Rottlerinisanaturalproductisolatedfrom
Mallotusphilippinensis.Thispolyphenoliccompound,originallydescribedasaselectiveinhibitorofPKCδ,caninhibitmanyotherPKC-unrelatedkinasesandhasanumberof
BIOLOGicalactions,includingmitochondrialuncouplingeffects.WerecentlyfoundthatRottlerininhibitsthetranscriptionfactornuclearfactorκBindifferentcelltypes,causingdownregulationofcyclinD1andgrowtharrest.ThepresentstudywascarriedouttoclarifythesurprisinglackofeffectofRottlerinonMCF-7cellvi
ABIlity,assessedbythe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)test.WefoundthatRottlerincausesoverestimationoftheMTTtest,le
ADIngtoinconsistentresultsbetweencellnumberandcellviability.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,apigmentedplantproductisolatedfrom
Mallotusphilippinensis(
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).
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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