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Journal of Ethnopharmacology 137 (2011) 1373– 1380

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

ogonin, an active ingredient of Chinese herb medicine Scutellaria baicalensis,nhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cellsy inducing the expression of maspin

ing Donga,1, Yong Zhanga,1, Jun Gua, Wenguang Wub, Maolan Lia, Jiahua Yanga, Lin Zhanga,ianhua Lua, Jiasheng Mua, Lei Chena, Songgang Lia, Jianwei Wangb, Yingbin Liua,b,∗

Department of General Surgery, Xinhua Hospital, Affiliated to School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, ChinaDepartment of Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China

r t i c l e i n f o

rticle history:eceived 8 May 2011eceived in revised form 19 July 2011ccepted 2 August 2011vailable online 6 August 2011

eywords:cutellaria baicalensis

ogoninaspinetastasisuman gallbladder carcinoma

a b s t r a c t

Ethnopharmacological relevance: A traditional Chinese medicine Scutellaria baicalensis is prescribed for thetreatment of a variety of inflammatory diseases and tumors in clinic in China. However, the detailed mech-anism of anti-metastasis effect of wogonin, a main active ingredient of Scutellaria baicalensis, remainselusive.Aim of the study: The present study was designed to investigate the action and mechanism of wogoninon the mobility and invasion of human gallbladder carcinoma GBC-SD cells.Materials and methods: Viability, apoptosis, mRNA and protein expression of tumor cells were analyzedby MTT, Annexin V staining, real-time PCR and Western blot, respectively. The migration and invasionassay was used to evaluate the anti-metastasis effect of wogonin. Knockdown of maspin was performedby specific small interference RNA.Results: Wogonin at the dose of 1–10 �M, which did not induce apoptosis, significantly inhibited themobility and invasion activity of human gallbladder carcinoma GBC-SD cells. In addition, the expressionsof matrix metalloproteinase (MMP)-2, MMP-9 and phosphorylated extracellular regulated protein kinase1/2 (ERK1/2) but not phosphorylated Akt were dramatically suppressed by wogonin in a concentration-dependent manner. Furthermore, the metastasis suppressor maspin was confirmed as the downstream

target of wogonin. Both maspin mRNA and protein were upregulated by wogonin. Interestingly, theknockdown of maspin resulted in almost completely blocking of wogonin-induced inhibition of MMP-2,MMP-9 and phosphorylated ERK1/2 as well as the mobility and invasion activity of GBC-SD cells.Conclusions: These findings suggest that wogonin inhibits cell mobility and invasion by upregulatingthe metastasis suppressor maspin. Together, these data provide novel insights into the chemoprotective

activ

effect of wogonin, a main

. Introduction

Tumor growth, invasion, and metastasis are multistep andomplex processes that include cell division and proliferation, pro-eolytic digestion of the extracellular matrix, cell migration throughasement membranes to reach the circulatory system, and remi-

ration and growth of tumors at the metastatic sites (Klein, 2008).t is firmly established that matrix metalloproteinase (MMP)-2nd MMP-9 secretion from cancerous cells leads to hydrolysis of

∗ Corresponding author at: Department of General Surgery, Xinhua Hospital, Affil-ated to School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road,hanghai 200092, China. Tel.: +86 21 65793206; fax: +86 21 65793206.

E-mail address: liuybphd@126.com (Y. Liu).1 These authors contributed equally to this work.

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.08.005

e ingredient of Chinese medicine Scutellaria baicalensis.© 2011 Elsevier Ireland Ltd. All rights reserved.

extracellular matrix, thus enabling cells to break out of their pri-mary site into the circulation and form the secondary metastaticsites. Invasiveness inhibitors may act by down-modulatingextracellular-barrier degrading proteinases such as MMP-2 andMMP-9 (Nozaki et al., 2003). Activation of extracellular regulatedprotein kinase 1/2 (ERK1/2) has been shown to be the mechanismfor promoting the production of MMPs (Beshir et al., 2010), whichare important for cell proliferation, invasion, and neovasculariza-tion. It has been shown that inhibition of ERK1/2 activation maylead to a reduction in the expression of MMP-2, as well as in theinvasion of tumor cells (Lev et al., 2004).

Maspin, a member of the serine protease inhibitor family, has

been shown to be a potent metastasis suppressor (Bailey et al.,2006; Khalkhali-Ellis, 2006; Lockett et al., 2006). The ectopicexpression of maspin suppresses the invasive potential of breastcancer cells in vitro and the growth and metastasis of prostate

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374 P. Dong et al. / Journal of Ethnop

nd breast cancers in a mouse model (Zou et al., 1994; Shi et al.,001). Maspin is expressed ubiquitously in multiple tissues andormal epithelial cells but reduced in tumor cells (Zou et al., 1994;halkhali-Ellis, 2006). Thus, inducing maspin expression in prema-

ignant and malignant cells may be an effective strategy for cancerrevention and treatment.

Scutellaria (HUANG QIN, Lamiaceae), which includes about 350pecies commonly known as skullcaps, is widespread in Europe,he United States and East Asia (Shang et al., 2010). A traditionalhinese medicine Scutellaria baicalensis is prescribed for the treat-ent of a variety of inflammatory diseases and tumors in clinic in

hina (Shang et al., 2010). The crude extract of Scutellaria baicalen-is has been reported to exert anti-cancer effect against someuman tumor cells (Ye et al., 2002; Kumagai et al., 2007). Wogo-in, a main active ingredient of Chinese herb medicine Scutellariaaicalensis, exhibits a variety of pharmacological activities includ-ng antioxidant (Gao et al., 1999), anti-inflammatory (Chi et al.,001), anxiolytic (Hui et al., 2002), anti-hepatitis (Guo et al., 2007),nticonvulsant (Park et al., 2007), anti-angiogenesis (Lu et al.,008), neuroprotective (Lee et al., 2003; Lim et al., 2010), anti-ancer (Wang et al., 2006; Baumann et al., 2008; Li-Weber, 2009)ctivities. Recent study has demonstrated that wogonin at the dosef more than 20 �M could effectively inhibit the proliferation andnduce the apoptosis in several cancer cell lines in vitro (Lee et al.,008; Zhang et al., 2008). However, little is known about the actionf wogonin on tumor cells at the sub-dose of apoptosis-inducingffect. Here we found that wogonin at the dose of 1–10 �M, whichid not induce apoptosis, significantly inhibited the mobility and

nvasion activity of human gallbladder carcinoma GBC-SD cells.urthermore, the metastasis suppressor maspin had been con-rmed as the downstream target of wogonin. Our data provideovel insights into the chemoprotective effect of wogonin, a mainctive ingredient of Chinese medicine Scutellaria baicalensis, againstighly metastatic tumor.

. Materials and methods

.1. Cell culture

All tumor cells used in the present study were maintained inG-Dulbecco’s modified Eagle’s medium (4.5 g/l glucose; Invitro-en) supplemented with 10% fetal calf serum (FCS, Invitrogen) plus

mM glutamine and 50 U/ml penicillin. Human gallbladder carci-oma GBC-SD, human prostate carcinoma LNCaP cells and human-937 leukemia cells were purchased from Cell Bank of Shang-ai Institute of Biochemistry and Cell Biology (Chinese Academyf Sciences, China) and they were grown at 37 ◦C in a 5% (v/v) CO2tmosphere.

.2. Drugs and reagents

Wogonin (5,7-dihydroxy-8-methoxyflavone, purity >99%,btained from Xi’an Tongjiang Biotechnology Co., Ltd., Xi’an,hina) was dissolved at a concentration of 50 mM in 100% DMSO as

stock solution, stored at −20 ◦C, and diluted with medium beforeach experiment. The final DMSO concentration did not exceed.1% throughout the study (all the control groups are composed of.1% DMSO). Dulbecco’s modified Eagle medium (DMEM), fetal calferum (FCS), Trizol Reagent and Lipofectamine 2000 Transfectioneagent were purchased from Invitrogen (Carlsbad, CA). 3-(4,5-imethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)

as purchased from Sigma Chemical Co. (St. Louis, MO). Transwell

nserts was purchased from Costar (Cambridge, MA, USA). Matrigelas purchased from BD Biosciences (Bedford, MA). SYBR Green

dye was purchased from Molecular Probes Inc. (Eugene, OR).

acology 137 (2011) 1373– 1380

Anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt (Thr 308)and anti-Akt were purchased from Cell Signaling Technology(Beverly, MA). Anti-MMP-2, anti-MMP-9, anti-maspin and anti-�Tubulin were purchased from Santa Cruz Biotechnology (SantaCruz, CA). All other chemicals were purchased from Sigma ChemicalCo. (St. Louis, MO).

2.3. Cytotoxicity assay

The cytotoxic effect was measured using the MTT assay. Cellswere seeded in 96-well microtiter plates with wogonin for indi-cated time, then MTT solution (5 mg/ml in RPMI 1640 medium;Sigma–Aldrich) was added (10 �l/well), and plates were incubatedfor a further 4 h at 37 ◦C. The purple formazan crystals were dis-solved in 100 �l of DMSO. After 5 min, the plates were read on anautomated microplate spectrophotometer (Sunrise, Tecan, Austria)at 570 nm. Assays were performed in triplicate on three indepen-dent experiments.

2.4. Cell apoptosis assay

The cells were stained with Annexin V-FITC/PI and measuredby FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).Annexin V+/PI− cells were considered as apoptotic cells. AnnexinV-FITC (fluorescein isothiocyanate)/PI (propidium iodide) kit waspurchased from BD Biosciences (San Jose, CA).

2.5. Cell migration assay and invasion assay

Cell migration and invasion assays were performed using 8.0-�m pore size Transwell inserts (Costar, Cambridge, MA, USA) aspreviously reported (Wang et al., 2009). For all migration assay,in brief, the undersurface of the membrane was coated withfibronectin (10 �g/mL) in PBS at 37 ◦C for 2 h. The membrane waswashed in PBS to remove excess ligand, and the lower chamberwas filled with 0.6 mL DMEM with 10% FCS. Cells were serum-starved overnight (0.5% FCS), harvested with trypsin/EDTA, andwashed twice with serumfree DMEM. Then, cells were resuspendedin migration medium (DMEM with 0.5% FCS), and 1 × 105 cells in0.1 mL were added to the upper chamber. After 24 h at 37 ◦C, thecells on the upper surface of the membrane were removed usingcotton swabs. The migrant cells attached to the lower surface werefixed in methanol at room temperature for 30 min and stained for20 min with a solution containing 0.5% crystal violet and 2% ethanolin 100 mM borate buffer (pH 9.0). The number of migrated cells onthe lower surface of the membrane was counted under a micro-scope in five fields at 100×. For cell invasion assay, all procedureswere carried out as in the migration assay except that Matrigelwas coated beforehand on the upper surface of the chambers (BDBiosciences) according to the manufacturer’s protocol.

2.6. Gelatin zymography

Gelatin zymography was performed in 10% SDS-PAGE that hadbeen cast in the presence of 0.1% gelatin. After a treatment withwogonin (1, 5 and 10 �M) for 24 h, the supernatants were collected.Samples were prepared in nonreducing loading buffer. After elec-trophoresis, the gels were washed twice with 50 mM Tris–HCl, pH7.6, containing 5 mM, CaCl2, 1 �M ZnCl2, and 2.5% Triton X-100(v/v) for 15 min, followed by a brief rinsing in washing buffer with-

out Triton X-100. The gels were then incubated at 37 ◦C for 24 h in50 mM Tris–HCl buffer containing 5 mM CaCl2, 1 �M ZnCl2, 1% Tri-ton X-100, and 0.02% NaN3, pH 7.6. The digestion was terminatedand the gels were stained with 0.1% Coomassie Brilliant Blue R250,

P. Dong et al. / Journal of Ethnopharmacology 137 (2011) 1373– 1380 1375

Fig. 1. Effect of wogonin on the vability and apoptosis of human tumor cells. (A) Wogonin at the dose of more than 10 �M could inhibit the viability of human tumor cells.Human gallbladder carcinoma GBC-SD cells, human prostate carcinoma LNCaP cells or human U-937 leukemia cells were treated with different concentrations of wogoninfor 24 h, respectively. *P < 0.05, **P < 0.01 vs. drug-untreated group. (B) Wogonin at the dose of more than 10 �M induced the apoptosis of human gallbladder carcinomaGBC-SD cells. Cells were treated with different concentrations of wogonin for 24 h. Cell apoptosis was estimated by the flow cytometry. The results shown are representativeo

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ollowed by destaining with 10% acetic acid, 10% methanol. Thenhe gels were observed by Gel-Pro analyzer.

.7. Western blot

Proteins were extracted in lysis buffer (30 mmol/L Tris, pH.5, 150 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10%lycerol, and phosphatase and protease inhibitors). The proteinsere then separated by SDS-PAGE and electrophoretically trans-

erred onto polyvinylidene fluoride membranes. The membranes

ere probed with antibodies overnight at 4 ◦C, and then incubatedith a horse radish peroxidase-coupled secondary antibody. Detec-

ion was performed using a LumiGLO chemiluminescent substrateystem (KPL, Guildford, UK).

2.8. Reverse transcriptase-polymerase chain reaction (RT-PCR)and real-time PCR analysis

RNA was extracted from liver tissue using Trizol Reagent(Invitrogen, Carlsbad, CA). One microgram RNA was reverse tran-scribed. The primer sequences used in polymerase chain reaction(PCR) were as follows: gapdh: 5′-AACGACCCCTTCATTGAC and 3′-CACGACTCATACAGCACCT; maspin: 5′-CTGACAACAGTGTGAACGACand 3′-TCTACTAACTAGGGTTCCGAAC. The PCR cycle conditionswere: 94 ◦C for 30 s, 56 ◦C for 40 s, and 72 ◦C for 40 s for 28cycles. After the amplification, PCR products were separated byelectrophoresis on 1.5% agarose gels and visualized by ethidium

bromide dying. For quantitative real-time PCR analysis, amplifi-cation was carried out for 40 cycles of the same PCR conditionmentioned above and product was detected using SYBR Green Idye (Molecular Probes Inc., Eugene, OR). Reactions were run in

1376 P. Dong et al. / Journal of Ethnopharmacology 137 (2011) 1373– 1380

F er carcinoma GBC-SD cells. Cells were treated with 1, 5, 10 �M wogonin for 12 h prior tot f Y-coordinate reflected the cell motility and invasion potency in vitro. *P < 0.05, **P < 0.01v

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Fig. 3. Effect of wogonin on the activities of MMP-2 and MMP-9 by gelatin zymogra-phy. (A) Gelatin zymography analysis of serum-free media conditioned by GBC-SDcells treated with wogonin for 24 h. (B) The relative enzyme activities of MMP-2 and

ig. 2. Effect of wogonin on the mobility and invasive potency of human gallbladdhe migration (A) or invasion (B) assay. The average invading cell number per field os. drug-untreated group.

riplicate using gapdh as the internal RNA control on an ABI 7300hermocycler (Applied Biosystems Inc., Foster City, CA).

.9. Small interference RNA

RNAi duplexes were designed to maspin using the RNAiesigner at http://www.dharmacon.com. Sequence informationsed in this study is as following. Maspin RNAi sequence, sense5′–3′) CCUAUGCAAAGGAAUUGGA; scrambled siRNA sense (5′–3′)CUUACGCUGAGUACUUCG. Maspin RNAi duplexes and scramblediRNA were synthesized and purchased from Genescript (Nan-ing, China). The siRNA transfection was performed as previouslyeported (Quan et al., 2010). Briefly, GBC-SD cells were plated in-well culture plates at density of 3 × 105 and transfected withiRNA by using Lipofectamine 2000 reagent (Invitrogen, Carls-ad, CA) according to the manufacturer’s protocol. Briefly, forach well, 5 �l Lipofectamine 2000 was diluted in 250 �l Opti-EMI medium (Invitrogen). This mixture was carefully added to

solution containing 200 nM siRNA in 250 �l Opti-MEMI medium.he solution was incubated for 20 min at room temperature, andhen gently dripped into the GBC-SD cells in 2 ml antibiotic free

edium. Regular growth medium was added 6–12 h after transfec-ion. Twenty-four hours after transfections, cells were treated with0 �M wogonin for 12 h. Then cells were harvested and performedo RT-PCR or Western blot or invasion assay.

.10. Statistical analysis

Data are expressed as mean ± SEM. Statistical significance wasetermined by one-way analysis of variance followed by the Bon-erroni post-hoc test for multiple comparisons or the two-tailedtudent’s t-test. A P value of less than 0.05 was considered statisti-ally significant.

. Results

.1. Wogonin inhibited the mobility and invasive potency ofuman gallbladder carcinoma GBC-SD cells at the sub-dose ofpoptosis-inducing effect

As shown in Fig. 1A, wogonin at the dose of more than 10 �Milled human gallbladder carcinoma GBC-SD cells, human prostatearcinoma LNCaP cells and human U-937 leukemia cells in a

MMP-9 are expressed as percentages relative to control samples. *P < 0.05, **P < 0.01vs. drug-untreated group.

concentration-depedent manner. However, wogonin at the con-centrations of 1–10 �M did not affect the survival of cells. In

addition, wogonin at the dose of 1–10 �M did not induce apoptosisof GBC-SD cells (Fig. 1B).

To examine the effect of wogonin at the sub-dose of apoptosis-inducing effect (1–10 �M) on cancer cell mobility, we performed

P. Dong et al. / Journal of Ethnopharmacology 137 (2011) 1373– 1380 1377

Fig. 4. Effect of wogonin on the activation of ERK1/2 and Akt in human gallbladder carcinoma GBC-SD cells. (A) Cells were treated with 10 �M wogonin for the indicatedtimes, then the phosphorylations of ERK1/2 and Akt were examined by Western blot assay. (B) Cells were treated with 1, 5, 10 �M wogonin for 12 h, then Western blot assayswere used to examine total and phosphorylation of ERK1/2 and Akt. The results shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01 vs.drug-untreated group.

Fig. 5. Quantitative real-time PCR and Western blot analysis for maspin expression in GBC-SD cells induced by wogonin. (A and B) Cells were treated with 10 �M wogoninfor the indicated times, then the mRNA and protein levels of maspin were determined by real-time quantitative PCR (A) and Western blot (B), respectively. (C) Cells weret ined1 showv

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reated with 1, 5, 10 �M wogonin for 6 h, then the mRNA level of maspin was determ2 h, then the protein level of maspin was determined by Western blot. The resultss. drug-untreated group.

igration assay using GBC-SD cells. As shown in Fig. 2A, cellu-ar migration was inhibited by up to 23%, 51% and 73% at 24 h

ith 1, 5 and 10 �M wogonin, respectively. To further explorehether the invasive activity was affected in response to low dose

f wogonin, we examined the invasive activity using a matrigel-oated membrane. As a result, wogonin dramatically reducedhe number of invaded cells, and this inhibition occurred in aoncentration-dependent manner (Fig. 2B). The inhibition rate of, 5 and 10 �M wogonin was about 20%, 55% and 71%, respec-ively.

.2. Wogonin inhibited the activities of MMP-2 and MMP-9 andown-regulated the expressions of phosphorylated ERK1/2 inBC-SD cells

MMPs are known to be crucial for degrading extracellularatrix components and for promoting tumor cellular invasion

n vitro and in vivo. We first examined whether the anti-invasive

by real-time quantitative PCR. (D) Cells were treated with 1, 5, 10 �M wogonin forn are representative of at least three independent experiments. *P < 0.05, **P < 0.01

activity of wogonin was correlated with the inhibition of activ-ities of gelatinolytic MMPs. It was shown that the activities ofMMP-2 and MMP-9 were reduced with the increasing concentra-tions of wogonin (Fig. 3). To determine which upstream signalingpathway was influenced by wogonin, we examined the expres-sion and phosphorylation of ERK1/2 and Akt. Treatment withwogonin inhibited phosphorylation of ERK1/2 in a time-dependent(Fig. 4A) and concentration-dependent (Fig. 4B) manner. How-ever, wogonin did not inhibit the activation of Akt. In all cases,the total steady state of protein levels remained unchanged(Fig. 4B).

3.3. Wogonin up-regulated maspin expression in GBC-SD cells

Quantitative real-time PCR and Western blot analysis revealedthat the mRNA and protein expressions of maspin were time-dependently (Fig. 5A and B) and dose-dependently (Fig. 5C and D)increased by wogonin in GBC-SD cells.

1378 P. Dong et al. / Journal of Ethnopharmacology 137 (2011) 1373– 1380

Fig. 6. Effect of maspin-specific siRNA on the invasive activity and MMP-2/9 signaling of wogonin-treated GBC-SD cells. (A–C) Cells were transfected with maspin-specificsiRNA and luciferase siRNA. Twenty-four hours after transfection, cells were treated with 10 �M wogonin for 12 h. The mRNA and protein levels of maspin were determinedby RT-PCR (A), real-time quantitative PCR (B) and Western blot (C), respectively. The results shown are representative of at least three independent experiments. *P < 0.05,**P < 0.01. (D and E) Maspin-specific siRNA significantly blocked the inhibition of mobility (D) and invasive (E) activity of GBC-SD cells by wogonin. Representative phasecontrast micrographs are shown. Cells were transfected with maspin-specific siRNA and luciferase siRNA, respectively. Twenty-four hours after transfection, cells weretreated with 10 �M wogonin for 12 h prior to the migration or invasion assay. *P < 0.05, **P < 0.01. (F) Maspin-specific siRNA prevented the inhibition of MMP-2, MMP-9 andphosphorylated ERK1/2 in response to wogonin. Cells were transfected with maspin-specific siRNA and luciferase siRNA, respectively. Twenty-four hours after transfection,cells were treated with 10 �M wogonin for 12 h. The protein levels of MMP-2, MMP-9 and phosphorylated ERK1/2 were determined by Western blot. The results shown arer

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.4. RNA interference of maspin reversed wogonin-inducednhibition of migration and invasion in GBC-SD cells

Considering the important role of maspin in metastasis, RNAnterference-mediated maspin knock-down was used. The messageNA level (Fig. 6A and B) and protein level (Fig. 6C) of maspin wereuccessfully silenced by maspin-specific siRNA on GBC-SD cells.onsequently, the inhibition of migration (Fig. 6D) and invasionFig. 6E) abilities of cells by wogonin were remarkably reversed byhe knockdown of maspin.

.5. Knockdown of maspin resulted in blocking ofogonin-induced inhibition of MMP-2, MMP-9 andhosphorylated ERK1/2 in GBC-SD cells

Furthermore, the knockdown of maspin using maspin-specificiRNA also resulted in almost completely blocking of wogonin-nduced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2Fig. 6F).

4. Discussion

The highlight of this work is the revelation that in the 1–10 �Mrange wogonin does not induce apoptosis but significantly inhibitsthe mobility and invasion activity of human gallbladder carcinomaGBC-SD cells by upregulating the metastasis suppressor maspin.Previously, wogonin has been reported to effectively inhibit theproliferation and induce the apoptosis at the dosages of more than10 �M in several cancer cell lines (Lee et al., 2008; Zhang et al.,2008). We also found that wogonin at the dosages of more than10 �M could significantly inhibit the viability of several humantumor cells including human gallbladder carcinoma GBC-SD cells,human prostate carcinoma LNCaP cells and human U-937 leukemiacells. However, the non-apoptosis-inducing action of wogonin ontumor cells at the dosages of less than 10 �M remains unclear.In the present study, 1–10 �M range wogonin did not inhibit the

proliferation and induce the apoptosis in human gallbladder car-cinoma GBC-SD cells (Fig. 1). Whether wogonin at the sub-dose ofapoptosis-inducing effect (1–10 �M) could influence other func-tions of tumor cells? Results from Fig. 2 showed that 1–10 �M

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P. Dong et al. / Journal of Ethnop

ogonin significantly inhibited the mobility and invasion activityf human gallbladder carcinoma GBC-SD cells in a dose-dependentanner. Thus, the mechanisms of wogonin on the metastasis

otentiality of tumor cells, at a sub-dose of apoptosis induction1–10 �M) needed to be further investigated.

ERK1/2 activation has been shown to be the major mechanismor promoting the production of MMP-2 and MMP-9 (Beshir et al.,010), which are important for promoting tumor cellular inva-ion. We presented evidence indicating that wogonin was able tonhibit human gallbladder carcinoma cell migration and invasionn vitro in parallel with down-regulation of MMP-2 and MMP-9 as

ell as ERK1/2 activation. Previously, Lee et al. also reported thatogonin significantly suppressed tumor necrosis factor-�-inducedMP-9 expression in human aortic smooth muscle cells (Lee et al.,

006). Together, these findings suggest wogonin has potential anti-etastatic effect in vitro and shed light on the investigation ofogonin on gallbladder carcinoma metastasis in vivo.

To further dissect the molecular mechanisms underlying thenti-invasive activity of wogonin, we focused on the role of maspin,

tumor metastasis suppressor. First, we found that both maspinRNA and protein were remarkably upregulated by wogonin. Then,

o validate the functional significance of maspin upregulation inogonin-treated cells, we knocked down maspin by siRNA and

xamined the invasive activity. As shown in Fig. 6D and 6E, knock-own of maspin restored the inhibition of cell invasion inducedy wogonin. Moreover, knockdown of maspin blocked wogonin-

nduced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2xpression. Thus, maspin may be responsible for the anti-invasivectivity of wogonin. Accumulated functional studies have demon-trated that maspin reduces tumor metastasis in vivo and tumorell motility and invasion in vitro (Hong et al., 2009; Lonardot al., 2010; Yeom et al., 2010). Thus, in addition to the functionf suppression of cancer cell growth at the dose of 25–100 �M,ogonin may be involved in the inhibition of metastasis throughpregulating maspin at the dose of 1–10 �M. Furthermore, maspinas also been shown to be involved in cell apoptosis as well asumor growth and metastasis. Jiang et al. have been reported thatndogenous maspin expression sensitizes breast carcinoma cellso staurosporine-induced apoptosis in vitro (Jiang et al., 2002). Inhe present study, we found that wogonin could induce significantpoptosis in GBC-SD cells at the higher dose (Fig. 1B). The relation-hip between maspin expression and apoptosis in wogonin-treatedells needs further investigation.

. Conclusion

In conclusion, wogonin upregulates the expression of metasta-is suppressor maspin, which is involved in anti-invasive activityf wogonin in human gallbladder carcinoma GBC-SD cells. Theresent study provides a novel insight into the anticancer activity ofogonin, a main active ingredient of Chinese medicine Scutellaria

aicalensis.

onflict of interest statement

The authors have no conflicts of interest.

cknowledgements

This study was supported by National Natural Science Foun-

ation of China (Nos. 30640060, 30872502, 30972918), Naturalcience Foundation of Zhejiang Province for Outstanding Youngesearcher Groups (No. R2080452) and Foundation of Medicalchool, Shanghai Jiao Tong University (No. 09XJ21012).

acology 137 (2011) 1373– 1380 1379

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Glossary

DMEM: Dulbecco’s modified Eagle mediumERK1/2: extracellular regulated protein kinase 1/2FCS: fetal calf serumFITC: fluorescein isothiocyanateMMP: matrix metalloproteinase

MTT: 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromidePI: propidium iodideRT-PCR: reverse transcriptase-polymerase chain reaction