GSK3β_β-catenin signaling is correlated with the differentiation of glioma cells induced by wogoni

Toxicology Letters 222 (2013) 212–223Contents lists available at ScienceDirectToxicologyLettersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /t o x l etGSK3␤/␤-catenin signaling is correlated with the differentiation of glioma cells induced by wogonin ଝYajing Wang a ,1,Yi Zhang a ,1,Chen Qian a ,Min Cai a ,Yan Li b ,Zhiyu Li c ,Qidong You c ,Qing Wang d ,Rong Hu a ,∗,Qinglong Guo a ,∗aState Key Laboratory of Natural Medicines,Jiangsu Key Laboratory of Carcinogenesis and Intervention,Department of Physiology,China Pharmaceutical University,24Tongjiaxiang,Nanjing 210009,PR China bJiangsu Centers for Diseases Prevention and Control,Nanjing 210009,PR China cDepartment of Medicinal Chemistry,China Pharmaceutical University,Nanjing 210009,PR China dDepartment of Neurosurgery,Wuxi Second Hospital Affiliated Nanjing Medical University,Wuxi 214002,PR Chinah i g h l i g h t s•Wogonin inhibited C6cells growthand arrested at G0/G1phase of the cell cycle.•Wogonin induced typical morpho-logical changes and an increase in GFAP level.•Induction of cell differentiation by wogonin ascribed to the restrain of GSK-3␤/␤-catenin pathway.•Blocking GSK-3␤/␤-catenin signaling promotes the glial differentiation of glioma cells.g r a p h i c a la b s t r a cta r t i c l ei n f oArticle history:Received 3February 2013Received in revised form 9July 2013Accepted 10July 2013Available online 19 July 2013Keywords:WogoninGlioblastoma cells DifferentiationGSK-3␤/␤-catenin pathway Cell cyclea b s t r a c tMalignant gliomas are the most common and most aggressive primary brain tumor,and for which dif-ferentiation therapy has emerged as a promising candidate strategy.In this study,we used in vitro and in vivo assays to examine the differentiation effects of wogonin,a major active constituent of Scutellaria baicalensis ,on glioma C6and U251cells.We found that wogonin can suppress cell proliferation and induce G0/G1arrest under a concentration-dependent manner.Wogonin also triggered significant reduction in the G1cell-cycle regulatory proteins cyclin D1,cyclin-dependent kinase 2and 4along with overexpress-ion of cell-cycle inhibitory proteins p27.Immunofluorescence and western blot analysis indicated that wogonin increased the expression of lineage-specific differentiation marker glial fibrillary acidic protein (GFAP).In mechanisms,we verified that wogonin significantly diminished the phosphorylated level of protein kinase B (AKT),and maintenance of low ␤-catenin expression level was dependent on glyco-gen synthase kinase 3␤(GSK3␤)activation at Ser9.Blocking GSK3␤/␤-catenin pathway was required for wogonin-induced proliferation inhibition and terminal differentiation by using canonical activator lithium chloride (LiCl)and inhibitor dickkopf-1(Dkk1).Moreover,intravenous administration of wogonin delayed the growth of C6glioma in the intracranial tumor model.These findings provide the evidence and mechanistic support for wogonin-based differentiation therapies for malignant glioblastoma.Further-more,inhibition of GSK3␤/␤-catenin pathway may be a key and requisite factor in glioma differentiation.© 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.ଝThis is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License,which permits non-commercial use,distribution,and reproduction in any medium,provided the original author and source are credited.∗Corresponding authors.Tel.:+862583271055;fax:+862583271055.E-mail addresses:michelhu@ (R.Hu),anticancer drug@ (Q.Guo).1These two authors contributed equally to the manuscript.0378-4274/$–see front matter © 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved./10.1016/j.toxlet.2013.07.013Y.Wang et al./Toxicology Letters222 (2013) 212–2232131.IntroductionGlioblastoma multiforme(GBM)is the most common primary tumor of the brain and one of the most lethal malignant tumors, whose highly invasion and metastasis leave rare opportunity for cure.In spite of advances in surgery,optimized radiotherapy and chemotherapy,patients face a dismal prognosis and the median survival rates range from12to15months(Louis et al.,2007). Therefore,great efforts have been made to develop novel adjuvant therapeutic strategies.Differentiation therapy,an approach to the treatment of advanced or aggressive malignancies,involves using agents,alone or in combination,that promote cancer cells maturation and dif-ferentiation into mature cells(Leszczyniecka et al.,2001).This therapeutic protocol is based on the concept that cancer cells dis-play alterations in differentiation states and growth control that can be corrected by appropriate differentiation inducers.More and more studies confirm that this therapy is effective in the treatment of several types of cancer cells.Thefirst differenti-ation agent found to be successful was all-trans-retinoic acid (ATRA)in the treatment of acute promyelocytic leukemia(APL), demonstrating the remarkable efficient to induce glioma cells dif-ferentiation(Bianchi et al.,2008;Kiningham et al.,2008;Redova et al.,2010;Siddikuzzaman Guruvayoorappan and Berlin Grace, 2011;Zhang et al.,2008d).Subsequently,several differentiation-inducing agents,including cholera toxin,capsaicin,valproic acid, neomycin,HMBA,saikosaponins,were reported to be capable of inhibiting cells growth and inducing glioma cells differentiation (Benitez et al.,2008;Cuevas et al.,2004;Gil and Kang,2008; Shirsat et al.,2001;Tsai et al.,2002;Xu et al.,2009).In addition, neuroblastoma cells exhibited effective differentiation with eleva-tion of intracellular adenosine3 ,5 -cyclic monophosphate(cAMP) concentration after treatment with cyclic nucleotide phosphodi-esterase inhibitors or adenylate cyclase activators(Chang et al., 2011;Hai et al.,2009).Although the roles of differentiation inducers obtained growing concern,effective differentiation agents for the treatment of malignant glioma have not yet been found(Kawamata et al.,2006).Wogonin,5,7-dihydroxy-8-methoxyflavone,is a naturally monoflavonoid extracted from Scutellaria baicalensi Georgi,which has been shown to have anti-inflammatory,anticancer,antivi-ral and neuroprotective effects.We have previously reported that wogonin could suppress promyelocytic leukemia U-937cells growth,inducing the granulocytic differentiation and G1arrest of U-937and NB4cells by up-regulating the expression of PLSCR1 gene(Zhang et al.,2008a,b).However,the potential effect of wogo-nin on the induction of solid tumors differentiation has not yet been explored.In addition,a recent study has indicated that wogonin can induce differentiation of neural precursor cells into neuronal cells both in culture and in vivo(Lim et al.,2010).These data prompt us to investigate the possible roles of wogonin on glioma cells differ-entiation.In this study,we investigated the effects of wogonin on cell survival and differentiation in glioma C6and U251cells and explored the underlying molecular mechanisms,in order tofind the potential therapeutic function of wogonin in the treatment of ing a syngeneic rat glioma tumor model,com-prising orthotopic transplantation of C6glioma cells into SD rats,we reported delayed growth of tumor following intravenous administration of wogonin.And we also examined whether the glioma C6cells pretreated with wogonin prior to transplanta-tion into the adult rat brain can be efficiently differentiated into mature astrocytes cells.Our results demonstrated that wogonin effectively induced G0/G1phase cell cycle arrest and the dif-ferentiation of glioma cells focusing on the GSK-3␤/␤-catenin signaling.2.Materials and methods2.1.Chemicals and antibodiesWogonin(purity>99%)was isolated from S.baicalensis Georgi according to the procedure reported previously(Hui et al.,2002).Stock solutions of wogo-nin were dissolved in dimethyl sulfoxide(DMSO),stored at20◦C and diluted with medium until needed.Thefinal DMSO concentration in all drug-treated cells was<0.1%.All-trans retinoic acid(ATRA,used as positive control),DAPI, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)were all pur-chased from Sigma–Aldrich(St.Louis,MO,USA).Stock solutions of ATRA were prepared in DMSO at10mM and stored at−20◦C wrapped with aluminum foil to avoid direct light irradiation.Dulbecco’s Modified Eagle Medium(DMEM), penicillin–streptomycin and trypsin–EDTA were purchased from Life Technologies Corporation(Grand Island,NY,USA).Sources of the antibodies are as follows:GFAP (sc-33673),CDK2(sc-163),CDK4(sc260)and␤-actin(sc-1616)were from Santa Cruz Biotechnology(Santa Cruz Biotechnology Inc.,CA);Cyclin D1(#2922),p27 Kip1(#2552),Phospho-Akt(Ser473,#9271),Akt(#9272),␤-catenin(#9562)and Phospho-␤-catenin(Ser33/37/Thr41,#9561)were from Cell Signaling Technology (Beverly,MA);GSK3␤(BS1402)and p-GSK3␤(S9,BS4084)were from Bioworld Technology Inc.(St.Louis,MN,USA).IRDyeTM800conjugated secondary antibodies were obtained from Rockland Inc.(Bedford,PA).2.2.Cell cultureRat C6and human U251glioma cell lines were obtained from the American Type Culture Collection(Manassas,VA,USA),and routinely cultured in DMEM containing 10%fetal bovine serum,1%penicillin and streptomycin,maintained in a humidified atmosphere of95%air,5%CO2at37◦C.Cells were plated in6-well plates or plastic incubatingflask and then treated with the various concentrations of wogonin and ATRA.2.3.Proliferation assayC6and U251cells were seeded into6-well plates at a density of5×105per well and were treated as indicated.Cells were collected after trypsin digestion and counted with a cell counting chamber(Qiujing,Shanghai,China)and micro-scope(YS100;Nikon,Ibaraki,Japan).For cell viability assay,cells were plated into 96-well plates at approximately8000/well in100␮l medium.Various compounds were added to the cells at indicated concentrations and durations.Cell viability was measured using an MTT assay following the manufacturer’s protocol.2.4.Clonogenic assayCells were plated in35-mm dishes at5000cells per well in DMEM culture medium.Plates were further incubated in incubator for14days until colonies were large enough to be visualized.Colonies were stained with0.01%Crystal violet for 1h and counted under inverted microscope.Experiments were done in triplicate.2.5.Cell cycle analysisCells were starved in serum-free medium for24h to be synchronized,and after treatment with wogonin,cells were harvested and resuspended with70%ethanol overnight at4◦C.Cells were spun down and incubated with the DNA-binding dye propidium iodide(PI)solution[0.1%sodium citrate(w/v),0.1%TritonX-100(v/v), and50mg/L PI in deionized water]for30min at room temperature.Finally,cells were analyzed by FACSCaliburflow cytometry(Becton Dickinson,San Jose,CA). 2.6.Immunofluorescent stainingCells werefixed with4%paraformaldehyde–PBS for30min,washed,and blocked with PBS containing1%BSA for1h at37◦C.The primary antibodies were diluted at certain concentration according to instructions.After being incubated with primary antibody for2h at37◦C,cells were treated with the secondary anti-body at appropriate dilution in1%BSA for1h at37◦C.Nuclei were stained with DAPI (1␮g/ml).Immunofluorescence photomicrographs were captured usingfluorescent microscope(Olympus IX51,Olympus Corporation,Tokyo,Japan).2.7.Western blot assayCells were collected,washed twice with PBS and lysed in extraction buffer (50mM Tris–HCl pH7.4,150mM NaCl,1mM PMSF,1mM EDTA,1%Triton X-100 and0.1%SDS,200mM b-mercaptoethanol,1mM phenylmethylsulfonylfluoride and 1g/ml aprotinin)for30min on ice.The lysates were centrifuged at13,000×g for 15min,and protein concentration in the supernatants was quantified by BCA assay. Proteins were separated by10%SDS–polyacrylamide gel electrophoresis and elec-troblotted onto nitrocellulose membranes by standard procedures.After blocking with1%BSA in PBS for1h at37◦C,the membranes were incubated for2h with pri-mary antibodies at37◦C followed by IRDye800conjugated secondary antibody for214Y.Wang et al./Toxicology Letters222 (2013) 212–2231h at37◦C.Immunoreactive protein bands were detected with an Odyssey Scanning System(LI-COR Inc.,Lincoln,NE).2.8.Real-time quantitative PCRTotal RNA were extracted from C6and U251cells using TRIzol®Reagent(Invi-trogen,Carlsbad,CA)and then the quality of total RNA was assessed by the ratio of A260/A280using a BioPhotometer(Eppendorf,Hamburg,Germany).1␮g of total RNA was reverse-transcribed using PrimeScript®RT Reagent Kit with gDNA Eraser(Perfect Real Time).Quantitative real-time PCR(q-PCR)was conducted fol-lowing the protocol supplied by TaKaRa SYBR Green Master Mix kit.All the PCR kits were purchased from TaKaRa Biotechnology Co.,Ltd.(Dalian,China).The rel-ative amount of target mRNA was determined using the comparative threshold (Ct)method by normalizing target mRNA Ct values to those for GAPDH( Ct).The primer sequences are as follows:rat GFAP,forward:5 -ACCTCGGCACCCTGAGGCAG-3 ,reverse:5 -CCAGCGACTCAACCTTCCTC-3 ,and human GFAP,forward:5 -CTGC-GGCTCGATCAACTCA-3 ,reverse:5 -TCCAGCGACTCAATCTTCCTC-3 ;rat GAPDH,for-ward:5 -GTTACCAGGGCTGCCTTCTC-3 ,reverse:5 -GGGTTTCCCGTTGATGACC-3 , and human GAPDH,forward:5 -TGGGTGTGAACCATGAGAAG-3 ,reverse:5 -GCTAAGCAGTTGGTGGTGC-3 .2.9.Rat intracranial glioma modelsTo investigate whether wogonin facilitates the differentiation of C6cells in vivo as well as in cultured cells,C6cells treated with25␮M wogonin,10␮M ATRA or not for7days were transplanted into the adult rat brain.For identification within the host tissue,C6cells were labeled with afluorescence dye,DiI-C18-(3),followed by exposure to wogonin for2h prior to transplantation.Before transplantation,C6 cells were pretreated,and then collected,suspended in phosphate buffered saline, complemented with2%of glutamine and1%of penicillin streptomycin.Sprague-Dawley rats weighing from150to180g were group-housed in a temperature-controlled room with a12h:12h light–dark cycle.Animal treatment and maintenance were carried out following the directions in the ethics approval from the Experimentation Ethics Review Committee of China Pharmaceutical Uni-versity.Rats(n=24)were accommodated for one week before experiment and then were randomly divided into three groups(control,wogonin and ATRA)with intracranial injection of different treatment conditions cells.After anesthetized with equithensin(2mL/kg i.p.),animals were placed in the stereotaxic device.A scalp inci-sion was made along the midline and a high-speed drill was used to create a0.5mm diameter burr hole in the skull.For each rat,a10␮l cell suspension(1×106C6cells) was injected over10min at a depth of6.0mm into the right striatum(3.0mm lat-eral to midline,0.5mm posterior to bregma)using automatic microinjector.5min after the injection,the needle was slowly removed,and the burr hole wasfilled with bone wax.The wound was sutured together,and the rats were allowed to recover. All rats were examined each day.Dead animals were studied,their brains removed and kept in frozen pipes at−80◦C.After20days,the remaining animals were killed. The brains were sectioned and prepared for histology and immunohistochemistry.Then,for study the anti-glioma activity of wogonin in vivo,C6cells were stereo-tactically injected into the right striatum of rats following the above method.Three days after tumor transplantation,animals were randomly divided intofive groups: saline control group,cisplatin(DDP)positive control group,30mg/kg wogonin group,60mg/kg wogonin group and120mg/kg wogonin group.After injecting C6 cells for3days,rats were treated by i.v.with saline,5mg/kg DDP,30mg/kg,60mg/kg and120mg/kg wogonin,respectively,every other day and continued for20days. Tumor volume was estimated following gadolinium enhanced T1-weighted MRI(GE Medical Systems)on day8,13and18.Number of voxels in each slice(slice thickness 1mm,total18slices)were counted using Image J software.Volume=Total number of voxels×0.015625mm3.The brains were excised and thenfixed in4%PFA–PBS for immunofluorescence.2.10.General morphology and immunohistochemistryBrain sections were cut using a cryostat(Leica CM3050S)at15␮m and collected on glass slides coated with silane and poly-l-lysine.The sections werefixed in4% paraformaldehyde solution and then stored at−80◦C before processing.Microscopic morphology of the brain was assessed by hematoxylin–eosin staining of cryostat sections.The tumor cross sectional areas were measured based on H&E stained cryostat sections from perfusion-fixed brains using computer-assisted image analy-sis as previously described.Tumor sizes were determined according to the following formula:tumor volume=(square root of maximum cross sectional area)3.For immunofluorescence,brain cryostat sections were processed for indirect immunofluorescence.Blocking was performed by incubating60min at room tem-perature with PBS containing5%bovine serum albumin(PBS–BSA).Then,sections were incubated with primary antibodies overnight at4◦C,with3%BSA and0.1% Triton-X.Slides were washed three times in PBS and exposed to FITC-conjugated secondary antibodies for2h at room temperature.Nuclei were counterstained with DAPI.The immunoreactivity wasfinally examined under an Olympus confocal microscope.The PCNA staining was performed using a PCNA cell proliferation detection kit (Keygen,Nanjing,China)according to the manufacturing instructions.Following above-mentioned procedures,the frozen sections were blocked with3%hydrogen peroxide and10%methanol for20min with subsequent rinses with PBS.The spec-imens were incubated with primary antibodies and second antibodies,subsequent treated with the appropriate ABC-complex as chromogenic agent,resulting in brown staining of antigen-expressing cells.2.11.Statistical analysisData were analyzed using post hoc tests included the Student’s t-test and the Tukey multiple comparison tests.All experiments reported here represent at least triplicate independent replications.All data are represented as mean value±SD and the significant difference is indicated as*P<0.05,**P<0.01.3.Results3.1.Wogonin’s effect on the cell viability of glioma C6and U251 cellsTo investigate the cytotoxicity of wogonin on glioma C6and U251cells,we examined cell viability using MTT assay for24h.As shown in Fig.1A and B,wogonin inhibited the growth of C6and U251cells in a concentration-dependent manner.We additionally performed cell count assay showing that the effects of low con-centration of wogonin(25␮M,50␮M)on cell growth over longer period of time showed significant proliferation inhibition(Fig.1C and D).Approximately50%growth inhibition was observed when C6and U251cells were exposed to50␮M wogonin for72h.This is comparable to the results observed with10␮M ATRA,a known differentiation agent for glioma cells.Next,wogonin-induced cell reproductive death was also examined by evaluating the colony-formation ability using clonogenic assay.As data shown in Fig.1E and F,the clone numbers of C6and U251cells treated with50␮M wogonin were62%and57%,respectively,as much as that in control. Obviously,treatment with wogonin showed significant decrease in cell survival in a dose-dependent manner.3.2.Wogonin induces glioma cells at G1/G0phase arrestWe carried out FACS analysis on C6and U251cells treated with various wogonin.As shown in Fig.2A and B,wogonin treatment (12.5,25,50␮M)for24h obviously depleted the G2/M population in a dose-dependent manner.To further investigate whether cells re-enter the cell cycle after wogonin treatment was removed for 4,8,12,and24h,we respectively examined the sub-G1groups of C6and U251cells at each time point,which were pretreated with 50␮M wogonin for24h.As shown in Fig.2D,after wogonin was removed for24h,cells began to re-enter new cell cycle,indicating that the effect of wogonin inducing sub-G1arrest was reversible. Next,we analyzed expression levels of principal cell-cycle regu-latory proteins including cyclin D1,cyclin-dependent kinase2,4 and the cyclin-dependent kinase inhibitors p27.When cells were treated with various concentrations(0–50␮M)of wogonin for24h, the proteins levels of cyclin D1,CDK4,and CDK2all decreased as the wogonin concentration was raised.In contrast,the50␮M wogo-nin treatment resulted in an obvious increase in p27protein levels (Fig.2E and F).These data indicate that wogonin induces G1/G0 phases arrest on glioma cancer cells.3.3.Wogonin promotes the glial differentiation of glioma cellsWogonin induced a dramatic change in glioma C6cell morphol-ogy.As shown in Fig.3A,many smaller and spindle-shaped cells with long synaptic-like structure formed after treatment of wogo-nin(25and50␮M).Immunofluorescence indicated that a dramatic increase in GFAP production in C6and U251cells treated with wogonin(25␮M)were remarkably similar to those treated withY.Wang et al./Toxicology Letters222 (2013) 212–223215Fig.1.Wogonin inhibits cell growth in glioma cells.Wogonin-induced cell deaths of two different glioma cells are shown in C6(A)and U251(B).Cells were treated within 0–200␮M wogonin for24h and cell viability was examined by MTT assay.(C and D)The effect of wogonin on the growth of glioma cells compared with all-trans retinoic acid(ATRA).Cells were treated with different concentrations of wogonin or10␮M ATRA for72h.(E and F)The effect of wogonin(0–50␮M)on the colony formation ability of glioma cells.Data are presented as means±SD(n=3).Statistical differences compared with the controls are given as*P<0.05,**P<0.01.ATRA(10␮M),that is known to induce glioma cell differentiation (Fig.3B).Western blot analysis also showed that wogonin signifi-cantly increased GFAP level after treatment of wogonin(25␮M)for 3days.This increased expression of GFAP was observed with wogo-nin concentrations ranging from0to50␮M in C6and U251cells (Fig.3C and D).In addition,gene expression level of GFAP was mea-sured by real time-PCR and it was found that the mRNA expression of GFAP increased in the wogonin treatment group as compared to the control(Fig.3E).All these results suggest that wogonin induces the differentiation of glioma cells into mature glial cells.3.4.Wogonin exerts its differentiation activity via the inhibitionof GSK-3ˇ/ˇ-catenin pathway in glioma cellsSince theflavonoid wogonin has been shown to induce decrease in phosphorylated Akt in several types of cells including malignant216Y.Wang et al./Toxicology Letters222 (2013) 212–223Fig.2.Wogonin induces growth arrest at G0/G1phase of cell cycle in glioma cells.(A and B)The effect of wogonin on cell cycle profile(C6(A)or U251(B))was examined byflow cytometry analysis.Cells were treated with wogonin at indicated concentration(0–50␮M)for24h.The experiments were repeated three times,and the result of a representative experiment was presented.The quantitative data of cell cycle phase are shown in the(C).(D)The recovery effect of glioma cells to re-enter into cell cycle after50␮M wogonin treatment was removed for4,8,12and24h.(E and F)Western blotting analysis on principal cell-cycle regulatory proteins in C6and U251cells.Cells were incubated with different concentration wogonin(0–50␮M)for24h.The quantitative data of cell-cycle regulatory protein levels are shown in the(G and H).Y.Wang et al./Toxicology Letters222 (2013) 212–223217Fig.3.Wogonin promotes the differentiation of glioma cells into mature glial cells.Cells were incubated with0,25and50␮M wogonin for3days.(A)Wogonin promotes morphological transformation of C6cells that indicated cell differentiation.(B)Immunocytochemistry of GFAP proteins expression.C6cells were exposed to25and50␮M wogonin at indicated concentration for3days.(C)The effect of wogonin compared with ATRA on GFAP protein by western blot analysis.Cells were treated with25␮M wogonin or10␮M ATRA for3days.(D)Concentration-dependent effect of wogonin on GFAP protein.Cells were incubated with0,12.5,25,and50␮M wogonin for3days.(E)The mRNA level of GFAP protein.Cells were incubated with wogonin(0–50␮M)for24h.GFAP mRNA levels were measured by realtime-PCR.Data(mean±SD)were representative of at least three independent experiments;**P<0.01vs control,*P<0.05vs control.glioma(Huang et al.,2011;Lu et al.,2008;Parajuli et al.,2011),and GSK3␤,a downstream target molecule of Akt,plays an important role in astrocytic differentiation of human glioblastoma cells(He et al.,2011;Korur et al.,2009;Li et al.,2010a,b),we hypothe-sized that wogonin induced the differentiation in glioma cells was associated with decreased activity of Akt/GSK3␤signaling. To test this hypothesis,we analyzed both phosphorylated and non-phosphorylated forms of Akt in C6and U251cells treated with indicated doses wogonin or25␮M wogonin for various lengths of time(Fig.4A and B).Our data indicated that Akt was constitutively phosphorylated in glioma cells,which was significantly inhibited by the wogonin under a dose-and time dependent manner.Wogo-nin caused a more pronounced decrease in phosphorylation of Akt in a time-dependent manner.As proto-oncoproteins␤-catenin is putative GSK-3␤substrates for phosphorylation-dependent inactivation,we tested whether wogonin mediated Akt inhibition could lead to inhibition of GSK-3␤/␤-catenin signaling activity. Western blot analysis also confirmed up-regulated expression of GSK3␤and down-regulated expression of␤-catenin in glioma cells treated by wogonin as time and concentration increased218Y.Wang et al./Toxicology Letters222 (2013) 212–223Fig.4.Akt/GSK-3␤/␤-catenin pathway is involved in wogonin induced the differentiation of glioma cells.Wogonin decreased the level of phosphorylated Akt,depend on dose(A)and duration(B).Dose-and time-dependent inhibition of GSK-3␤and␤-catenin activities by wogonin was shown in(C)and(D).The ratios of the quantitative data are shown in the under panels.Cells were cultured for24h with various doses of wogonin,or with50␮M wogonin for indicated time.The Akt,GSK-3␤and␤-catenin protein expression and their phosphorylated levels were examined by western blot analysis.Results are expressed as the mean±S.E.M.of three independent experiments.**P<0.01 vs control,*P<0.05vs control.Y.Wang et al./Toxicology Letters222 (2013) 212–223219(Fig.4C and D).In addition,the phosphorylation levels of GSK3␤and␤-catenin presented reverse change tendency corresponding to their each protein levels.To further study␤-catenin pathway regulating the differentia-tion of glioma cells,20mM LiCl and20ng/ml DKK-1were separately added into wogonin treated glioma cells for3days.LiCl,as a known activator of canonical␤-catenin signaling,can inhibit GSK3␤activ-ity and consequently induce accumulation of␤-catenin.Dkk1can bind to LRP5/6co-receptors for Wnt ligands,and inhibit␤-catenin signaling.We found that C6cells became round and lose the synap-tic sample structure after LiCl treatment,but Dkk1significantly promoted C6cells differentiating into mature glial cells(Fig.5A). The result shown in Fig.5B also illustrated that the expression level of␤-catenin was increased by LiCl and decreased by DKK-1. Actually,wogonin caused significant inhibition of␤-catenin pro-tein compared to the separate drug groups.Treatment of C6and U251cells with LiCl in the presence of wogonin(50␮M)resulted in apparent down-regulation of GFAP compared to treatment with wogonin alone.The expression of GFAP was upregulated after Dkk1 treatment,and this activation by Dkk1was increase by the com-bined with wogonin treatment.These results suggest that blocking the␤-catenin signaling treated by Dkk1promotes the differen-tiation of glioma cells,while activating this pathway stimulated by LiCl inhibits glioma cells differentiating into mature glial cells. We thus concluded that wogonin suppressed the GSK3␤/␤-catenin signaling through attenuating the phosphorylation of Akt,and fur-ther exerted differentiation-inducing and anti-proliferative effect in glioma cancer cells.3.5.Wogonin promotes differentiation of C6glioma cells in vivoGlioma C6cells were pretreated with wogonin for72h before intracranial implantation to determine whether wogonin facilitates the differentiation of C6cells in vivo as well as in cultured cells,andFig.5.Inhibition of␤-catenin signaling promotes the glial differentiation in glioma cells.Cells were incubated with50␮M wogonin,20mM LiCL or20ng/ml Dkk1for72h, respectively.(A)Wogonin,LiCl,and DKK-1induced morphological changes of C6cells(a,control;b,LiCl;c,DKK-1;d,wogonin;e,wogonin and LiCl;f,wogonin and Dkk1).(B)␤-Catenin and GFAP protein levels were examined by western blot analysis.(C)The ratios of the quantitative data are shown in the under panels.Results are expressed as the mean±S.E.M.of three independent experiments.**P<0.01vs control,*P<0.05vs control.。