-mail address: gonskim@gnu.ac.kr (G. Kim).

Journal of Ethnopharmacology 155 (2014) 847–851

baicalensis Georgi which is grown in different countries is neces-sary to figure out the exact ethno pharmacological effects.

Human Forkhead Box M1 (FOXM1) is a family of forkhead boxtranscription factor which consist of more than 50 transcriptionfactors (Myatt and Lam, 2007). FOXM1 is proliferation relatedfactor which is up regulated in human solid tumor and it isoverexpressed in hepatocellular carcinoma (Okabe et al., 2001).Interestingly, there is growing evidence that increased expressionof FOXM1 correlates with metastasis in various malignant tumors(Chandran et al., 2007; Dai et al., 2007). Previous studies showedthat inhibition of FOXM1 transcription factor lead to decreasemetastasis and invasion in breast and pancreatic cancer cells(Wang et al., 2007; Ahmad et al., 2010). Metastasis is a funda-mental property of malignant tumors and complex and multipleprocesses involving the overexpression of matrix metalloprotei-nases (MMPs). Thus, effective control of FOXM1 can be anexcellent strategy in HCC treatment.

2. Materials & methods

2.1. Sample preparation and high-performance liquidchromatography- tandem mass spectrometry (HPLC-MS/MS)

Korean Scutellaria baicalensis Georgi were purchased from theAnimal Bio Resources Bank (Jinju, Korea). The voucher specimen(#00100B) was deposited at the Animal Bio Resources Bank,Gyeongsang National University. The sample was prepared accord-ing to the method of Seo et al. (2013).

2.2. Cell culture and treatment

HepG2 human HCC cells were obtained from the Korean CellLine Bank (Seoul, Korea). The cells were maintained in DMEMsupplemented with 10% FBS and 1% penicillin/streptomycin (p/s)in a 5% CO2 atmosphere at 37 1C.

2.3. Cell proliferation assay

HepG2 cells or Chang liver cells (1�104 cells/well) were platedonto 12-well plates and treated with flavonoids at concentrationsof 50, 100, 200 and 400 μg/mL or vehicle alone for 48 h. Cellviability was estimated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT). Cell viability was expressed as apercentage of proliferation versus controls.

2.4. Wound healing assay

Cell migration was measured by a wound healing assay. HepG2cells were cultured on 12-well plates (1�106 cells/well) for 34 h.After confluent growth, the cells were scratched with a yellow tipto obtain a constant straight line devoid of cells. Suspended cellsand media were aspirated and phenol-free medium containingvarious concentrations of flavonoids (0, 50, 100, 200 and 400 mg/mL) were added. A negative control containing only cells and 10 μLDMSO was used as a reference for each sample. Three randomlyselected spaces along the scratched line were photographed usingan inverted microscope. The area of the wound was quantified byImage J software using the polygon selection mode. The migrationof cells towards the wounds was expressed as a percentage ofwound closure: % of wound closure¼[(At¼0 h�At¼Δh)/At¼0 h]�100% (Yue et al., 2010), where At¼0 h is the area of the woundmeasured immediately after scratching, and At¼Δh is the area ofthe wound measured for 48 h after scratching.

2.5. Gelatin zymography

HepG2 cells (2�105 cells/well) were seeded in wells of a 6-wellplate and were treated with various concentrations of flavonoidsfor 48 h at 37 1C. After that, conditioned media were collected andgelatin zymography was performed to confirm matrix metallo-proteinase-2 (MMP-2) activity according to the methods ofNewcomb 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 manufac-turer's instructions. PCR primers used to amplify the Homosapiens FOXM1, MMP-2 and glyceraldehyde 3-phosphate dehydro-genase (GAPDH) cDNA were purchased from Bioneer (Seoul,Korea). The oligonucleotids used and RT-PCR conditions were thesame as previously described (Park et al., 2012).

2.7. Plasmid construction and cell transfection

To further investigate the mechanism of the G2/M cell cyclearrest, we overexpressed FOXM1 in the HepG2 cells. To makeoverexpression in HepG2 cells, FOXM1 DNA were isolated by PCRfrom single-stranded cDNA harvested from HepG2 cells usingfollowing specific primers with restriction enzymes (dashedunderline: EcoR I and Hind III): 50-GATCAAGCTTATGAAAAC-TAGCCCCCGTCGGC-30 and 50-GATCGAATTCCTACTGTAGCTCAG-GAATAAACT-30. PCR products were digested with EcoR I andHind III, cloned into the corresponding restriction sites ofpcDNA3.1 (Invitrogen, USA), and verified by DNA sequencing.HepG2 cells were transiently transfected with 10 mg of FOXM1-expressing constructs or empty vector (negative control) usingLipfectamine LTX Reagent (Invitrogen) according to the manufac-turer's instruction. Transfected cells were cultured for 24 h inserum-free DMEM at 37 1C in 5% CO2, and treated with variousconcentrations of flavonoids (0, 50, 100, 200 and 400 μg/mL) for48 h until western blot analysis.

2.8. Western blot analysis

Total cell lysates were prepared using lysis buffer (1 M Tris–HCl[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 by10% polyacrylamide gel and then electrophoretically transferredonto a polyvinylidene (PVDF) membrane. After blocking in Trisbuffered saline-Tween containing 5% skimmed milk powder for30 min, each membrane was incubated or 14 h at 4 1C withprimary antibodies against each target protein. After washing fourtimes, the membranes were incubated with horseradishperoxidase-conjugated secondary antibodies, and protein levelswere detected using an enhanced chemiluminescence reagent(Anigen, Seoul, Korea).

2.9. Statistical analysis

Data are analyses as the mean7standard deviation (SD) of aminimum of three independent experiments. A Student's t-testwas performed using Instats software (Graphpad, San Diego, CA).P valueso0.05 were considered statistically significant.

3. Results and discussion

Recent studies have been shown that Scutellaria baicalensis rootextract or major components inhibit cancer cell metastasis through

H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847–851848

various signal pathways {131 Dong, P. 2011; 478 Zhang, Z. 2014}.However, there are only few studies related with anti- FOXM1activities and flavonoids. Especially, this is first report on flavonoidswhich are originated from Korean Scutellaria baicalensis Georgiinhibited FOXM1 protein expression. The main purpose of this workis to reveal the effects and underlying molecular mechanism offlavonoids isolated from Korean Scutellaria baicalensis Georgi treat-ment on human HCC HepG2 cell lines. The results showed that 50–400 mg/mL of flavonoids from Scutellaria baicalensis Georgi signifi-cantly inhibited metastasis of HepG2 cells. As shown in Fig. 1A, the

flavonoids decreased proliferation rate to 44.4% at 400 μg/mL after48 h incubation. Moreover, flavonoids did not decreased the Changliver cells viability lesser than 80%. The anti-metastatic effects offlavonoids on HepG2 cells were evaluated using gelatin-zymographyand wound healing assay. After treatment with various concentra-tions of flavonoids for 48 h, MMP-2 activity was remarkably downregulated at dose dependent manner (Fig. 1B). As shown in Fig. 1C,flavonoids inhibited wound closure in dose dependent manner,specifically, wound closure were 44.173.0%, 21.570.8% and13.972.8% at 100, 200 and 400 μg/mL, respectively. Most hepatic

Fig. 1. Effects of flavonoids on cell viability and cell migration in HepG2 cells. (A) Cell proliferation was determined by a standard MTT assay. (B) HepG2 cells were treatedwith indicated concentrations of flavonoids for 48 h and then subjected to gelatin zymography. (C) Confluent cultured HepG2 cells were wounded with a micropipette tipand incubated at 37 1C for 48 h. Cell migration score was calculated as wound closure percentage. The data represent the mean7SD of three replicates independentexperiments. The asterisk (*) indicates a significant difference from the control group (*po0.05).

H.S. Park et al. / Journal of Ethnopharmacology 155 (2014) 847–851 849

cancer is diagnosed at late advanced tumor stage and because ofmetastasis to near other organs patients abandon surgical removal(Katyal et al., 2000; Kummar and Shafi, 2003). For these reason,inhibition of metastasis can be excellent strategy in HCC treatment.Previous in vitro studies on FOXM1 implicated its involvement in theearly stage of metastasis. FOXM1 stimulate invasion and angiogenesisof pancreatic cancer cells via induction of MMP-2 and -9 (Wang et al.,2007). Interestingly, our results also showed that flavonoid inhibitsMMP-2 expression and cell migration. To examine the effect offlavonoids on FOXM1 and MMP-2 gene expression, HepG2 cells werepretreated with various concentrations of flavonoids for 48 h. Thelevel of FOXM1 and MMP-2 gene expression was decreased signifi-cantly following flavonoids treatment in dose dependent fashion(Fig. 2A). We then examined FOXM1 expression pattern of HepG2cells. Clearly, FOXM1 protein was much lower in the flavonoidstreated groups compared to the control (Fig. 2B). Extracellularprotease like matrix metalloprotease-2 is important marker to theacquisition of the metastatic ability. Hence, we examined the patternof MMP-2 to confirm the effect of FOXM1 cell metastasis. Transientover-expression of FOXM1 increased MMP-2 level by 1.3 fold. How-ever, flavonoids treatment decreased MMP-2 level by 0.86 fold(Fig. 2C). These data suggest that FOXM1 regulates the cell metastasisthrough increasing the level of MMP-2. These findings corroboratewith crucial results that flavonoids decreased metastasis throughinhibition of FOXM1 and these results correlated with MMP-2inhibition. Further investigations will be needed to understand themolecular mechanism of flavonoids in interrelation with FoxM1 andMMP-2 inhibition. The concentration of major components ofScutellaria baicalensis root extract such as wogonin, baicalin andnorwogonin might be different depending on various climatic

conditions which differ from country to country. Thus, comparativeexperiment of Scutellaria baicalensis root extract from differentcountries is required to study its effect on cancer cell line. Inconclusion, our study suggests that flavonoids identified from KoreanScutellaria baicalensis Georgi could inhibit HCC cell metastasis, andmay provide alternative offers in the treatment for HCC. Lastly, weexpect our study will benefit the verification of anti-cancer effects ofScutellaria baicalensis Georgi for Ethnopharmacological evidence.

Acknowledgments

This work was supported by a grant from the National ResearchFoundation (NRF) of Korea funded by the Ministry of Science, ICT &Future Planning (Nos.2012M3A9B8019303 and 2012R1A2A2A06045015) and National R&D Program for Cancer Control, Ministry forHealth, Welfare and Family Affairs, Republic of Korea. (No. 0820050).

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