2 0 1 0 년 2 월 석사학위논문
I nduct i on ofApopt osi s
by Angel i cadecur si vaExt r acti n Gl i omaCel l s
조선대학교 대학원
치의생명공학과
조 선 호
I nduct i on ofApopt osi s
by Angel i cadecur si vaExt r acti n Gl i omaCel l s
Angel i cadecur si va추출물에 의한
신경교종 세포의 Apoptosis유도2010년 2월 일
I nduct i on ofApopt osi s
by Angel i cadecur si vaExt r acti n Gl i omaCel l s
지도교수 김 도 경
이 논문을 이학 석사학위신청 논문으로 제출함.
2009년 10월 일
조선대학교 대학원
치의생명공학과
조 선 호
조 선 호의 석사학위 논문을 인준함
위원장 조선대학교 교 수 김 흥 중 인
위 원 조선대학교 교 수 국 중 기 인
위 원 조선대학교 교 수 김 도 경 인
2009년 11월 일
TABLE OF CONTENT
TABLE OF CONTENT
---ⅰLI ST OF TABLE
---ⅲLI ST OF FI GURES
---ⅳABSTRACT
---ⅴI .I NTRODUCTI ON
---1I I .MATERI ALS AND METHODS
---31.Materials--- 3
2.Plantmaterialandextractpreparation--- 3
3.Cellcultures--- 4
4.Inhibitionofcellgrowth--- 4
5.DNA fragmentationanalysis--- 4
6.Immunoblotting--- 5
7.Determinationofcaspaseactivation--- 5
8.Dataanalysis---6
Ⅲ.RESULTS
---71.CytotoxiceffectofEEAD inC6cells---7
2.DNA fragmentation--- 8
3.ActivationofcaspasesbyEEAD ---8
Ⅳ.DI SCUSSI ON
---9Ⅴ.REFERENCES
---12Ⅵ.FI GURE LEGENDS
---17LI ST OF TABLE
TABLE 1.ANTIPROLIFERATIVE EFFECT OF EEAD IN C6CELLS---7
LI ST OF FI GURES
Fig.1.CytotoxiceffectsofEEAD inC6cells---19
Fig.2.FragmentationofinternucleosomalDNA byEEAD inC6cells---20
Fig.3.Proteolyticcleavageofprocaspase-3,procaspase-7andprocaspase-9 byEEAD treatmentinC6cells---21
Fig.4.Activationofcaspase-3/-7byEEAD treatmentinlivingC6cells---22
ABSTRACT
Induction ofApoptosis
by Angel
i cadecur si vaExt
ractin GliomaCellsCho,Seon-Ho
Advisor:Prof.Kim,DoKyung,Ph.D.
DepartmentofBiodentalEngineering,
GraduateSchoolofChosun University
The Angelica decursiva has been used in Korean traditionalmedicine as an antitussive,ananalgesic,anantipyreticandacoughremedy.However,ithasbeen completely unknown its anti-tumor activity.In this study,therefore,we have examined the cytotoxic activity of ethanolextract of Angelica decursiva root (EEAD),and the mechanism ofcelldeath exhibited by EEAD in C6 ratglioma cells.
The cytotoxic effect of EEAD on cell growth inhibition in C6 cells was examined using MTT assay. And the cell death mechanism by EEAD was
examined using DNA fragmentation analysis, immunoblotting and caspase activationmeasurement.
Treatment of EEAD in C6 cells induced the apoptotic cell death in a concentration- and a time-dependentmanneras determined by MTT assay and DNA fragmentation analysis.The proteolytic processing ofcaspase-3,-7 and -9 wasincreasedby EEAD treatmentin C6cells.In addition,activationofcaspase-3 and-7wasdetectedinlivingC6cellsbyfluorescencemicroscopy.
TheseresultssuggestthattheEEAD caninducethesuppressionofcellgrowth andcellapoptosisin C6ratgliomacells,andthatitmay havepotentialproperties foranti-tumordrugdiscovery.
---
KEY WORDS: anti-tumortherapy,apoptosis,celldeath,EEAD,gliomacells
I .I NTRODUCTI ON
In recentyears,there has been a globaltrend toward the use ofnatural substances present in fruits, vegetable, oilseeds and herbs as medicine and functional food. Several of these substances, such as Taxol, Oncovin and captothecin,are shown to have potentialvalues as tumor chemo-preventive or therapeutic agents within the human body.1-4Mostofthese bioactive substances exerttheirtumorchemotherapeuticactivity by blocking cellcycleprogression and triggering apoptotic celldeath.1-4Therefore,induction ofapoptosis in tumorcells hasbecomeanimportantindicatorofthetumortreatmentresponseinemployinga plant-bioactivesubstancetoreduceandcontrolhumanmortalityduetotumor.5,6
Apoptosis, which is a major way of programmed cell death, plays an important role in the regulation of tissue development and homeostasis in eukaryotes.7-9Apoptosis may occurvia a death receptor-dependentextrinsic ora mitochondria-dependentintrinsicpathway andapoptosisisinducedby treatmentof chemotherapeuticagents.10,11
Thereareseveralmedicinalplantsthatareconsidered to possesssignificant anti-tumoractivity.12-16Angelicadecursiva,oneofKorean traditionalmedicine,has been used mainly as a folk remedy for treatment of antitussive, analgesic, antipyretic and a cough.However,the anti-tumoractivity ofAngelica decursiva wasnotreportedatall.
Gliomaisthemostcommontypeofmalignantbraintumorsandaccountsfor 40–50% of primary brain neoplasms.17 Despite advances in microsurgical
techniques,radiotherapy and chemotherapy,there has been little improvementin the clinicaloutcome ofpatients suffering from these kinds oftumors.18 In this study,therefore,weexamined theeffectofethanolextractofAngelicadecursiva root(EEAD)oncellgrowthandthemechanism ofcelldeath elicitedby EEAD in C6ratgliomacells.
I I .MATERI ALS AND METHODS
1.Materials
TheC6 ratglioma cells wereprovided by American TypeCulture Collection (ATCC,Rockville,MD,USA).3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)waspurchasedfrom Sigma(StLouis,MO,USA).Anti-caspase-3, anti-caspase-7 and anti-caspase-9 antibodies were purchased from CellSignaling Technology, Inc. (Danvers, MA, USA). Cell-permeable fluorogenic substrate PhiPhiLux-G1D2waspurchasedfrom OncoImmunin,Inc.(Gaithersburg,MD,USA).
2.Plantmaterialandextractpreparation
Dried rootpartsofAngelicadecursiva werepurchased from Jeonnam herbal medicinefarmer'scooperative,RepublicofKorea.Thebotanicalidentification was madeby Prof.Su-In Cho,SchoolofOrientalMedicine,Pusan NationalUniversity, Republic ofKorea.The roots were ground with a Wiley millto pass a 1-mm screenandwereextractedwith95% ethylalcohol(EtOH)at40℃ for5hours.The extractwas then filtered through a Advantec No.1 filter paper.The collected filtratewasdried by evaporation undervacuum at40℃ using arotary evaporator (N-1000V-W, Eyela, Japan). After evaporation, the concentrated extract was freeze-driedat-40℃ for3daysandstoredinarefrigeratorat2℃ untilused.
3.Cellcultures
The C6 rat glioma cells were grown in the Dulbecco's Modified Eagle’s Medium (DMEM)supplemented with 10% FBS and theappropriateconcentrations ofantibiotics(100U/mlpenicillinand100㎍/mlstreptomycin).19TheC6cellswere maintained as monolayers in plastic culture plates at 37oC in the humidified atmospherecontaining5% CO2.
4.Inhibition ofcellgrowth (MTT assay)
The cellviability testwas performed according to the previously described methodwithminormodifications.20,21Thecellswereseededataconcentrationof5 X 103cells/wellin 24-wellplates.After24 hours growth,the cells were treated with EEAD atvariousconcentrationsand incubation times.Thecellviability was assessed using MTT assay.Threeseparateexperimentswereperformed foreach concentration/exposuretimecombination.
ethanol.The DNA was resuspended in Tris-EDTA buffer,pH 8.0 containing 5 ug/mlDNase-freeRNaseandincubatedat37oC for1hr.TheDNA wasvisualized on1.5% agarosegelinthepresenceof0.5㎍/mlethidium bromide.
6.Immunoblotting
The cells were treated with 1 ㎍/ml EEAD for 0, 1 and 2 days. Immunoblotting wasperformed according tothepreviously described method with minor modifications.222222 The anti-caspase-3, anti-caspase-7 or anti-caspase-9 antibody (1:1000dilution,CellSignaling Technology,Inc.,Danvers,MA,USA)was usedastheprimaryantibody.
7.Determination ofcaspaseactivation
The activity of caspase-3/-7 was determined using the cell-permeable fluorogenicsubstratePhiPhiLux-G1D2(OncoImmunin,Inc.Gaithersburg,MD,USA), which was used according to the manufacturer’s instructions.The cells were treatedwith 1㎍/mlEEAD for24hoursandincubatedwithPhiPhiLux-G1D2.The activity of caspase-3/-7 was visualized by fluorescence microscopy (IX71, Olympus,Japan).
8.Dataanalysis
Allexperimentswereperformedin triplicate.Resultsarepresented asmean ± S.E.M.Statisticalsignificance was analyzed by using Student's t-test for two groups and oneway analysisofvarianceformulti-group comparisons.P<0.05 is consideredstatisticallysignificant.
Ⅲ.RESULTS
1.CytotoxiceffectofEEAD in C6cells
ToanalyzetheeffectofEEAD ontheviabilityofcells,thecellsweretreated with EEAD atvarious concentrations for 1,2 and 3 days,and then the MTT assaywasperformed.From 0.01to300㎍/mltreatmentofEEAD,theinhibitionof C6 cellgrowth depended on the EEAD treatmenttime (Fig.1).When the cells weretreatedwithEEAD (0.01-300㎍/ml),EEAD inhibitedtheproliferationofC6 cellsin a dose-dependentmanner(Fig.1).TheIC50valuesofEEAD on thecell viabilityareshowninTABLE 1.
TABLE 1. ANTIPROLIFERATIVE EFFECT OF EEAD IN C6CELLS
days IC50(㎍/ml)
1day > 300
2days 1.05± 0.113
3days 0.19± 0.026
TheIC50valuesrepresentthemean± S.E.M.forthreeexperiments.
2.DNA fragmentation
Increased cellularapoptosis is only one among severalpossible mechanisms involved in reduced cellproliferation.To determine if apoptosis is indeed the underlying mechanism forthe reduced cellproliferation we had observed,the C6 cellstreatedwith EEAD weresubjected toDNA fragmentation.Asshown in Fig.
2,theformation ofDNA ladderin theC6 cellstreated with 1㎍/mlEEAD was observedinatime-dependentmanner.
3.Activation ofcaspasesby EEAD
Thelevelsofprocaspase-3,procaspase-7andprocaspase-9wereexaminedby immunoblotting and thelevelsofprocaspase-3andprocaspase-7weredetectedby fluorescence microscopy using a selective fluorogenic substrate since caspase-3, caspase-7 and caspase-9 are effectorcaspases ofapoptotic celldeath.Treatment with 1 ㎍/mlEEAD significantly promoted proteolytic cleavages ofprocaspase-3,
Ⅳ.DI SCUSSI ON
Recently,scientific attentions increased to orientalmedicine forthe discovery ofnoveldrugsincluding anti-tumoragents.12-16Chemotherapeuticdrugsareknown to induce cytotoxicity in tumor cells through diverse mechanisms, in which signaling events play an important role depending upon the cell type and stimulus.23,24Thereisaneedtofindnew anti-tumordrugsthatcankillcancerous cellswith minimaltoxicity.The Angelica decursiva has been used asa material formedicaltreatmentinKoreantraditionalmedicine.However,itsanti-tumoreffect has been unknown atall.The main goalofthis study was to investigate the effectofEEAD on cellgrowth andthecelldeath mechanism by EEAD in C6rat gliomacells.
InMTT assay,EEAD inhibitedgrowthofC6cellsinaconcentration- anda time-dependentmanner (Fig.1).This corresponded with the results ofseveral extracts(Echinacearoot,Toonasinensis,Willow bark)thathaveanti-tumoreffects viathesuppression oftumorcellgrowth in aconcentration-dependentmanner.25-27 Therelatively lowerconcentration (1 ㎍/ml)ofEEAD was enough to inhibitthe C6 cellgrowth compared with other extracts.25-27 These results speculated that EEAD has specific cytotocity fortumorcells and potentialvalue foranti-tumor drugdiscovery.
Apoptosisisan importantway tomaintain cellularhomeostasisbetween cell division and celldeath.7-9And,theinduction ofapoptosisin tumorcellsisoneof
useful strategies for anti-tumor drug development.28 So, many studies were performed forscreening ofapoptosisincluding extractsfrom plants.In thisstudy, treatmentwith EEAD induced internucleosomalDNA fragmentation in C6 cells, suggesting apoptoticcelldeath(Fig.2).TheseresultsindicatedthatEEAD inhibits thegrowthofthesecellsbyactivatingcellapoptosis.
Theactivation ofafamily ofintracellularcysteineproteases,called caspases, is known to play an importantrole in the initiation and execution ofapoptosis induced by variousstimuli.29,30Among thecaspasesidentified in mammalian cells, caspase-3,caspase-7 and caspase-9 may serve as effectorcaspases ofapoptotic celldeath.29-31 Caspase-3,caspase-7 and caspase-9 are synthesized as inactive proenzymes (ofsizes 32 kDa,35 kDa and 47 kDa,respectively),which require proteolytic activation.29-31 Our results show that high levels of procapase-3, procapase-7 and procapase-9 were presentin EEAD-untreated C6 cells,and the amountofprocaspase-3,procapase-7andprocaspase-9wasdecreasedafterEEAD treatmentin the C6 cells (Fig.3).In addition,the activity ofcaspase-3/-7 was increased by EEAD treatmentin C6 cells compared with DMSO treatmentas a control(Fig.4).These results suggested thatEEAD induces apoptotic celldeath
growth.However,to elaborate thisnascentpossibility,furtherinvestigation ofits activity including in vivo and purification of bioactive compounds is now in progress.
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4. MukherjeeAK,BasuS,SarkarN,GhoshAC:Advancesincancertherapywith plantbasednaturalproducts.CurrMedChem 2001;8:1467–1486.
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8. HengartnerMO:Thebiochemistryofapoptosis.Nature2000;407:770–776.
9. Kaufmann SH,HengartnerMO:Programmed celldeath:aliveand wellin the new millennium.TrendsCellBiol2001;11:526–534.
10. KaufmannSH,Earnshaw WC:Inductionofapoptosisby cancerchemotherapy.
ExpCellRes2000;256:42–49.
11. Reed JC:Apoptosis-regulating proteinsastargetsfordrug discovery.Trends MolMed2001;7:314–319.
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13. LeeSM,LiML,TseYC,Leung SC,LeeMM,TsuiSK:Paeoniae Radix,a chineseherbalextract,inhibithepatomacellsgrowthbyinducingapoptosisina p53independentpathway.LifeSci2002;71:2267–2277.
14. ChengYL,LeeSC,LinSZ,Chang WL,ChenYL,TsaiNM,LiuYC,TzaoC, Yu DS,Harn HJ:Anti-proliferativeactivity ofBupleurum scrozonerifolium in A549human lung cancercellsinvitroandinvivo.CancerLett2005;222:183–
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15.Park DI,LeeJH,Moon SK,Kim CH,LeeYT,Cheong J,ChoiBT,ChoiYH:
Induction ofapoptosisand inhibition oftelomeraseactivity by aqueousextract from Platycodon grandiforum in human lung carcinama cells.PharmacolRes 2005;51:437–443.
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MethanolicextractofPereskiableo(Kunth)DC.(Cactaceae)inducesapoptosis inbreastcarcinama,T47-D cellline.JEthnopharmacol2005;96:287–294.
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Ⅵ.FI GURE LEGENDS
Fig.1. CytotoxiceffectsofEEAD in C6cells.
TheC6cellsweretreatedwith0,0.01,0.03,0.1,0.3,1,3,10,30,100and 300 ㎍/mlEEAD for1 day (filled circle),2 days (filled square)and 3 days(filled triangle).Cellviabilitiesweredetermined by theMTT assay.
Thepercentageofcellviability wascalculated asaratioofA570nm of EEAD treatedcellsanduntreatedcontrolcells.Eachdatapointrepresents the mean ± SEM for three experiments.**P<0.01 vs. control and
***
P<0.001vs.control(thecontrolcellsweremeasured in theabsenceof EEAD treatment).
Fig.2. FragmentationofinternucleosomalDNA by EEAD in C6cells.
The cells were treated with 1 ㎍/mlEEAD for 0,1 and 2 days and nuclearDNA wassubjectedtoagarosegelelectrophoresis.
Fig.3. Proteolyticcleavageofprocaspase-3,procaspase-7andprocaspase-9 by EEAD treatmentinC6cells.
Activity ofprocaspase-3,procaspase-7 and procaspase-9 by EEAD was measuredinC6cells.Thecellsweretreatedwith1㎍/mlEEAD for0,1
and2days.Thecelllysatewaspreparedandanalyzedbyimmunoblotting asdescribedin“MATERIALS AND METHODS”.
Fig.4. Activation ofcaspase-3/-7by EEAD treatmentinliving C6cells.
The cells were treated with 1 ㎍/mlEEAD for 24 hours and added specific cell-permeable substrate Phiphilux G1D2.Active ofcaspase-3/-7 wasvisualizedbyfluorescencemicroscopy.
Ⅶ.FI GURES
Fi g.1.
EEAD treatment (1 ㎍/㎖) Time (days)
M 0 1 2
EEAD treatment (1 ㎍/㎖) Time (days)
M 0 1 2
Ⅶ.FI GURES
EEAD treatment (1 ㎍/㎖)
Time (days) 2
0 1
Caspase-3 Caspase-7 Caspase-9
β -actin 35 KDa →
35 KDa → 37 KDa → 47 KDa →
EEAD treatment (1 ㎍/㎖)
Time (days) 2
0 1
Caspase-3 Caspase-7 Caspase-9
β -actin 35 KDa →
35 KDa → 37 KDa → 47 KDa →
Ⅶ.FI GURES
Fi g.3.
Caspase-3/-7 activity by EEAD treatment
Mock (DMSO treated)
Bright Field Caspase-3/-7 activity
EEAD treated
Bright Field Caspase-3/-7 activity
Caspase-3/-7 activity by EEAD treatment
Mock (DMSO treated)
Bright Field Caspase-3/-7 activity
EEAD treated
Bright Field Caspase-3/-7 activity
Ⅶ.FI GURES
ABSTRACT i n KOREAN
Angel i cadecursi va추출물에 의한 신경교종 세포의 Apopt osi s유도
조 선 호
조선대학교 대학원 치의생명공학과 ( 지도교수:김 도 경)
우리나라에서 오래전부터 의약품으로 사용되어져 온 전통 약제 중 하나인 Angelica decursiva는 민간요법으로 진해제,진통제,해열제 또는 기침약 등으로 사용되어져 왔 다.그러나 Angelicadecursiva의 항암효과에 관한 자료는 거의 없다.본 연구는 흰쥐 신경교종 세포 C6 세포주를 이용하여 Angelicadecursiva의 암세포 성장억제에 미치 는 효과와 세포성장 억제기전을 밝히기 위해,C6세포주에서 Angelicadecursiva의 에 탄올 추출물(EEAD)을 이용하여 MTT 분석, DNA fragmentation 분석 및 immunoblotting등을 시행하였다.
EEAD는 C6세포의 성장을 시간과 농도에 의존적으로 억제하였다.EEAD를 처리 한 C6 세포주 실험군에서 DNA fragmentation 현상을 확인할 수 있었다.C6세포에
EEAD를 처리한 실험군에서 procaspase-3, procasapse-7 및 procaspase-9의 proteolyticcleavage현상을 확인할 수 있었다.C6세포에 EEAD를 처리한 실험군에 서 caspase-3/-7의 활성화를 확인할 수 있었다.
본 연구의 결과로 Angelicadecursiva에탄올 뿌리 추출물 EEAD는 흰쥐 신경교종 세포 C6세포주의 apoptosis를 유도하여 암세포 성장을 억제시키는 것으로 사료된다.
또한 본 연구의 결과로,EEAD를 이용한 암세포의 성장억제에 관한 하나의 방향을 제 시할 수 있을 것으로 사료된다.
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논문제목
한글:Angelicadecursiva추출물에 의한 신경교종 세포의 Apoptosis유도
영문: InductionofApoptosisbyAngelicadecursivaExtract inGliomaCells
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4.저작물에 대한 이용기간은 5년으로 하고,기간종료 3개월 이내에 별도의 의사 표시가 없을 경우에는 저작물의 이용기간을 계속 연장함.
5.해당 저작물의 저작권을 타인에게 양도하거나 또는 출판을 허락을 하였을 경우에는 1개월 이내에 대학에 이를 통보함.
6.조선대학교는 저작물의 이용허락 이후 해당 저작물로 인하여 발생하는 타인에 의한 권리 침해에 대하여 일체의 법적 책임을 지지 않음
7.소속대학의 협정기관에 저작물의 제공 및 인터넷 등 정보통신망을 이용한 저작물의 전송ㆍ출력을 허락함.
동의여부 : 동의(O ) 반대( ) 2010년 2월 일
저작자: 조 선 호 (서명 또는 인)
