Differential induction of apoptosis and autophagy by pyrrolizidine alkaloid clivorine in human

23 Pages·2017·8.59 MB·English

RESEARCHARTICLE
Differential induction of apoptosis and
autophagy by pyrrolizidine alkaloid clivorine in
human hepatoma Huh-7.5 cells and its toxic
implication
WenjuLiu1☯,XuLi2☯,BoZhou2☯,ShoucaiFang2,WenzheHo3,HuiChen4,HaoLiang2,
LiYe2*,JunTang1*
a1111111111 1 KeyLaboratoryofCombinatorialBiosynthesisandDrugDiscovery(WuhanUniversity),Ministryof
Education,andWuhanUniversitySchoolofPharmaceuticalSciences,Wuhan,P.R.China,2 GuangxiKey
a1111111111
LaboratoryofAIDSPreventionandTreatment&GuangxiCollaborativeInnovationCentreforBiomedicine,
a1111111111
SchoolofPublicHealth,GuangxiMedicalUniversity,NanningCity,Guangxi,P.R.China,3 Departmentof
a1111111111
PathologyandLaboratoryMedicine,TempleUniversitySchoolofMedicine,Philadelphia,PA,UnitedStates
a1111111111 ofAmerica,4 GeriatricsDigestionDepartmentofInternalMedicine,TheFirstAffiliatedHospitalofGuangxi
MedicalUniversity,NanningCity,Guangxi,P.R.China
☯Theseauthorscontributedequallytothiswork.
*[email protected](JT);[email protected](LY)
OPENACCESS
Citation:LiuW,LiX,ZhouB,FangS,HoW,Chen Abstract
H,etal.(2017)Differentialinductionofapoptosis
andautophagybypyrrolizidinealkaloidclivorinein
Growingevidencesuggeststhatthepyrrolizidinealkaloids(PAs)-inducedhepatotoxicityis
humanhepatomaHuh-7.5cellsanditstoxic
mediatedbymultiplecelldeath/defencemodalities.However,thedetailedmechanismsare
implication.PLoSONE12(6):e0179379.https://
doi.org/10.1371/journal.pone.0179379 stilllacking.Inthisstudy,thehepatotoxiceffectsoffourPAsincludingthreeretronecine-
typeones(senecionine,seneciphyllineandmonocrotaline)andoneotonecine-type(clivor-
Editor:SalvatoreV.Pizzo,DukeUniversitySchool
ofMedicine,UNITEDSTATES ine)ontheproliferationofHuh-7.5cellsandthepossiblemechanismswereinvestigated.
TheresultsshowedthatallthePAscouldinhibitcellproliferationandinduceapoptosisina
Received:November3,2016
concentration-dependentmanner.Amongthemclivorinewasthemostsignificantone.In
Accepted:May30,2017
additiontoitseffectonapoptosis,clivorinetreatmentcouldpromoteautophagyinHuh-7.5
Published:June26,2017
cells,asevidencedbytheaccumulationofautophagosomes,theenhancementofLC3B
Copyright:©2017Liuetal.Thisisanopenaccess expressionattheconcentrationsclosetoitsIC value,andtheincreasedconversionof
0
articledistributedunderthetermsoftheCreative
LC3B-ItoLC3B-IIinthepresenceoflysosomalinhibitor(chloroquine)anddecreasedforma-
CommonsAttributionLicense,whichpermits
tionofgreenfluorescentprotein(GFP)-LC3positivepunctainthepresenceofautophagic
unrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalauthorand sequestrationinhibitor(3-methyladenine).AmongtheothertestedPAs,senecionineand
sourcearecredited. seneciphyllinealsoactivatedautophagyatthesameconcentrationsusedforclivorinebut
DataAvailabilityStatement:Allrelevantdataare monocrotalinedidnot.Furthermore,ourstudydemonstratedthatsuppressionorenhance-
withinthepaperanditsSupportingInformation mentofautophagyresultedintheremarkableenhancementorsuppressionofsenecionine,
files.
seneciphyllineandclivorine-inducedapoptosisattheconcentrationclosetotheIC forcli-
10
Funding:Thisworkwassupportedbygrantsfrom vorine,respectively,indicatingaprotectiveroleofautophagyagainstthePA-inducedapo-
theNationalNaturalScienceFoundationofChina
ptosisatthelowlevelofexposure.Collectively,ourdatasuggestthatPAsindifferent
(Nos.31270401toJT,81271851toLY,and
81460305toHC)(http://npd.nsfc.gov.cn/ structuresmayexertdifferenttoxicdisturbancesonthelivercells.Apoptosismaybeoneof
fundingProjectSearchAction.action); themostcommonmodelsofthePA-inducedcytotoxicity,whileautophagymaybeastruc-
GuangxiNaturalScienceFoundation
ture-dependentdefencemodelintheearlystageofPAintoxication.Differentialinductionof
(2013GXNSFCB019004toLY)(http://www.gxst.
gov.cn/wsbs/kjxm/xmlxgg/689094.shtml);Project
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 1/23
ClivorineinducesautophagyinHuh-7.5cells
ofGuangxiUniversitiesScientificResearch apoptosisandautophagyprobablydependingontheconcentrationisessentialforthecyto-
((2013ZD012toLY,HC);GuangxiUniversity“100- toxicpotencyofclivorine.
Talent”Program&GuangxiUniversityInnovation
TeamandOutstandingScholarsProgram(GuiJiao
Ren2014[7]toLY).Thefundershadnorolein
studydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript.
Competinginterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Pyrrolizidinealkaloids(PAs)arewidelydistributedinabout3%oftheworld’sfloweringplants
mainlyfromthreefamilies:Boraginaceae,Asteraceae,andFabaceae[1,2].Broadly,morethan
660PAsandPAN-oxideshavebeenidentifiedsofar,whicharemainlydividedintothree
types:retronecine-type,otonecine-typeandplatyphylline-type[3,4].Theformertwotypes
(Fig1)arebelievedtohavehepatotoxicity[4–6],genotoxicity[5],neurotoxicity[7,8],pneu-
motoxicity[5,9]andembryotoxicity[10].Theapparentinsultsinhumansafterexposureof
thesePAshavebeenmanifestedashepaticsinusoidalobstructivesyndrome(HSOS)accompa-
niedbyhepaticmegalocytosis,ascites,jaundice,andchroniccirrhosis[5,6].Todata,there
havebeennumerousHSOScasereportsofhumanintoxicationthroughPA-containingherbs
orfoodstuffsconsumptionsinmanycountries,butthereisyetnodefinitetreatmentforit[6,
11–13].
Senecionine,seneciphyllineandmonocrotalinearethreerepresentativesforretronecine-
typePAs.TheformertwosarecommonlyfoundingenerasuchasSenecioandGynuraofthe
familyAsteraceae,whilemonocrotalineismainlypresentinCrotalariaofthefamilyFabaceae
[3,11].Forexample,senecionineandseneciphyllinearetwomajorPAcomponentsinthe
rootsofGynurajaponica(Thunb.)Juel.,whichisusedforthetreatmentofphysicalinjuries
andtraumatichaemorrhagesinChineseherbalmedicines(CHM)[11,12];monocrotalineisa
predominantPAintheseedsofCrotalariaassamicaBenth.,whichhaseffectsofdispelling
wind-dampsyndrome,stoppingbleedinganddiminishingswelling[9].Asarepresentative
otonecine-typePA,clivorineisabundantinquiteafewLigulariaspeciesfromAsteraceae,
whoserootsandrhizomesareusuallyusedtorelievecoughandremoveexcessivephlegmin
CHM[13–15].Currently,intakeormisuseofPA-containingherbsformedicalpurposesisstill
commoninChina,creatingagrowingsafetyconcernwiththepotentialexposuretoPAs[3,
11–16].
InviewofthemechanismofPA-inducedhepatotoxicity,PAsmaysuffermetabolicactiva-
tionbycytochromesP450(CYP450s)toproducethehighlyreactivepyrrolicmetabolitesand
thencausehepatotoxicity[17–19].However,howthePAs,eitherretronecineorotonecine-
type,inducehepatotoxicityatthecellularandmolecularlevelsisnotwellknown.Inthepast
twentyyears,afewmodesofcelldeath,e.g.,necrosis(oncosis)andapoptosishavebeenfound
tomediatethePA-inducedcytotoxicity[13,20–22].Severalretronecine-typePAsincluding
senecionine,monocrotaline,isoline,retrorsineandriddelliineandtheotonecine-typePAcli-
vorinehadbeenreportedtocauseapoptosisinvitroand/orinvivo[20–23].Forinstance,Ji
etal.[20,24]foundthatclivorinecouldremarkablyinhibitcellproliferationandinducemito-
chondria-mediatedapoptosisinhumanliverL-02cells.Thiseffortinvolvedthepathwayby
degradationofBcl-xLprotein,mitochondrialreleaseofcytochromec,andactivationofcas-
pases-3/-9.Thefurtherstudyindicatedthatsenecioninealsoinvolvedthesameapoptoticsig-
nallingpathwayasclivorine[20].Monocrotalinecouldinducebothoncosisandapoptosisof
hepaticparenchymalcellsofratsinvivowhichbutoccurreddifferentlyindistribution[22].
TheoncoticlesionsprimarilyoccurredinthecentrilobularregionswithabundantCYP450s,
whilethecaspaseinhibitioncouldpreventthedevelopmentofbothoncosisandapoptosiswith
littleeffectsonthebioactivationofmonocrotaline.Ahypoxia-regulatedcell-deathfactor,
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ClivorineinducesautophagyinHuh-7.5cells
Fig1.Thechemicalstructuresofsenecionine,seneciphylline,monocrotalineandclivorine.
https://doi.org/10.1371/journal.pone.0179379.g001
BNIP3,wasfoundtobeup-regulatedandimplicatedinswitchingthemodeofcelldeathfrom
apoptosistooncosisaftermonocrotalineexposure.Thestudyonretrorsineshowedthatits
cytotoxicmodeonHuh-7cellsmaybedose-dependentwithapoptosisatlowdosesandnecro-
sisathighdoses[21].ArecentstudyalsofoundthatthePC12cellsafterclivorineexposure
involvedtheapoptoticdeathattheconcentrationshigherthan50μMwhilesuppressedneuro-
naldifferentiationviaTrkA/Aktsignallingpathwayatlowerdosesthanit[7].Alltheevidence
suggeststhatthemodesofPA-inducedcelltoxicitieswerecomplexanddiversewithinvolve-
mentsofmanycellularfactorsand/orevents,whichmaydependonchemicalstructure,con-
centration,treatmenttimeandcelltypesorevencellularcompartment.
Autophagy(hereafterreferringtomacroautophagy)isthenaturallydestructivemechanism
thatdisassembles,througharegulatedprocess,unnecessaryordysfunctionalcellularcompo-
nents[25].Duringthisprocess,targetedcytoplasmicconstituentsareisolatedfromtherestof
thecellwithinadouble-membranedvesicleknownasanautophagosome.Theautophagosome
thenfuseswithalysosomeandthecontentsaredegradedandrecycled[26].Inthecontextof
disease,autophagyhasbeenseenasanadaptiveresponsetostress,whichpromotessurvival,
whereasinsomeothercasesitappearstostimulatecellmorbidityanddeath,orsometimes
calledautophagiccelldeath[27].Manystudieshaveshowedthatprogressofmanydiseases,e.
g.,tumour,diabetesanddrug-inducedliverinjuries(DILI)arecloselyrelatedtotheautopha-
gicprocessincells[28–30].InthecaseofDILI,autophagywasfoundtoprotectthelivercells
byremovalofthedamagedmitochondria,whichregulationmaybeanewapproachforthe
treatmentofliverdiseases[31,32],butiftoomuchdamagedmitochondriaoverloadautop-
hagyorelicit“autophagicstress”,theDILImaybeenhanced[33,34].InChina,thePAs-con-
tainingherbalmedicineshavebeenanimportantcauseofDILIowingtonumerousHSOS
cases[6,16].Eventhoughsomemodesofcelldeathandsignalpathways,e.g.oncoticnecrosis
andmitochondrial-mediatedapoptosishavebeenreported[20–24],thereisstilllimited
knowledgeofthelivercell-intrinsicresponsemodesandmachineriesforthePA-induced
toxicity.
Inourpreliminarystudy,clivorinecouldmodulatethemRNAexpressionoftheautop-
hagy-associatedgeneLC3(correspondingtomicrotubule-associatedprotein1lightchain3)in
Huh-7cells.TheevidenceimpliesthatautophagymayhaveanimpactonthetoxicityofPAs.
Inthepresentstudy,wecontinuedtostudythetoxiceffectsofPAsonthehumanhepatoma
Huh-7.5cellswiththreeretronecine-typePAs(senecionine,seneciphylline,monocrotaline)
andoneotonecine-typePAclivorineatdifferentconcentrations.Theireffectsoncellprolifera-
tionandunderlyingmechanismespeciallyinvolvingautophagywereinvestigated.Ourfind-
ingsdemonstratethatallPAshavecytotoxicpotency,amongthem,themostdistinctoneis
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ClivorineinducesautophagyinHuh-7.5cells
clivorine.Thesameapoptoticpathwaymayberesponsiblefortheirtoxicities,whileautophagy
mayplayaprotectiveroleintheearlystageoftoxicinsultsbyPAsespeciallyclivorine.
Materialsandmethods
Chemicalsandreagents
SenecionineandseneciphyllinewereisolatedfromGynurajaponica(Thunb.)Juel.,clivorine
wasfromLigulariahodgsoniiHook.,andmonocrotalinewasfromCrotalariaassamicaBenth.
aspreviouslydescribed[3,9,14].AllPAs’structureswereconfirmedusingMSandNMRspec-
troscopyandtheirpuritiesfurtherdeterminedtobemorethan98%byHPLCanalyses.Dul-
becco’smodifiedeaglemedium(DMEM)werepurchasedfromCorningCo.,Ltd.(Corning,
NY,USA);fetalbovineserum(FBS)werepurchasedfromGibco/ThermoFisherScientific
China(Shanghai,China).Chloroquine(CQ),rapamycin(Rapa)and3-methyladenine(3-MA)
werepurchasedfromSigma-Aldrich(Shanghai,China).MTTcellproliferationandcytotoxic-
ityassaykitswerepurchasedfromBosterCo.Ltd.(Wuhan,China).AnnexinV-kFluor488/PI
doublestainingApoptosisDetectionKitwerepurchasedfromKeyGenBioTechCo.Ltd.
(Shanghai,China).BothreversetranscriptasekitandqPCRkitwerepurchasedfromTAKARA
biotechnologyCo.Ltd.(Dalian,China).TheprimerswerepurchasedfromSangonBiotech
Co.,Ltd.(Shanghai,China).AllotherreagentswerepurchasedfromSigma-Aldrich(St.Louis,
MO)orSinopharmChemicalReagentCo.Ltd.(Shanghai,China),unlessotherwiseindicated.
Priortotheexperiments,the0.1Mstocksolutionsofsenecionine,seneciphylline,monocrota-
lineandclivorinewerepreparedbydissolvingin10%DMSOaqueoussolution.
Cellline
HumancancerliverHuh-7.5cellswerekindlyprovidedbyProfessorWenzheHoatTemple
UniversitySchoolofMedicine,USA.ThecellsweremaintainedinDulbecco’smodifiedEagle’s
medium(DMEM)supplementedwith10%fetalbovineserum(FBS),100U/mLpenicillin,
and100μg/mLstreptomycinat37˚Cwith5%CO .
2
Cellviabilityassay
Cellsinlog-phasewereseededin96wellplates(10,000cells/well)overnightandthentreated
withsenecionine,seneciphylline,monocrotalineandclivorinefor48h.ThecellswithoutPA
treatments(withonlysolvent)wereusedasthenegativecontrols.Elevenconcentrationswere
setforeachPA,including0μM,3.125μM,6.25μM,12.5μM,25μM,50μM,100μM,200μM,
400μM,800μMand1000μM;eachrepeatedfor6wells.Cellviabilitywasassayedusingthe
MTTassaykitaccordingtothestandardprocedureprovidedbythesupplier.Briefly,22μLof
MTTstainingfluidwasaddedineachwell,andthenincubatedfor4h.Afterwards,100μLof
theformazancrystalsweredissolvedbyadding2-propanolineachwell.Theabsorptionofthe
formazansolutionwasmeasuredbyaMultiskanGOspectrophotometer(ThermoScientific
China,Shanghai,China)atawavelengthof570nm[35].Thecellviabilitywascalculatedas
thefollowingformula.
TheODvalueof PA (cid:0) treatedgroup
Thecellviabilityð%Þ¼ (cid:2)100
TheODvalueof controlgroup
Basedonthevaluesofcellviability,thevaluesofIC (theconcentrationthatinhibitedcell
50
viabilityto50%ofthecontrol),IC (theconcentrationthatinhibitedcellviabilityto20%of
20
thecontrol),IC (theconcentrationthatinhibitedcellviabilityto10%ofthecontrol)andIC
10 0
(themaximalconcentrationwithoutinhibitiononthecellviability)werealsodetermined
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 4/23
ClivorineinducesautophagyinHuh-7.5cells
usingnonlinearregressionanalysisinGraphPadPrism4(GraphPadSoftware,Inc.,LaJolla,
USA).
Flowcytometry
Forflowcytometryanalysis,fourconcentrationswereselectedforeachPA,including
6.25μM,25μM,100μM,and400μM.Mostoftheconcentrationswerelowerthanthecalcu-
latedIC valueforeachPA(except400μMforclivorine)toavoidthenon-apoptoticcell
50
death.Aftertreatment,thecellswerecollected,washedwithPBSanddigestedby0.25%trypsin
solution(withoutEDTA),andthenfurtherincubatedatroomtemperaturefor2minutes.
Afterthat,thecellswerere-collectedandsuspendedwithbindingbuffer.Thesuspensionwas
incubatedwithAnnexinV-kFluor488andstainedwithPI(KaijiBiologyCorp.,China)at
roomtemperaturefor20minutesandthenanalyzedonflowcytometer(CytoFlexz,Beckman,
USA).TheannexinV-positive/PI-negativecellsindicatedapoptoticcellsattheearlyperiod,
andtheannexinV-positive/PI-positivecellsshowedthoseatthelaterperiod[36].Theratioof
apoptosisorapoptosisindex(AI)wascalculatedusingthefollowingequation:
Apoptosiscellnumbers
AIð%Þ¼ (cid:2)100
Apoptosiscellnumbersþthenormalcells
Real-timePCRanalysis
SYBRGreenquantitativereal-timePCR(RT-PCR)wasusedtoinvestigatethemRNAexpres-
sionlevelsoftheautophagy-associatedgenesincludingLC3,ATG3,ATG5,andATG7.Forthis
analysis,24wellplateswereusedbyseeding50,000cells(500μLofcellsuspension)perwell,
whichwerethentreatedwithorwithout56μLofeachPAfor24h.Threeconcentrationsfor
eachalkaloid,respectively,including3.125μM,6.25μMand12.5μMwereusedfortheexperi-
ments.AllconcentrationswereinorlowerthantherangeofthecalculatedIC andIC values
0 10
foreachPAtoavoidtheundesirablecytotoxicity.TotalRNAwasextractedwithTRIzol,and
thefirststrandcDNAwassynthesizedwithTAKARARR036Areversetranscriptasekitfollow-
ingthemanufacturer’sinstructions.Real-timePCRwasperformedwiththeBio-RadCFX96
thermalcycler(Bio-RadLaboratories,Hercules,CA,USA)accordingtothemanufacturer’s
protocol.PrimersforallautophagicgenesandtheinternalreferenceGAPDHweredesigned
byPrimerexpress2.0,whichsequenceswereasfollows:
• GAPDH(internalreference)sense,5’-GGTGGTCTCCTCTGACTTCAACA-3’andanti-
sense5’-GTTGCTGTAGCCAAATTCGTTGT-3’;
• LC3sense,5’-AGCAGCATCCAACCAAAATC-3’andantisense5’-CTGTGTCCGTTC
ACCAACAG-3’;
• ATG3sense,5’-CCAACATGGCAATGGGCTAC-3’andantisense5’-ACCGCCAGCATC
AGTTTTGG-3’;
• ATG5sense,5’-TGGGATTGCAAAATGACAGA-3’andantisense5’-TTTCCCCATCTT
CAGGATCA-3’;
• ATG7sense,5’-CACTGTGAGTCGTCCAGGAC-3’andantisense5’-CGCTCATGTCCC
AGATCTCA-3’.
Eachcyclethreshold(Ct)wasmeasuredandnormalizedtotheaverageCtvalueofGAPDH
(n=6).Theresultswerecalculatedby2-44ctmethodandgivenastherelativetranscriptional
levelsoftargetgenes.
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ClivorineinducesautophagyinHuh-7.5cells
Western-blotanalysis
Inthisexperiment,threeconcentrations(3.125μM,6.25μM,and12.5μM)werealsochosen
foreachPA,respectively,accordingtoRT-PCRresults.Totalproteinconcentrationswere
determinedbyNanoDrop2000spectrophotometer(ThermoScientific,USA),forwhicheach
samplewasnormalizedtotheequaltotalproteinconcentration.Proteinswereseparatedby
SDS-PAGEandprobedwithappropriateprimaryandsecondaryantibodiesaspreviously
describedbyLietal.[37].Thefollowingantibodiesincludinganti-humanantibodiesagainst
LC3B(1:3000,CellSignalling),andinternalreferenceβ-actin(1:5000,Millipore)wereused.
TheproteinlevelsincludingLC3B-I,LC3B-IIandβ-actinwerequantifiedbyQuantityOne
(Chemicallightemittinggelimagingsystem).
Transmissionelectronmicroscopeanalysis
ElectronmicroscopywasutilizedtodetecttheautophagyresponseinHuh-7.5cellstreated
withorwithoutsenecionine,seneciphylline,monocrotalineandclivorine(each6.25μM).The
cellsweredigestedwithtrypsinandcentrifuged,andthenimmediatelyfixedwith2.5%glutar-
aldehyde/0.1Msodiumcacodylate.Thecellswerethenpost-fixedwith1%osmiumtetroxide,
followedbydehydrationwithanincreasingconcentrationgradientofethanolandpropylene
oxide.ThecellswerethenembeddedwiththeepoxyresinEpon812andcuttoultrathinsec-
tions(50–60nm)usinganultramicrotome(LKB-I,Bromma,Sweden).Imageswereexamined
usingaH7650electronmicroscope(HitachiLtd.,Tokyo,Japan)at80kVafterthesamples
werestainedwith3%uranylacetateandleadcitrate.
Fluorescencemicroscopicanalysis
TransientGFP-LC3-expressingHuh-7.5cellsweregeneratedusinglentivirus-mediated
GFP-LC3over-expression.AlentiviralvectorcontainingGFP-LC3fusiongeneandlentivirus
werepurchasedfromGenechemCompany(Shanghai,China).InfectionofHuh-7.5cellswith
lentiviruswascarriedoutatamultiplicityofinfection(MOI)of10.0.Autophagywasassessed
usingGFP-LC3redistributionincellsthatwasdetectedbyaninvertedfluorescencemicro-
scope(NikonTi-s,Japan).ThenumberofGFP-LC3punctapercellwasdeterminedinthree
independentexperiments(30cellswerecountedperexperiment).
Autophagyinhibitorandactivatortreatments
ToaddressthePA-inducedautophagyfluxinHuh-7.5cells,theautophagyinhibitorswere
used.Briefly,CQ(5μM)and3-MA(5mM)wereaddedtothecellsnaiveandexpressing
GFP-LC3fromlentiviruses,respectively,2hbeforesenecionine,seneciphyllineandclivorine
treatments(each6.25μM).ThePA-treatedcellswerethensubjectedtothemeasurementof
autophagy-relatedproteinlevelsbyWesternblottingorofgreenfluorescencesignalbyfluores-
cencemicroscopeasdescribedabove.Asafurtherefforttoaddresstherelationshipbetween
apoptosisandautophagy,boththeautophagyinhibitor3-MAandautophagyactivatorRapa
wereused.Huh-7.5cellswerepre-treatedwiththe3-MA(5mM),orRapa(100nM)for2h,
andthenincubatedinthepresenceofsenecionine,seneciphylline,clivorineandmonocrota-
line(each12.5μM)for24h.Finally,thecellswereharvestedandtreatedfortheflowcytometry
analysis.
Statisticalanalysis
Valuesareexpressedasmean±SDofatleastthreeindependentexperiments.Statisticalanaly-
siswasperformedusingSPSS17.0software(SPSSInc.,Chicago,IL)orGraphPadPrism4
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 6/23
ClivorineinducesautophagyinHuh-7.5cells
(GraphPadSoftware,Inc.,LaJolla,USA),usingone-wayanalysisofvariancefollowedbytwo-
tailedt-testforevaluation,P<0.05wasconsideredtobesignificantlydifferent.
Results
Cytotoxicityofsenecionine,seneciphylline,monocrotalineandclivorine
inHuh-7.5cells
Senecionine,seneciphylline,monocrotalineandclivorine(Fig1)arefourrepresentativesof
hepatotoxicPAspresentinmanyChineseherbalmedicines[3,9,11–15].AsshowninTable1
andFig2,allfouralkaloidscouldinhibittheHuh-7.5cellviability.ForeachPA,thecellsur-
vivalratedecreasedwiththeincreaseofconcentration,theefficiency,however,wasdifferent.
Theinhibitoryeffectsofsenecionineandseneciphyllineintherangeoftheconcentrations
from100μMto1000μM,clivorinefrom12.5μMto400μM,monocrotalinefrom50μMto
1000μM,weresubstantiallyhigherthanthenegativecontrol.Comparatively,theotonecine-
typePAclivorineshowedthehighestanti-proliferativepotency.At100μMforexample,its
averageinhibitionrate(51.4%)wassignificantlyhigherthan16.7%,19.3%and28.4%ofother
threeretronecine-typePAssenecionine,seneciphyllineandmonocrotaline,respectively.
Moreover,theIC valuecalculatedinaverageforclivorinewas141.7μM,whichwasremark-
50
ablylowerthanthoseobservedwiththeotherthreePAs(509.7μM,491.9μMand413.2μM).
Thisresultwascomparabletothatreported[4,20],inwhichclivorineexhibitedthehigher
cytotoxicitythanothertypesofPAs.BecauseMTTassayisacommonmethodforassessing
theinhibitoryeffectoncellviability,ourresultfurtherdemonstratedthatdirectcelldeathmay
playanimportantroleinthetoxicityofclivorine.ItisworthofnotethattheIC values
50
obtainedindifferentstudiesmaybelargelydifferentfromeachother,e.g.,theIC valuefor
50
clivorineinthisstudywasmuchhigherthan40.8μMinanotherstudybyJietal.[20].Thedis-
crepanciesmaybeduetodifferencesincelllinesbeingusedorinvitromodelforcytotoxicity
screening[4,20].TheHuh-7.5celllineusedinthepresentstudywasderivedfromHuh-7cell
line,whichhasbeenreportedtohavenoormutantexpressionofbcl-2andp53genes,indicat-
ingthatthiscelllinemaybesusceptibletotoxinsbutinsensitivetosomecytotoxicagents-
inducedapoptosis[20,21].BecausedifferenttypesofPAsmayhavedifferentmodesofcyto-
toxiceffects[4,20,23],thetoxicresponsesandunderlyingmechanismsofHuh-7.5celllineto
variousPAswithdifferenttypesornecineacidmoietiesmaybedifferent.Ontheotherhand,
consideringthatthePAswithhighconcentrationsmayhavemoredirectcytotoxicity[4,21],it
isalsointerestingtoknowhowthecellsrespondtoPAsatlowornontoxicconcentrations.
Accordingly,themaximalnon-toxicconcentrations(IC s)andthosewith10%and20%of
0
Table1. InhibitoryeffectsofdifferentPAsoncellviabilityinHuh-7.5cells.
PA IC (μM) IC (μM) IC (μM) IC (μM)
0 10 20 50
Senecionine 22.7±10.0**## 57.2±22.1**## 136.7±59.3**# 509.7±122.6***
Seneciphylline 21.0±18.3 47.5±28.8* 113.4±38.1**# 491.9±78.0***
Monocrotaline 5.9±2.6 20.1±5.1 59.9±12.2** 413.2±80.6***
Clivorine 5.4±3.6 13.4±8.4 27.2±14.7 141.7±28.3
AllvaluesexpressedasMean±SD(n=3).
*P<0.05
**P<0.01and
***P<0.001comparingwithclivorine
#P<0.05
##P<0.01comparingwithmonocrotaline.
https://doi.org/10.1371/journal.pone.0179379.t001
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ClivorineinducesautophagyinHuh-7.5cells
Fig2.Cytotoxiceffectsofsenecionine,seneciphylline,monocrotalineandclivorineincreasewiththeincreaseoftheirconcentrationsin
Huh-7.5cellsasdeterminedbyMTT.Cellswereincubatedwithindicatedconcentrationsofsenecionine(closedrectangle),seneciphylline(closed
diamond),monocrotaline(closedtriangle)andclivorine(opencircle)for48h,respectively.CellviabilitywasmeasuredbyMTTassayasdescribedin
MaterialsandMethods.Valuesareshownasmean±SD(n=3),ofwhichthesolventcontrolareat0μM;Δ,#P<0.05,**P<0.01,***,ΔΔΔ,
###P<0.001,comparingclivorinewithsenecionine(*),seneciphylline(Δ),andmonocrotaline(#),respectively,atthecorrespondingconcentrations.
https://doi.org/10.1371/journal.pone.0179379.g002
inhibitionrates(IC sandIC s)werecalculatedforallfourPAs(Table1).Incomparison,
10 20
clivorinehadthelowestIC ,IC andIC valuesof5.4μM,13.4μMand27.2μMinaverage,
0 10 20
respectively.TheresultsespeciallyattheconcentrationsofIC oraboveshowedremarkable
20
discrepancybetweenclivorineandallotherretronecine-typePAs.Ofnote,monocrotaline
showedcomparablecytotoxicpotentialtothatofclivorineatthelowerconcentrationsthan
IC .Thepotencyorderofhepatotoxicitywasthusclivorine(cid:21)monocrotaline(cid:21)seneciphyl-
20
line(cid:21)senecionine.
Senecionine,seneciphylline,monocrotalineandclivorineinduce
apoptosisofHuh-7.5cells
AsforthemechanismofcytotoxicityforPAs,manystudiessuggestedthattheapoptoticcell
deathmayplayavitalroleinit[13,20–23].Senecionine,monocrotalineandclivorinehad
beenfoundtoinduceapoptosisinseverallivercelllines,totheexclusionofHuh-7.5cells.In
thisexperiment,theeffectsoffourPAsincludingsenecionine,seneciphylline,monocrotaline
andclivorineontheapoptosisofHuh-7.5cellswerefurtherinvestigatedandcomparedby
usingflowcytometry.Basedontheaboveanalyses,aseriesofconcentrationsaroundorlower
thanIC swereinvestigated.Asaresult,theoverallapoptosisratiosorAIsofHuh-7.5cells
50
increasedwithincreasingconcentrationsofeachPA(Fig3).Allthreeretronecine-typePAs,
senecionine,seneciphyllineandmonocrotalineshowedalmostsamepotencytoinducethe
apoptosisinHuh-7.5cells.Generally,theirAIsagainstHuh-7.5cellsatallconcentrations
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 8/23
ClivorineinducesautophagyinHuh-7.5cells
Fig3.Evaluationofsenecionine,seneciphylline,monocrotalineandclivorine-inducedapoptosisof
Huh-7.5cellsbyflowcytometryanalysis.Cellsweretreatedbyfourconcentrationsofsenecionine,
seneciphylline,monocrotalineandclivorinefor24h,respectively.Afterwards,thecellswerecollectedand
stainedwithAnnexinV-kFluor488andPI.Theflowcytometryanalysiswasperformed.Thepercentagesof
apoptoticcellsatboththeearlyandlateperiodswereshownasQ1-LRandQ1-UR,respectively,intheform
ofscatterplots(a-1toa-4).Theresultsintherepresentativeofthreeindependentexperimentsweremarked
ineachdiagramandsummarizedinthehistograms(b-1tob-4).a-1andb-1showtheresultsfrom
senecionine-treatedcells;a-2andb-2,fromseneciphylline-treatedcells;a-3andb-3,frommonocrotaline-
treatedcells;a-4andb-4,fromclivorine-treatedcells.N0,F0,M0andC0,negativecontrols;N6.25,N25,
N100andN400representsenecionine-treatedcellsattheconcentrationsof6.25μM,25μM,100μMand
400μM;F6.25,F25,F100andF400representseneciphylline-treatedcellsattheconcentrationsof6.25μM,
25μM,100μMand400μM;M6.25,M25,M100andM400representmonocrotaline-treatedcellsatthe
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 9/23
ClivorineinducesautophagyinHuh-7.5cells
concentrationsof6.25μM,25μM,100μMand400μM;andC6.25,C25,C100,andC400representclivorine-
treatedcellsattheconcentrationsof6.25μM,25μM,100μMand400μM,respectively.*P<0.05;**P<0.01
vs.negativecontrol.
https://doi.org/10.1371/journal.pone.0179379.g003
determined(6.25μM,25μM,100μMand400μM)weremarkedlyhigherthanthatoftheneg-
ativecontrol(4.2%inaverage),butallthesevalueswerenotveryhigh(around20%inaverage
at400μM)andshowednodifferencesfromeachotheratthesameconcentrations.Thisresult
suggeststhattheconcentrationmaycontributetotheirpotenciestoinduceapoptosis.
Incomparison,clivorineproducedrelativelyhigherapoptosisratiosofHuh-7.5cellsthan
otherthreeretronecine-typePAsatthecorrespondingconcentrations.Thediscrepancies
becomemuchmoreobviousattheconcentrationshigherthan25μM,inwhichtheAIvalueat
100μM(22.8±1.7%)wasalmostequaltoorhigherthanthoseofsenecionine,seneciphylline
andmonocrotalineat400μMandconsiderablyhigherthanthoseofthematthesameconcen-
tration.Thiseffectmaypartiallybederivedfromthedirectcytotoxicityofclivorine,butforthe
retronecine-typePAsmaymainlyfromtheinhibitionofcellproliferation[4].Exceptforthe
concentration,thechemicalstructureespeciallynecinebasesmayalsobeakeydeterminant
forthePA-inducedapoptosis.Basedonaboveexperiment,clivorinemayhavehigherpotency
toinduceapoptosisinHuh-7.5cellsthanotherPAs,butwhicheffectstendtobeconsistentat
thelowconcentrationsof25μM(16.2±0.9%)and6.25μM(11.2±1.7%).Also,monocrotaline
didnotshowapparenthigheffectscomparabletothoseinMTTassayattheconcentrations
lessthanIC .TheseresultssuggestthatapoptosismaynotbemeredeterminantforthePA-
20
inducedcytotoxicity,inwhichsomeothermechanismofactionmaybeinvolved.Combined
theresultsfromMTT,thePAsintoxicationmaydependontheirchemicalstructuresandcon-
centrations,whichwasingoodagreementwiththosereportedbypreviousinvestigators[4,
21].
Senecionine,seneciphyllineandclivorineenhancethemRNA
expressionofLC3inHuh-7.5cells
ThePA-inducedcytotoxicityinHuh-7.5cellsmarkedlydecreasedatloworevennontoxic
concentrations,whichmaynotbecausallyinvolvedbyapoptosisornecrosis.Inthisscenario,
somereportssuggestedthatthecelldefencebutnotdirecttoxicpathwaymayplayapivotal
role[32–34].Understressfulconditions,autophagyisusuallyactivatedtoprotectthecellfrom
damage[38].Avarietyofkeyautophagy-relevantcomponentshavebeenfoundtoparticipate
intheactivationbyinvolvinginubiquitin-likeconjugationsystems,forinstance,Atg3,Atg5,
Atg7andLC3involvedintheLC3lipidationandATG12-ATG5conjugationsystemsthatare
essentialfortheautophagosomeformationandmaturation[39].Theirtranscriptionaland
translationalregulationespeciallythatofLC3,themostwidelyusedmarkerformonitoring
autophagyispreferredtoprovidecorrelativedatarelatedtoautophagy[27,37–39].Accord-
ingly,fourautophagy-associatedgenesincludingATG3,ATG5,ATG7andLC3wereexamined
byRT-PCRtoobserveifsenecionine,seneciphylline,monocrotalineandclivorinecouldmod-
ulateautophagyatthetranscriptionallevel.Toavoidtheundesirabledirecttoxiceffects,the
choiceoftheconcentrationwasmainlybasedonthevaluesintherangeofIC andIC ofcli-
0 10
vorineforeachPA.AsshowninFig4,therelativemRNAlevelsofLC3asthefoldofcontrol
were1.27±0.06,1.92±0.05and1.75±0.10,inthecellstreatedbyclivorineat3.125μM,
6.25μMand12.5μM,1.73±0.08and1.69±0.06bysenecionineat6.25μMand12.5μM,and
1.81±0.11and1.79±0.09byseneciphyllineatthesametwoconcentrationsassenecionine,
respectively,allofwhichweresignificantlyhigherthanthatofnegativecontrol.Forthethree
PAs,theeffectsat6.25μMand12.5μMwerealsoremarkablyhigherthanthoseatthelower
PLOSONE|https://doi.org/10.1371/journal.pone.0179379 June26,2017 10/23