花青素相关论文.docx

花青素相关论文.docx

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花青素相关论文

FoodandChemicalToxicology43（2005）1557–1566

Eectsofanthocyanidinontheinhibitionofproliferationandinductionofapoptosisinhumangastricadenocarcinomacells

Ping-HsiaoShih,Chi-TaiYeh,Gow-ChinYen*

DepartmentofFoodScience,NationalChungHsingUniversity,250KuokuangRoad,Taichung40227,Taiwan

Received15December2004;accepted4May2005

Abstract

Anthocyaninsarenaturallyoccurringreddishpigmentsthatabundantinfruitsandvegetables.Toinvestigatethemechanisticbasisfortheanti-tumorpropertiesofanthocyanins,fiveaglycone（cyanidin,delphinidin,malvidin,pelargonidin,andpeonidin）andfourglycosylated（cyaniding-3-glucoside,malvidin-3-glucoside,pelargonidin-3-glucosideandpeonidin-3-glucoside）anthocyaninswereusedtoexaminetheireectsoncellcycleprogressionandinductionofapoptosisinhumangastricadenocarcinomaAGScells.Thedatafromcellviabilityassayshowedthatmalvidinexhibitedthemostpotentanti-proliferationeectonAGScellsinatime-anddose-dependentmanner（P<0.05）.ThiseventisaccompaniedthearrestofAGScellsattheG0/G1phasebymalvidinatthetestedconcentrationsof0–200lM.CellularuptakeofanthocyaninandanthocyanidinwasconfirmedbyHPLCanalysisandtheintracellularaccumulationofmalvidin（24.9±1.1lM/mgprotein）wasobservedwhentreatmentofAGScellswithmalvidinfor12h.Inaddition,anaccumulationofAGScellsinsub-G1phase（20%and30%increasefor100and200lMofmalvidin,respectively）wasobservedaswellasbytheappearanceofafractionofcellswithananeudiploidDNAcontent.Theoccurrenceofapoptosisinducedbymalvidinwasconfirmedbymorphologicalandbiochemicalfeatures,includingapoptoticbodiesformation,caspase-3acti-vationandpoly（ADP-ribose）polymeraseproteolysis.Furthermore,themitochondrialmembranepotentialofapoptoticcellsaftertreatmentwithmalvidinwassignificantlylostandresultedintheelevationofBax/Bcl-2ratiofor1.6-foldagainstcontrolfor

100lMtreatment.Inaddition,themalvidintreatmentsignificantlyincreasedthep38kinaseexpressionandinhibitedtheERKactiv-ity,andtheeectsofmalvidinoncaspase-3activationwereblocked,respectively,bytheERKandp38inhibitors.Thesefindings

suggestthatgrowthinhibitionandcytotoxicityofAGScellsbymalvidinisinvolvedintheinductionofapoptosisratherthannecrosis.

2005ElsevierLtd.Allrightsreserved.

Keywords:

Anthocyanidin;Apoptosis;AGScells;MAPKpathway;Bcl-2family

1.Introduction

Followingthechangeofdietaryhabitsandtheexces-siveprocessoffood,thetendencyofgastrointestinaldis-easeisontherise.Gastriccancerisakindofgastrointestinal（GI）tractcancerthatistheleadingcauseofcancer-relatedmortalityintheworldandapproximately90%ofstomachcancersareadenocarci-

*Correspondingauthor.Tel.:

+886422879755;fax:

+886422854378.

E-mailaddress:

gcyen@dragon.nchu.edu.tw（G.-C.Yen）.

0278-6915/$-seefrontmatter2005ElsevierLtd.Allrightsreserved.doi:

10.1016/j.fct.2005.05.001

nomas（KelleyandDuggan,2003）.Studieshaveshownthatahighintakeofsmoked,salted,nitratedfoodsandcarbohydrates,butalowintakeofvegetables,fruits,andmilks,arelinkedtocancerincidence.Thesedietshavebeenshowntosignificantlyincreasetheriskforstomachcancer（Serafinietal.,2002）.Epidemiologicalstudieshaveprovidedconvincingevidencethatdietaryfactorscanmodifytheprocessofcarcinogenesis,includ-inginitiation,promotionandprogressionofseveraltypesofhumancancer（Ray,2005）.Theoccurrenceofgastrointestinal（GI）cancershasincreasedstrikinglyduringthepastdecade.Despiteadvancesinearly

1558P.-H.Shihetal./FoodandChemicalToxicology43（2005）1557–1566

diagnosisandtreatmentmodalities,thesideeectsofchemicaldrugsandrecurrencearestilltheproblems.Therefore,thedevelopmentofchemotherapeutic/che-mopreventiveagentsforgastriccancerisimportantforreducingthemortalitycausedbythisdisease.

Anthocyaninsareglycosidesofanthocyanidinsuniver-sallyassociatedwithattractive,colorful,andflavorfulfruits.Recently,therehasbeenaresurgenceofinterestinanthocyaninsduetotheirpotentialbiologicalandphar-macologybenefit,suchas:

antioxidant（Moyeretal.,2002）,anti-inflammatory（SubarnasandWagner,2000）,reducingtheriskofcardiovasculardiseases（WangandMazza,2002）,andanti-tumorproperties（Katsubeetal.,2003）.Animalstudieshavealsodemonstratedthatfeedingwithanthocyanin-richextractprotectedagainsttert-butylhydroperoxide-inducedhepatictoxicity（Wangetal.,2000）anddecreaselipidperoxidationandDNAdamageinvitaminE-depletedrats（Ramirez-Tortosaetal.,2001）.Morerecently,anthocyaninshavebeenshowntobeaneectivechemopreventiveagentagainst1,2-dimethyhydrazine-and2-amino-1-methyl-6-phenylimi-dazo[4,5-b]pyridine-inducedmammarycarcinogenesisinrats（Hagiwaraetal.,2002）.Inaddition,anthocyaninscanbedirectlyabsorbedanddistributedtotheblood（Tsudaetal.,1999）,andtobeincorporatedincellcultures,bothintheplasmamembraneandinthecytosol（Youdimetal.,2000）.Extensivestudiesindicatethatanthocyaninshavestrongfreeradicalscavengingandantioxidantactivi-ties（Wangetal.,1997）,suggestingthattheyplayanimportantroleinpreventingagainstmutagenesisandcar-cinogenesis（Omenn,1995）.Anthocyaninsalsoshowedinhibitoryeectsonthegrowthofsomecancercells（Kameietal.,1995）.Ourrecentstudyhasreported（YehandYen,2005）thattheinductionofapoptosisbytheanthocyanidinsthroughregulationofBcl-2geneandactivationofc-junn-terminalkinasecascadeinhepatomacells.However,theresearchconcerningtheprotectiveeectofanthocyaninsagainstgastricadenocarcinomaislimited;thus,theobjectiveofthepresentstudywastodeterminewhetheranthocyaninswouldbeusefulinthepreventionofhumangastricadenocarcinomacar-cinogenesis.Inthepresentstudy,humanculturedgastricadenocarcinoma（AGS）cellswerechosenforstudy.

AGScellslinehasbeenshowntogrowinathymicmiceandtohavethesamehistochemicalandcytologicalcharacteristicsasthespecimentakenfromthepatient.Itisimportanttocharacterizehumantumorcellsinvitrointhisdetailedmanner,sincetheyserveasexcellentmodelsystemsforotherstudiesinvolvingtheheterogeneousre-sponsestoanticancerdrugs（Barrancoetal.,1983）,andrecentlythiscelllinehasbeenwidelyusedasamodelsys-temforevaluatingcancercellapoptosis（Liuetal.,2003）.

Apoptosisisadefinedtypeofcelldeathanddiersfromtraditionalcelldeath,necrosis.Manyrecentstud-ieshaveindicatedthatanticancerdrugsorcancerche-mopreventiveagentsactthroughtheinductionof

apoptosistopreventtumorpromotion,progression,andtheoccurrenceofcellularinflammatoryresponsesotherthannecrosis.Apoptosisisalsoagene-directedformofcelldeathwithwell-characterizedmorpholo-gicalandbiochemicalfeatures（BrownandAttardi,2005）.Initiationofapoptosisappearstobeacommonmechanismofmanycytotoxicagentsusedinchemother-apy.Therefore,apoptosisinducingagentsareexpectedtobeidealanticancerdrugs.Inaddition,someflavo-noids,suchasquercetin,apigenin,andphloretin,inhibitcancercellgrowththroughtheinductionofapoptosis（Wangetal.,1999）.Therefore,understandingthemech-anismofapoptosishasimportantimplicationsinthepreventionandtreatmentofmanydiseases.

Inthisstudy,weinvestigatedtheinductionofapop-tosisbyfivetypicalanthocyaninsandtheirrelativeantho-cyanidinsinhumangastricadenocarcinomacells.Inaddition,themolecularmechanismsoftheapoptoticeectsinducedbymalvidinwerealsodetermined.

2.Materialsandmethods

2.1.Materials

Anthocyanidins[cyanidinchloride,delphinidinchloride,malvidinchloride,pelargonidinchloride,peonidinchloride],anthocyanins[cyanidin-3-O-gluco-sidechloride,malvidin-3-O-glucosidechloride（oenin）,pelargonidin-3-O-glucosidechloride,andpeonidine-3-O-glucosidechloride]wereobtainedfromExtrasynthese（Genay,France）.HumanadenocarcinomaAGScellswerepurchasedfromtheBioresourceCollectionandResearchCenter（BCRC,FoodIndustryResearchandDevelopmentInstitute,HsinChu,Taiwan）.Goatanti-rabbitIgGpolyclonalantibodyconjugatedtoperoxidasewereobtainedfromSigmaChemicalCo.（St.Louis,MO,USA）.Anti-ERK,anti-phospho-ERK,anti-JNK,anti-phospho-JNK,anti-p38,anti-phospho-p38,anti-capsase-3,anti-poly（ADP-ribose）polymerase,andanti-b-actinantibodieswereobtainedfromCellSignalingTechnology（Beverly,MA）.Anti-Bcl-2,andanti-BaxantibodieswereobtainedfromPharmingen（SanDiego,CA）.MolecularmassmarkersforproteinswereobtainedfromPharmaciaBiotech（Saclay,France）.Polyvinyldi-fluoride（PVDF）membraneforWesternblottingwasobtainedfromMillipore（Bedford,MA,USA）.

2.2.Cellculture

TheAGScellswereculturedinHamsF-12Kmediumsupplementedwith10%heat-inactivatedfetalbovineserum（FBS）andantibiotics（100U/mlpenicillin,

100lg/mlstreptomycin）at37LCinahumidifiedatmo-sphereof5%CO2.Duringtheexperimentstheconcen-trationofFBSwereadjustedto5%.

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2.3.Cellmorphologicalobservation

Morphologyofcontrolcellsandsamples-treatedcellsweredetectedbyphasecontrastmicroscope（Nicon）.

2.4.Cellviabilityassay

CellviabilityassaywasperformedwithMTTphoto-metricanalysisandtrypanbluedyeexclusionmethod.IntheMTTassay,5·104cellswereseededin96-wellmicrotiterplate.Cellsweretreatedwithoutorwithva-riousconcen