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RottlerinisanaturalproductisolatedfromMallotusphilippinensis.Thispolyphenoliccompound,originallydescribedasaselectiveinhibitorofPKCδ,caninhibitmanyotherPKC-unrelatedkinasesandhasanumberofBIOLOGicalactions,includingmitochondrialuncouplingeffects.WerecentlyfoundthatRottlerininhibitsthetranscriptionfactornuclearfactorκBindifferentcelltypes,causingdownregulationofcyclinD1andgrowtharrest.ThepresentstudywascarriedouttoclarifythesurprisinglackofeffectofRottlerinonMCF-7cellviABIlity,assessedbythe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)test.WefoundthatRottlerincausesoverestimationoftheMTTtest,leADIngtoinconsistentresultsbetweencellnumberandcellviability.Rottlerin,however,stronglydiffersfromotherantioxidantpolyphenols,whichdirectlyreducetetrazoliumsalts,sinceitdoesnotexhibitanyreactivitytowardthetetrazoliumsaltsinvitronordoesitmodulatelactatedehydrogenaseactivity.TheinterferenceintheMTTassayoccurredonlyinculturedcells,concomitantlywithadecreaseintheenergycharge.BecausethesameMTToverestimationwasobservedinthepresenceofuncouplingagents,weconcludethattheRottlerinartifactislinkedtoitsuncouplingactionthat,byacceleratingoxidativechain,accidentallyresultsinenhancedMTTreduction.TheseresultssuggestcautionintheuseoftheMTTassayinthepresenceofRottlerinanduncouplersingeneral.

KeyWords:Rottlerin-MCF-7cell-MTT-LDH-FCCP-mitochondrialuncoupling

Introduction

Rottlerin(alsocalledmallotoxinorkamala),isa5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-l,2-chromene,apigmentedplantproductisolatedfromMallotusphilippinensis(Fig.1).Since1994,RottlerinhasbeenusedasaPKCδinhibitor(1)althoughtheselectivityofRottlerinininhibitingthePKCδisoformhasbeenrecentlyquestioned(2,3)andascribedtoalikelyindirecteffectmediatedbymitochondrialuncouplinganddecreaseinATPcontent(4).OurlaboratoryshowedforthefirsttimethatRottlerincanprevent,independentlyfromPKC,theactivationofthetranscriptionfactornuclearfactorκB(NFκB),inducedbyeitherphorbolesteroroxidativestressinMCF-7cells(5),HaCaTkeratinocytes(6)andHT-29cells(unpublishedresults),whosegrowthresultedtobearrestedbecauseofdownregulationofcyclinD1,atboththeproteinandmRNAlevels.Althoughthemolecularmechanismisnotdefinitivelyclarified,thepreventionoftheNFκBactivationprocesswaslikelyachievedthroughbothRottlerininhibitionofproteinkinases(7,8)andRottlerinfreeradicalsscavengingactivity(9).Indeed,NFκBcanbeactivatedbyanumberofpathwaysandisaredox-sensitivetranscriptionfactorforkeymoleculesinvolvedininflammation,cancerprogression,cellcyclecontrol,andprotectionagainstapoptosis(10).

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Fig.1Rottlerinstructure.

However,inourpreviouspaper(5),wefoundthatMCF-7cellviabilitywasnotalteredbya24-hRottlerintreatment,aresultthatwasinevidentconflictwiththeinhibitionofNFkBandcellproliferation,asevaluatedby[³H]-thymidineincorporationintoDNA.Sincethemeasurementofcellviabilitywasbasedonthereductionof3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)toformazancrystalbymitochondrialdehydrogenases(11),inthecurrentstudywerevisitedourpreviousresults,checkingforapossIBLeinterferenceofRottlerinintheMTTassay.

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