舞茸Dfraction 通过激活BAK1基因诱导乳腺癌细胞凋亡.docx

舞茸Dfraction 通过激活BAK1基因诱导乳腺癌细胞凋亡.docx

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舞茸Dfraction通过激活BAK1基因诱导乳腺癌细胞凋亡

声明：

本文转载自JournalofMedicinalFood，即《药物强化性食品杂志》，是由美国MaryAnnLiebert出版公司出版发行的一本有关功能食品及保健食品的国际性杂志。

为方便广大读者阅读，我们对论文摘要进行了中文翻译，译文仅供参考，如有不足之处，敬请指正。

---本论文由杭州正树保健食品有限公司整理编辑

【参考译文】

Maitake（DFraction）MushroomExtractInducesApoptosisin

BreastCancerCellsbyBAK-1GeneActivation

舞茸D-fraction通过激活BAK-1基因诱导乳腺癌细胞凋亡

摘要：

根据多年经验，菇类一直被用作传统药物治疗多种疾病。

基于对舞茸固有的免疫调节和抗肿瘤特性的研究，人们进一步分离出多种具有生物活性的复合物，其中之一的舞茸D-fraction已被确认具有削弱肿瘤细胞生存能力的功能。

本研究验证了舞茸D-fraction对人类乳腺癌细胞（MCF7）生存能力和细胞凋亡的影响。

这些细胞分别用浓度为18μg/ml,36μg/mL,91μg/m,183μg/mL,367μg/mL的舞茸D-fraction进行处理,另有一组未经处理的细胞作为对照组，实验持续24小时。

根据舞茸D-fraction的剂量，处理过的细胞生存能力（根据3-（4,5-二甲基）-5-（3-羧甲基苯环）-2-（4-硫基苯）-2H-四唑盐复合物检测法）依次有所降低。

细胞凋亡的统计数依剂量都有明显上升（终端脱氧核糖核酸末端转移酶介导的三磷酸脱氧尿嘧啶缺口端标记法）。

细胞经D-fraction恒温培养后，进行微阵列检测，结果显示能直接体现细胞凋亡途径的两种蛋白质——BAK-1基因和细胞色素C的转录产物上升。

反转录聚合酶链反应也证实了微阵列检查的结果：

BAK-1是最过表达的基因之一。

这诸多发现更加肯定了舞茸D-fraction对乳腺癌细胞凋亡的影响，并进一步凸显了释放于细胞质的细胞色素C的干预作用。

释放在细胞质中的细胞色素C，是细胞凋亡途径中另一个主要因素，其产量在经舞茸D-fraction培养后有所增加，且随D-fraction浓度的增加而上升。

这一发现显示，舞茸D-fraction的作用在于诱导线粒体功能障碍。

因此，阐明舞茸D-Fraction发挥作用的分子机理对于癌症预防和治疗方法的发展至关重要。

关键词：

细胞凋亡，BAK-1,乳腺癌，细胞色素C，舞茸，蘑菇

论文原文

JournalofMedicinalFood

2011,1-10

Maitake（DFraction）MushroomExtractInducesApoptosis

inBreastCancerCellsbyBAK-1GeneActivation

RaquelSoares,ManuelaMeireles,1AnaRocha,1AnaPirraco,1DiegoObiol,2ElianaAlonso,2

GiselaJoos,2andGabrielaBalogh2

1DepartmentofBiochemistry,FacultyofMedicine,UniversityofPortoFoundation,Porto,Portugal.

2CenterforScientificandTechnicalInvestigation,Cerzos-Conicet,BahıáBlanca,Argentina.

ABSTRACTFormanyyearsmushroomshavebeenusedempiricallyintraditionalmedicinetotreatseveraldiseases.Studyofthemaitakemushroom,withitsimmunomodulatoryandantitumoralproperties,hasledtotheisolationofseveralbioactivecompounds.Oneofthese,Dfraction,isknowntoreducetumorcellviability.ThisstudyexaminedtheeffectofisolatedDfractiononviabilityandapoptosisofhumanbreastcancercells（MCF7）.Thesecellsweretreatedwithmaitake（Dfraction）extractat18μg/mL,36μg/mL,91μg/mL,183μg/mL,or367μg/mLorwereleftuntreated（control）for24hours.MCF7incubationwiththemaitakeextractresultedindecreasedcellviability[3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophe-nyl）-2H-tetrazoliumassay]inadose-dependentmanner.Apoptosiswasstatisticallysignificantlyincreasedinadose-dependentmannerateveryconcentrationtested（terminaldeoxynucleotidyltransferase-mediateddeoxyuridinetriphosphatenick-endlabelingassay）.UponincubationwithDfraction,amicroarrayassayrevealedupregulationofBAK-1andcytochromectranscripts,2proteinsdirectlyinvolvedintheapoptoticpathway.Reversetranscriptasepolymerasechainreactionstudiesconfirmedthesefindings;BAK-1wasoneofmostoverexpressedgene,asobservedbymicroarrayassay.ThesefindingsconfirmtheapoptoticeffectofmaitakeDfractioninbreastcancercellsandfurtherhighlighttheinvolvementofcytochromecreleasetothecytoplasm.Cytoplasmicreleaseofcytochromec,anotherplayerintheapoptoticpathway,wasalsoincreasedafterincubationwithDfractioninadose-dependentmanner.Thisfindingindicatesthattheeffectofthiscompoundinvolvesmitochondrialdysfunction.TheidentificationofthemolecularmechanismsbywhichDfractionexertsitseffectsiscrucialforthedevelopmentofpreventiveandtherapeuticstrategiesforcancer

.

KEYWORDS:

·apoptosis·BAK-1·breastcancer·cytochromec·maitake·mushroom

INTRODUCTION

Breastcanceristheleadingcauseofdeathfromcancerinwomen,themostcommoncanceramongwomenworldwide,andthesecondmostfrequentcancerintheworld.Itsincidenceisstillincreasing,withatotalnumberofcasesof1,050,100in2002comparedwith572,100in1980.Breastcancercauses370,000deathsannually,representing13.9%ofcancerdeathsinwomen,mainlyintheindustrializedcountries.1Whileresearcherssearchforthecauseofthedisease,aneffectiveandrapidtreatmentismandatoryforthesepatients.

Maitakemushroom（Grifolafrondosa）isagiantmushroomindigenoustonorthernJapanthathasbeenusedbyJapaneseherbalistsformanyyears.MostresearchonmaitakehasfocusedontheuseofmaitakeDfractionfortreatingvariousmalignancies.ThebioactiveDfractionextractedfrommaitakeisaprotein-boundpolysaccharidecompoundandispreparedbyastandardizedproceduredevelopedbyMaitakeProductsInc.Amongthevariousmaitakefractionsthathavebeenprepared,Dfractionwasfoundtobethemostpotentforenhancingtheimmunesystemviaoraladministrationorinjection（bothrouteswereeffective）,leadingtothehighestreductionintherateofcancerproliferation.2MaitakeDfractionhasbeenreportedtoexertitsantitumoreffectintumor-bearingmicebyenhancingtheimmunesystemthroughactivationofmacrophages,Tcells,andnaturalkillercells.3Inapreviousstudy,thecombinationofimmunotherapywiththemaitakeDfractionandchemotherapysuggestedthattheDfractionmightdecreasethesizeoflung,liver,andbreasttumorsinpatientswithcancer.2,4,5In1998,theFoodandDrugAdministrationgrantedMaitakeProducts,Inc.,aninvestigationalnewdrugapplicationtoconductaphaseIIpilotstudyusingmaitakeDfractionamongpatientswithadvancedbreastandprostatecancers.TheseongoingstudiesareevaluatingtheimmunestimulatoryeffectofDfractionontumorsize,immuneassays,clinicalsymptoms,andpatientqualityoflife.

ThepurposeofthecurrentstudywastoinvestigatethedirecteffectofmaitakeDfractiononbreastcancerMCF7cells.WefoundthatincubatingMCF7cellswithmaitakeDfractionsignificantlydecreasedcellviabilityandinducedcelldeath.GenomicanalysisusingcomplementaryDNA（cDNA）microarraysrevealedtheupregulationof22pro-apoptoticand7anti-apoptoticgenescomparedwithuntreatedMCF7cells（control）.BAK-1transcriptatthemessengerRNAlevelwasfoundtobeupregulated.AfterincubationwithDfraction,cytoplasmicreleaseofcytochromec,anotherplayerintheapoptoticpathway,wasalsoincreasedinadose-dependentmanner.

MATERIALSANDMETHODS

BioactivemaitakeDfraction

ThebioactiveDfractionwasextractedfrommaitakemushroom,correspondingtotheprotein-boundpolysaccharidecompound,andwaspreparedbyastandardizedproceduredevelopedbyMaitakeProducts,Inc.

Cellcultures

ThehumanbreastcancerMCF7celllinewasobtainedfromtheAmericanTypeCultureCollection.MCF7cellswereroutinelyculturedinEagleminimalessentialmediumcontaining10%inactivatedfetalbovineserumand1%penicillin/streptomycin.Cellculturemedia,fetalbovineserum,andpenicillin/streptomycinwerepurchasedfromInvitrogenLifeTechnologies.Cellsweregrownat37℃inahumidified5%CO2atmosphere.Inagreementwithpreviousstudies,incubationswereperformedfor24hoursinserum-freeconditions.6

LabelingandcDNAhumanmicroarrays

Weadoptedthedirectlabelingofprobeswithamine-modifiedrandomprimer,using5μgofamplifiedRNA（aaRNA）asstartingmaterial.The5μgofaaRNA（8μL）wascombinedwithamine-modifiedrandomprimer（2μg/μL,1μL）andribonucleaseinhibitor（5units/μL,1μL）.Themixwasincubatedat70℃for10minutesandthenchilledonicefor10minutes.Primer-RNAsolutionwasaddedtothereversetranscriptasemix（5×first-strandbuffer,6μL;50×amplifieddeoxyuridinetriphosphate[dUTP]/dinucleotidetriphosphate[25mMdeoxyadenosinetriphosphate,deoxyguanosinetriphosphate,anddeoxycytidinetriphosphate;15mMdeoxythymidinetriphosphate;and10mMaminoallyl-dUTP],0.6μL;dithiothreitol,0.1M,3μL;SuperscriptIIreversetranscriptase[Invitrogen/LifeTechnologies],2μL）andincubatedat42℃for2hours.ThereactionwasterminatedbyaddingEDTA（0.5M,10μL）,andtheRNAwashydrolyzedwithNaOH（1M,10μL）at65℃for30minutes.

Probepurification

ProbeswerecleanedwithaQIAquickpolymerasechainreaction（PCR）purificationkit（Qiagen）;theCy3-andCy5-labeledproductswerecombinedand30μLofwaterwasadded,followedby500μLofBufferPB.ThesampleswereappliedtoQIAquickcolumns,whichwerecentrifugedat13,000rpmfor1minute,afterwhichtheflowthroughwerediscarded.Towashthecolumns,750μLofBufferPEwasadded,columnswerespunagainfor1minute,andtheflow-throughwasdiscarded.Thewashingstepwasrepeatedoncemore,andcolumnswerespunagaintoremoveresidualethanol.Freshcollectiontubeswereplacedbeneatheachcolumn,30μLofBufferEBwasadded,andtubeswereincubatedfor1minuteatroomtemperature.Columnswerethencentrifugedat13,000rpmfor1minute,andtheelutionstepwasrepeatedonce.Eluteswerepartiallydriedinavacuumcentrifugeandthevolumeswereadjustedto23μLwithwater.

Hybridizationandwashingconditions

Thefollowingwereadded:

4.5μLof20×saline-sodiumcitrate,2μLofpoly（A）（10mg/mL）,and0.6μLof10%（wt/vol）sodiumdodecylsulfate（SDS）.Theprobeswerethendenaturedat98℃for3minutes.Theproductswerepipettedontoarrays,coverslipswereapplied,andtheslides

wereplacedinahybridizationchamber（Corning）.cDNAhumanmicroarrayscontaining25,000knowngeneswereincubatedina42℃waterbathfor16hoursandweresubsequentlywashedwith0.5×saline-sodiumcitrate,0.01%（wt/vol）SDS,followedby0.06×SSC,atroomtemperature

for10minuteseach.Slideswerespunfor5minutesat800rpm（130g）atroomtemperature.

Arrayscanningandanalysis

ArrayswerereadwithanAffymetrix428fluorescentscanner（MWGTechnologies）at10-μmresolutionandvariablephotomultipliertubevoltagesettingstoobtainthemaximalsignalintensitieswithlessthan1%（wt/vol）probesaturation.TheresultingimageswereanalyzedbyusingImaGene,version4.2,andG