肾细胞癌的新治疗方法维生素K3与舞茸Dfraction相结合诱导癌细胞凋亡.docx

肾细胞癌的新治疗方法维生素K3与舞茸Dfraction相结合诱导癌细胞凋亡.docx

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肾细胞癌的新治疗方法维生素K3与舞茸Dfraction相结合诱导癌细胞凋亡

声明：

本文摘自美国JournalofEndourology，即《内泌尿学杂志》，是微创泌尿外科学领域的主要杂志，为临床外科医生提供泌尿生殖道的良性和恶性疾病的最新治疗方法。

为方便广大读者阅读，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】

AlternativeTherapeuticApproachtoRenal-CellCarcinoma:

InductionofApoptosiswithCombinationofVitaminK3andD-fraction

肾细胞癌的替代治疗方法：

维生素K3与舞茸D-fraction相结合诱导癌细胞凋亡

ABSTRACT翻译参考

摘要

目的：

由于晚期肾细胞癌（RCC）的预测性差，因此我们研究了一种替代治疗方法，即联合使用维生素K3（VK3）和舞茸D-fraction（DF）。

VK3是一种人造维生素K的衍生物，而DF是一种菇类的生物活性提取物，这两者都被证实具有抗肿瘤活性。

因此，我们进行了体外实验来研究VK3与舞茸D-fraction联合对抗晚期肾细胞癌的效果。

材料与方法：

将ACHN细胞株（RCC）分别用不同浓度的VK3和D-fraction以及两者的混合物处理，72小时后用MTT法测定细胞生存率。

为了考察其可能存在的抗癌机制，我们对细胞周期，染色质修饰以及细胞凋亡等部分进行研究。

结果：

当VK3浓度为4μM时，可导致癌细胞生存率减少约20%，而当舞茸D-fraction浓度大于等于500μg/mL时，癌细胞生存率减少20%至45%。

然而，当VK3（4μM）与舞茸D-fraction（300μg/ml）联合使用时，癌细胞生存率降低了90%以上。

细胞周期分析表明，VK3/DF疗法会引起G1细胞周期停滞，同时伴随着p21WAF1和p27Kip1增长。

在实验过程中，组蛋白脱乙酰基酶（HDAC）也明显被灭活（约60%），这指示染色质修饰的情况。

另外，蛋白质印迹分析表明，经VK3/DF处理的细胞中出现Bax上升以及（ADP-核糖）聚合酶（PARP）活化现象，这些都会诱发癌细胞的凋亡。

结论：

VK3和DF联合导致ACHN细胞生存率显著降低，并通过p21WAF1介导的G1细胞周期停滞最终诱导细胞凋亡。

因此，VK3/DF的联合应用可能具有临床意义，它能作为一种替代疗法用于改善晚期RCC的治疗模式。

论文原文

JournalofEndourology

Volume27,Number12,December2013,pp1499-1503

AlternativeTherapeuticApproachtoRenal-CellCarcinoma:

InductionofApoptosiswithCombination

ofVitaminK3andD-fraction

MichaelDegen,MD,BobbyAlexander,MD,MuhammadChoudhury,MD,

MajidEshghi,MD,andSensukeKonno,PhD

Abstract

Purpose:

Becauseofadismalprognosisforadvancedrenal-cellcarcinoma（RCC）,analternativetherapeuticapproach,usingvitaminK3（VK3）andD-fraction（DF）wasinvestigated.VK3isasyntheticVKderivativeandDFisabioactivemushroomextract,andtheyhavebeenshowntohaveantitumoractivity.WeexaminedifthecombinationofVK3andDFwouldexhibittheimprovedanticancereffectonRCCinvitro.

MaterialsandMethods:

HumanRCC,ACHNcellline,weretreatedwithvaryingconcentrationsofVK3,DF,oracombinationofthetwo.Cellviabilitywasassessedat72hoursbyMTTassay.Toexplorethepossibleanticancermechanism,studiesoncellcycle,chromatinmodifications,andapoptosiswereconducted.

Results:

VK3aloneledtoa~20%reductionincellviabilityat4μM,whileDFaloneinduceda20%to45%viabilityreductionat≥500μg/mL.AcombinationofVK3（4μM）andDF（300μg/mL）ledtoadrastic>90%viabilityreduction,however.CellcycleanalysisindicatedthatVK3/DFtreatmentinducedaG1cellcyclearrest,accompaniedbytheup-regulationofp21WAF1andp27Kip1.Histonedeacetylase（HDAC）wasalsosignificantly（~60%）inactivated,indicatingchromatinmodifications.Inaddition,Westernblotanalysisrevealedthattheup-regulationofBaxandactivationofpoly-（ADP-ribose）-polymerase（PARP）wereseeninVK3/DF-treatedcells,indicatinginductionofapoptosis.

Conclusions:

ThecombinationofVK3andDFcanleadtoaprofoundreductioninACHNcellviability,throughap21WAF1-mediatedG1cellcyclearrest,andultimatelyinducesapoptosis.Therefore,thecombinationofVK3/DFmayhaveclinicalimplicationsasanalternative,improvedtherapeuticmodalityforadvancedRCC.

Introduction

Renal-cellcarcinoma（RCC）isthethirdmostcommongenitourinarytumorintheUnitedStates,1andnearly30%ofpatientswithRCCwillpresentwithmetastaticdisease.2,3StandardtreatmentfornonmetastaticRCCiscompleteresectionofthetumorbyeitheraradicalorpartial

nephrectomy4,5;however,20%to30%ofthesepatientswillprogresstometastaticdisease.Whileamajorityofthesepatientsinwhommetastsisdevelopsaftersurgerypresentedwithlargertumorsnecessitatingradicalnephrectomies,arecentstudysuggestedthatdistantmetastasisdevelopedin4%ofpatientswhohadpositivesurgicalmarginsafternephron-sparingsurgery,commonlyperformedlaparoscopicallyorrobotically.6Witha5-yearsurvivalrateof<10%formetastaticRCC,7thesepatientsareleftwithadismalprognosis,makingRCConeofthemostfatalgenitourinarycancers.

Unfortunately,conventionaltherapeuticmodalitiesformetastaticRCC,includingcytoreductivesurgery,radiotherapy,chemotherapy,orimmunotherapy,werenotveryeffective.3,8Newer,specificallytargetedtreatmentoptionsfordistantmetastasis,includingtyrosinekinaseinhibitors（sunitinib,etc.）,mammaliantargetofrapamycininhibitors（temsirolimus,etc.）,andmonoclonalantibodies（G250,etc.）,arepromising,butthesurvivalresultsarestillpoor.9ThesereasonsthuspromptedustoexploreanoveltreatmentforpatientswithmetastaticRCC.

Wehavebeenworkingwithvitaminsandnaturalagents/substancesasanalternativeapproachtosuchmetastaticRCCcases.Particularly,wewereinterestedinvitaminK3（menadione;VK3）asapotentialtherapeuticagent.VK3isasyntheticderivativeofnaturallyoccurringfat-solublevitaminK（VK）10andhasbeenshowntohaveanantitumoreffectonseveralcancercells.11-13BecauseVK3requiresarelativelyhighdosetobeeffective,11combinationsofVK3andotheragentshavebeenpostulatedtoimproveitsefficacyatalowerdose.

Atthesametime,wehavealsobeenstudyingthebioactiveextractfrommaitake（Grifolafrondosa）,knownasD-fraction（DF）.DFhasbeenextensivelystudiedforthepast30yearsandhasbeenshowntohaveimmunomodulatoryandantitumoractivities.14–16Forinstance,antitumoractivityofDFhasbeendemonstratedintumor-bearingmicethroughactivationofvariousimmuneeffectors.14,15Inaddition,inductionofapoptosisbyDFwasalsoreportedinbreastcancercells.16DFthusappearstobeaninterestingandpromisingnaturalagentthatcouldbeusedincancertreatment.

ThepurposeofthisstudywastoinvestigatewhetheracombinationofVK3andDFwouldexhibittheimprovedandenhancedanticancereffectonRCCinvitro,asanalternativetherapeuticapproach.Thesestudieswereperformedfocusingmainlyoncellviability,cellcycle,chromatinmodifications,andapoptosis.

MaterialsandMethods

Cellculture

ThehumanrenalcarcinomaACHNcells（withaggressivegradeIVproperty）weremaintainedinRPMI1640mediumcontaining10%fetalbovineserum,penicillin（100units/mL）,andstreptomycin（100μg/mL）.Forexperiments,ACHNcellswereseededattheinitialcelldensityof2×105cells/mLinsix-wellplatesorT-75flasksandculturedwithvaryingconcentrationsofVK3,DF,ortheircombinations.Cellviabilitywasthenassessedat72hoursby3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazoliumbromide（MTT）assay.

Cellviabilityassay（MTTassay）

CellviabilitywasdeterminedbyMTT（3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide）assayfollowingtheprotocolofthevendor（Sigma-Aldrich,St.Louis,MO）.Briefly,attheharvesttime,MTTreagent（0.5mg/mL）wasaddedtoeachwellinthesix-wellplate,followedby3-hourincubationat37℃.AfterremovingMTTreagent,dimethylsulfoxidewasaddedtoeachwellandabsorbanceofformazansolutionwasreadonamicroplatereader.Cellviabilitywasthenexpressedaspercentage（%）ofviablecellsrelativetothecontrolreading（100%）.

Cellcycleanalysis

ABDFACscanflowcytometer（FranklinLakes,NJ）,equippedwithadoublediscriminationmodule,wasusedforcellcycleanalysis.Approximately1×106cellswerere-suspendedin500μLofpropidiumiodidesolutionandincubatedatroomtemperaturefor1hour.Tenthousandnuclei

wereanalyzedforeachsample,quantifiedincellcyclecompartments,andestimatedascellcyclephasefractions.

Histonedeacetylase（HDAC）assay

HDACactivitywasmeasuredusingtheEpigenaseHDACAssayKitfollowingtheprotocolofthemanufacturer（Epigentek,Farmingdale,NY）.Briefly,10μgofnuclearextractsfromeachsamplewereaddedtothecoatedmicroplatewells.After90-minuteincubationat37℃,allwellsintheplateweretreatedwith1stantibodyfor60minutes,followedby30-minuteincubationwith2ndantibody.Theplatewasthentreatedwithreactionsolutionfor10minutes,andthereactionwasterminatedwithstopsolution.Absorbanceofsampleswasreadonamicroplatereader,andHDACactivitywascalculatedandexpressedbythe%relativetocontrols（100%）.

Westernblotanalysis

Thedetailedproceduresaredescribedelsewhere.17Anequalamountofcelllysates（7μg）,obtainedfromcontrolandagent-treatedcells,wasfirstsubjectedto10%SDSgelelectrophoresis

andtransferredtoanitrocellulosemembrane（blot）.Theblotwasincubatedfor90minuteswiththeprimaryantibodiesagainstp21WAF1,p27Kip1,Bax,orpoly-（ADP-ribose）-polymerase（PARP）（SantaCruzBiotechnology,SantaCruz,CA）,followedby30-minuteincubationwiththesecondaryantibodyconjugates.Thespecificimmunoreactiveproteinbandswerethendetectedbychemiluminescencefollowingtheprotocolofthemanufacturer（KPL,Gaithersburg,MD）.

Statisticalanalysis

Alldatawerepresentedasmean+standarddeviation,andstatisticaldifferencesbetweengroupswereassessedwitheitherone-wayanalysisofvarianceortheunpairedStudentttest.ValuesofP<0.05wereconsideredtoindicatestatisticalsignificance.

Results

EffectsofVK3,DF,ortheircombinationsonACHNcellgrowth

ACHNcellswereculturedseparatelywithvaryingconcentrationsofVK3（0–4μM）orDF（0–700μg/mL）.After72hours,MTTassayshowedthatVK3alonehadlittleeffectsbutledtoa~20%reductionincellviabilityat4μM（Fig.1A）,whileDFinduceda20%to45%viabilityreductionat

≥500μg/mL（Fig.1B）.WhetherthecombinationofVK3andDFmaypotentiatetheanticancereffectwasalsoexamined.Suchstudyshowedthataprofoundcellviabilityreduction,fromsevere（~90%）celldeath,wasattainedwiththespecificcombinationof4μMVK3and300μg/mLDF（Fig.2）.

EffectsofVK3/DFcombinationoncellcycle

ToexplorehowsuchVK3/DFcombinationwouldinduceaviabilityreductionorcell