w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Protective
effect
of
Rheum
turkestanikum
root
against
doxorubicin-induced
toxicity
in
H9c2
cells
Azar
Hosseini
a,∗,
Arezoo
Rajabian
baPharmacologicalResearchCenterofMedicinalPlants,SchoolofMedicine,MashhadUniversityofMedicalSciences,Mashhad,Iran
bDepartmentofPharmacology,SchoolofMedicine,MashhadUniversityofMedicalSciences,Mashhad,Iran
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received15October2015 Accepted7February2016 Availableonline26March2016
Keywords: Apoptosis Cardioprotective Doxorubicin H9c2
Lipidperoxidation Reactiveoxygenspecies
a
b
s
t
r
a
c
t
Doxorubicinisachemotherapydrugbutitsclinicalusingislimitedbecauseofitscardiotoxicity. Reac-tiveoxygenspeciesplayanimportantroleinthepathologicalprocess.Theaimofthisstudyistoevaluate theprotectiveeffectofRheumturkestanicumJanisch.,Polygonaceae,againstdoxorubicin-induced apo-ptosisanddeathinH9c2cells.ThecellswereincubatedwithdifferentconcentrationsofR.turkestanicum
extractandN-acetylcysteineaspositivecontrolfor2h,followedbyincubationwith5Mdoxorubicin
for24h.CellviabilityandapoptoticinductionweredeterminedbyusingMTTandPIassays,respectively. Thelevelofreactiveoxygenspeciesandlipidperoxidationwasmeasuredbyfluorimetricmethods. Doxo-rubicinsignificantlydecreasedcellviabilitywhichwasaccompaniedbyanincreaseinROSproduction andlipidperoxidation.PretreatmentwithR.turkestanicumincreasedtheviabilityofcardiomyocytesand coulddecreaselipidperoxidationandreactiveoxygenspeciesgeneration.Also,R.turkestanicum atten-uatedapoptoticinduction.N-acetylcysteineat100Mreducedthelevelsofreactiveoxygenspecies
andlipidperoxidation.But,treatingH9c2cellswithN-acetylcysteinedidlittletoprotectH9c2cellsfrom doxorubicin-inducedcelldeath.R.turkestanicumexertsprotectiveeffectagainstoxidativestress-induced cardiomyocytesdamage.OurfindingsshowedthatR.turkestanicumcouldexertthecardioprotective effectsagainstdoxorubicin-inducedtoxicitypartlybyanti-apoptoticactivity.Also,N-acetylcysteine pre-ventedoxidativestressviareductionofreactiveoxygenspeciesandlipidperoxidation.N-acetylcysteine inducedlessprotectiveeffectsthanR.turkestanicumextractagainstdoxorubicin-inducedcytotoxicity.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Doxorubicin(DOX)asaneffectivechemotherapydrughas
seri-ousside effectssuchasdilatedcardiomyopathy andcongestive
heartfailure(Turakhiaetal.,2007).Therefore,usingthisdrugmust belimited.ThemechanismsofDOX-inducedcardiotoxicityarenot completelyunderstood,butmostevidencesindicatethatthe gen-erationofreactiveoxygenspecies(ROS)isinvolved(Bryantetal., 2007).IncreasedlevelofROSleadstoproteinandlipid
peroxida-tion,DNA damageand irreversiblecelldamage(Ghorbanietal.,
2015; Asadpour et al., 2014). ROS can directly impair contrac-tilefunction,activatehypertrophysignalingpathways,stimulate cardiac fibroblast proliferation and induce extracellular matrix remodeling(TakimotoandKass,2007;Tsutsuietal.,2011). Exces-siveROSalsocauseaccumulationofintracellularCa2+incardiac cellswhichincreasesthemitochondrialpermeabilityandleadsto
∗ Correspondingauthor.
E-mail:hoseiniaz@mums.ac.ir(A.Hosseini).
thereleaseofcytochromecintothecytoplasmandthefollowing apoptoticcascades(Pacheretal.,2001).Interestingly,some
nat-uralfoodshavebeenreportedtocontainsubstantialamountsof
antioxidantsandfreeradicalscavengingagents.Thesecompounds
diminishsomeside effectsof chemotherapeuticagents on
nor-malcellsbyreducingtheirgenotoxicity(Bryantetal.,2007).H9c2 myoblasts,aratembryoniccellline,whichhastheabilityto
dif-ferentiatebetweena skeletal orcardiac musclephenotype,can
beinstrumental in understanding DOXcytotoxicity in different
stages(Brancoetal.,2012).Therecent studieshaveshownthat someoftheherbsactagainstoxidativeinjury-related cardiotoxic-ity.Rheumspecies,Polygonaceae,havealonghistoryasmedicinal plantsintraditionalChinesemedicine.Themainactiveingredients oftheRheumspeciesareaseriesofanthraquinones,dianthrones,
glycosides and tannins. The anthraquinone derivatives include
emodin,rhein,chrysophanol,physcion,alizarin,citreorosein,and aloe-emodin(DorseyandKao,2007).R.turkestanicumJanisch.isa plantthatgrowswidelyincentralAsiaandalsoinnortheastofIran.
Traditionally, people use roots of R. turkestanicum as an
anti-diabetic and anti-hypertensive as well as anticancer agent
http://dx.doi.org/10.1016/j.bjp.2016.02.004
(DorseyandKao,2007).Recentstudieshaveshownotherspeciesof
RheumsuchasR.undulatumiscontaininganti-oxidantcompounds.
Rhapontigenin and rhaponticin are isolated from R. undulatum
scavengeROS,the1,1-diphenyl-2-picrylhydrazyl(DPPH)radical,
andhydrogenperoxide(H2O2)(Zhangetal.,2007).Also,these
com-poundsdecreasemembranelipidperoxidationandcellularDNA
damage,whicharethemaintargetsofoxidativestress-induced
cel-lulardamage(Zhangetal.,2007).Therecentstudyshowedsome
ofisolatedanti-oxidantcompoundsfromRheumspeciessuchas
R. emodiprotected H9c2 cells against H2O2 (Chai et al., 2012). Inthis study,theprotective effectof hydro-alcoholicextract of
R.turkestanicumonDOX-inducedcardiotoxicitywasevaluatedfor firsttime.
Materialandmethods
Reagents
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT),
thiobarbituricacid(TBA),2,7-dichlorofluorescindiacetate (DCFH-DA),propidiumiodide(PI),N-acetylcysteine(NAC),sodiumcitrate
and Triton X-100 were purchased from Sigma (St. Louis, MO,
USA).High-glucoseDulbecco’sModifiedEaglesMedium(DMEM),
penicillin-streptomycinand fetal bovineserumwere purchased
fromGibco.Trichloroaceticacid(TCA)andmalondialdehyde
bis-(dimethylacetal)(MDA)wereobtainedfromMerck(Darmstadt,
Germany).
Preparationofextracts
TherootofRheumturkestanicum Janisch.,Polygonaceae,was
collected from Chenar, a village in Zavin Rural District, Kalat
County,RazaviKhorasanProvince,Iran.Theplantwasidentified byM.R.Joharchi,fromFerdowsiUniversityofMashhadHerbarium.
Voucherspecimen(No.21377)wasdepositedinFerdowsi
Univer-sityofMashhadHerbarium.DriedR.turkestanicumroot(20g×3)
wasgroundintofinepowderandthen 50gofthispowderwas
subjectedtoextractionwith70%ethanolinaSoxhletapparatusfor 48h.Thehydro-alcoholicextractwasthendriedonawaterbath andkeptfrozeninlessthan−18◦Cforthefollowinguse.Theyield ofextractwas19%(w/w).Theextractwasdissolvedin dimethyl-sulfoxide(DMSO)toafinalconcentrationof50mg/mlbeforebeing usedincytotoxicityandapoptosisassays.
Cellculture
Rat heart cell line H9c2 wasobtained from American Type
Culture Collection (ATCCCRL-1446)and maintainedat 37◦C in
ahumidifiedatmospherecontaining5%CO2.Thecellswere
cul-turedinDMEMsupplemented with10%fetalbovineserumand
100units/mlpenicillinand100g/mlstreptomycin.Forthe
exper-iments,theywereseededin96-welland 24-wellcultureplates
forMTT/ROSandMDAassays,respectively.Forapoptosisassay,
cellswereseededat100,000/wellina24-wellplate.Alltreatments werecarriedoutintriplicate.Thecellswerepretreatedwithextract
(6–200g/ml)for2handthenincubationwascontinuedinthe
presenceoftheextractwith5Mdoxorubicinfor24h.Also,NAC
wasappliedaspositivecontrolat10and100M.
Cellviability
The cell viability was determined using a modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay as
describedpreviously(Mosmann,1983;Malichetal.,1997).Briefly,
MTTsolutioninphosphate-bufferedsaline(5mg/ml),wasadded
toeach wellatfinalconcentrationof0.05%.After3h,the
form-azanprecipitatewasdissolvedinDMSO.Theabsorbanceat570
and620nm(background)wasmeasuredusingaStatFAX303plate
reader.Theexperimentwascarriedoutintriplicate.
Lipidperoxidationassay
TheleveloflipidperoxidationwasestimatedbymeasuringMDA whichistheendproductoflipidperoxidation.Attheendof incu-bation,thecellswerescrapedandcentrifugedat13,000×gat4◦C for30min(BuegeandAust,1978).Then,400lofTCA(15%)and
800lofTBA(0.7%)wereaddedto500lofcellsamples.The
mix-turewasvortexedandthenheatedfor40mininaboilingwater
bath.Then,200lofthesamplewastransferredto96-wellplate
andthefluorescenceintensitywasreadwithexcitation/emission of480/530nm.Theexperimentwascarriedoutintriplicate.
Measurementofreactiveoxygenspecies
IntracellularROSlevelwasevaluatedusingafluorescentprobe,
DCF-DA (Wu and Yotnda,2011). Atthe end of incubation, the
cellsweretreated(30min)withDCFH-DA(10M)at4◦Cinthe
dark.Then,thefluorescenceintensitywasdetectedwith
excita-tion/emissionof485/530nm.Theexperiment wasperformedin
triplicate.
Apoptosis
ApoptoticcellsweredetectedbyusingPIstainingofthetreated
cellsfollowedbyflow cytometrytodetecttheso-calledsub-G1
peak.Briefly,H9c2cellswereculturedovernightina24-wellplate andpretreatedbyR.turkestanicumfor4handthentreatedwith DOXfor24h.Floatingandadherentcellswerethenharvestedand incubatedat4◦Covernightinthedarkwith500lofahypotonic buffer(50g/mlPIin0.1%sodiumcitrateplus0.1%TritonX-100).
Theexperimentwascarriedoutintriplicate(Nicolettietal.,1991).
Statistics
Alldatawereexpressedasmean±SEM.Statisticalanalysiswas performedusingonewayanalysisofvariance(ANOVA)followedby Tamhane’sT2posthoctest.Differenceswereconsideredsignificant atp<0.05.
Results
EffectofDOXoncellviability
Incubation ofthecells withdifferent concentrationsof DOX
(2.5–20M) showed that the cell viability reduced in a dose
dependentmanner(Fig.1).
EffectofR.turkestanicumextractoncellviability
Incubationwith6MDOXsignificantlydecreasedcellviability
to55±4.5%ofcontrol(p<0.001).Pretreatmentwith12–200g/ml
ofR. turkestanicum couldincrease theviabilityof H9c2cellsto 71±0.68% (p<0.05), 74.6±2.5% (p<0.01), 79±1.3% (p<0.001), 84±3.6%(p<0.001)and 88±1.3%(p<0.001) ofcontrol, respec-tively(Fig.2).However,atdoseof6g/ml,R.turkestanicumwas
notabletoprotectH9c2cellsagainstDOX-inducedcytotoxicity.
AlsopretreatmentofcellswithNACatdosesof10and100M,
protectedcellsagainstDOX(5M)atdoseof100M(69±1.8%,
0 20 40 60 80 100 120
20 10
5 2.5
Cont
Cell viability (% of control)
∗
∗∗∗
∗∗∗ ∗∗∗
Concentration of DOX (µM)
Fig.1.Dose–responsecurveofDOXonviabilityofH9c2cells.Thecellsweretreated withdifferentconcentrations(2.5–20M)ofDOXfor24h.Thecellviabilitywas quantitatedbyMTTassay.Resultsaremean±SEMfromthreeindependent experi-ments.***p<0.001,*p<0.05versuscontrol.
0 20 40 60 80 100 120
Cell
viability
(%
of
control)
Concentration (µg/ml)
Cont DOX
NAC10µMNAC100µM
6 12 25 50
100 200 ###
∗∗∗ ∗∗ ∗
∗∗∗ ∗∗∗
DOX (5µM)
∗
Fig.2.EffectofRheumturkestanicumextractandNAC(positivecontrol)on via-bility of H9c2cells. The cells were pretreated with different concentrations (25–200g/ml)oftheextractfor2h,thenexposedtodoxorubicin(DOX)for24h. ThecellviabilitywasquantitatedbyMTTassay.Resultsaremean±SEMfrom threeindependentexperiments.###p<0.001versuscontrol,*p<0.05,**p<0.01and
***p<0.001versusDOX.
EffectofR.turkestanicumonROSproduction
Asexpected,DOXcausedasignificantincreaseinthelevelof ROS inH9c2 cells (217±4.6% ofcontrol, p<0.001).The extract
at concentrations of 25, 50, 100 and 200g/ml was able to
decreaseintracellularROSlevelto186±5.2%(p<0.01),171±4.6% (p<0.001),146±1.1%(p<0.001)and132±2.6%(p<0.001)of con-trol,respectively(Fig.3).Also,NACatdoseof100Msignificantly
decreasedROSproduction(165±2.3%,p<0.001)(Fig.3).
EffectofR.turkestanicumextractonMDAlevel
The level of lipid peroxidationwas evaluated by measuring
of MDA level, which is the end product of lipid peroxidation.
As shown in Fig. 4, exposure of the cells to DOX resulted
in a significant increase of MDA level (168±6.5%, p<0.001)
as compared to control cells cultured in the absence of DOX
(100±1.3%). The content of MDA was significantly decreased
in the cells pretreated with 25g/ml (137±1.5%, p<0.01), 50g/ml(129±2.1%,p<0.001),100g/ml(117±3.3%,p<0.001), or200g/ml(110±4.1%,p<0.001)ofR.turkestanicum.Also,NAC
0 50 100 150 200 250
200 100 50 25 NAC100µM DOX
Cont
RO
S
(% of
con
tr
ol)
###
∗∗ ∗∗∗
∗∗∗ ∗∗∗
DOX (5µM)
∗∗∗
Fig.3.TheeffectsofRheumturkestanicumextractandNAC(positivecontrol)on intracellularROSlevelofunderdoxorubicintreatmentinH9c2cells.Thecellswere pretreatedwithdifferentconcentrations(25–200g/ml)oftheextractfor2h,then exposedtodoxorubicin(DOX)for24h.Resultsaremean±SEMfromthree inde-pendentexperiments.###p<0.001versuscontrol,**p<0.01and***p<0.001versus
DOX.
0 40 80 120 160 200
200 100 50 25 NAC100µM DOX
Cont
MD
A
(%
of
co
nt
rol)
DOX (5µM)
###
∗∗ ∗∗∗
∗∗∗ ∗∗∗
∗∗∗
Fig.4.TheeffectsofRheumturkestanicumextractandNAC(positivecontrol)on MDAcontentunderdoxorubicintreatmentinH9c2cells.Thecellswerepretreated withdifferentconcentrationsoftheextract(25–200g/ml)for2h,thenexposedto 5Mdoxorubicinandincubatedfor24h.Resultsarethemeans±SEMfromthree independentexperiments.###p<0.001versuscontrol,**p<0.01and***p<0.001
versusDOX.
atdoseof100MsignificantlydecreasedMDAcontent(120±2.8%,
p<0.001)(Fig.4).
EffectofR.turkestanicumonapoptoticcelldeath
TheproportionofapoptoticcellswasmeasuredwithPIstaining
ofDNAfragmentationbyflowcytometry.Theextractinduceda
sub-G1peak(oneofthereliablebiochemicalmarkersofapoptosis) inflowcytometryhistogramoftreatedcellscomparedtocontrol (Fig.5).
Discussion
Our findings showed doxorubicin increased cell death, ROS
generation, MDA production and induced apoptotic cell death.
R.turkestanicumdecreasedcytotoxiceffectsofdoxorubicinin car-diomyoblast cells. The extract increased cell viability, reduced
ROS and MDA production. Also, NAC as positive control,
pro-tected the cells against DOX via reduction of ROS and MDA
levels.
DOX,asananthracyclineantibiotic,hasbeenused incancer
therapy for three decades. However,the clinicaluseof DOX is
limitedbecauseofitincreasedriskofcardiotoxicity(Wenckeretal., 2003;Xinetal.,2009).Inaddition,DOXaffectonspecificenzymes,
512 64 64
0 0 0
100 101 Gm: 28.20 CV: 67.59 [0-637] 216
Gm: 10.51 CV: 60.12 [0-592] 7265
Gm: 7.78 CV: 66.28 [0-519] 4018
Gm: 6.98 CV: 68.24 [0-540] 6087 (60.9%) (2.2 %)
Gm: 11.09 CV: 51.73
[0-540] 4827(48.3 %) (40.2 %) (72.7%)
M1
FL2-H FL2-H FL2-H
M1 M1
M1 M1
M1
Control (2.2%) DOX (72.7%) 25µg/ml (60.9%)
50µg/ml (48.3%) 100µg/ml (40.2%)
Gm: 66.99 CV: 21.85 [0-665] 5397 (27.0 %)
200µg/ml (27%)
102 103 100
101 102 103
64
0
100 101 102 103
64
0
512
0 100 101 102 103
100 101 102 103
100 101 102 103 104
Fig.5.TheeffectoftheR.turkestanicumextractonapoptosisunderdoxorubicintreatmentinH9c2cellsusingPIstainingandflowcytometry.Resultsarethemeans±SEM fromthreeindependentexperiments.###p<0.001versuscontrol,**p<0.01and***p<0.001versusDOX.
accumulationofthesedefectsmayultimatelyresultinirreversible cardiacfailure(Tokarska-Schlattneretal.,2006).Themain
mech-anismof DOX-inducedcardiotoxicity is production ofROS as a
derivativeoftheDOXmetabolism(Liuetal.,2009;Zhangetal., 2009). However,superoxideradicals are involved in other ROS andgeneratehydroxylradicalandhydrogenperoxide(Bastetal., 2007).Cardiactissueissensitivetooxidativedamagebecauseof itshighoxidativemetabolismandlowantioxidantdefensesinthis organcomparedwithothers(Pintoetal.,1986;Shug,1987).Inthe presentstudyH9c2cellswereusedasapharmacologicalmodelto evaluatethepotentialcardioprotectiveeffectofR.turkestanicum
against doxorubicin. The results showed that R. turkestanicum
hasprotectiveeffect onH9c2cells against DOX-induced
oxida-tivestress.H9c2cells aremorphologicallysimilar toimmature
embryoniccardiomyocytes.Consideringthatthesecellspreserve
electricaland hormonalsignalpathwaysfoundin adultcardiac
cells (Sheng et al.,2010), theyare a usefulmodel for studying
oxidativestress-inducedcardiomyocytedamage(Winsteadetal.,
2005).Inthismodel,DOXsignificantlyincreasedlipid peroxida-tionandinducedtheapoptoticrate.Thesechangesaresimilarto theDOX-induceddeleteriouseffectsonnormalcardiaccellswhich leadtothelossofcardiomyocytesviability.Accordingtocurrent
reports, generatingROS by DOX causes mitochondrial damage,
whichmayleadtocardiomyocyteapoptosis,necrosis,ordeath.
Thestudieshave shown,somenatural substances,suchas
cur-cumin,naringenin-7-O-glucoside,andplantainosideDprevented DOX-inducedmitochondrialinjuriesincardiomyocytes(Kimetal., 2007;Hosseinzadehetal.,2011).Inourstudy,weusedDCFH-DAas anintracellularmethodofROSdetection.Theresultsdemonstrated thatR.turkestanicumcansignificantlyreducetheintracellularROS
production byDOX in H9c2cells. We alsomeasuredlipid
per-oxidation(MDAformation)inH9c2cells.ThereductionofMDA
contentsuggestedthatR.turkestanicumcanattenuatethe oxida-tivestressinducedbyDOXinH9c2cells.AlsoresultsshowedNAC aspotentanti-oxidantprotectedcellsagainstDOXviareductionof oxidative-stress.TherecentstudieshaveshownNACinhibitedROS
production,lipidperoxidation,andrestoredantioxidantenzyme
activitiesbuthadmodesteffectontheprotectionofDOX-induced
cardiaccelldeathascomparedtoothernaturalsourcesof antiox-idants(Shietal.,2009).NACinducedlessprotectiveeffectsthan
R.turkestanicumextractagainstDOX-inducedcytotoxicity. How-everprotectiveeffectsofextractmaybemediatedviamechanisms similartoNAC.
Conclusion
ThisstudyshowsthatR.turkestanicumisanovelpotent pro-tectiveagentagainstDOX-inducedcardiotoxicity,theprotective effectsmaybeduetoitsantioxidantproperties.Thiseffectis cor-relatedwithreducingoxidativestressandinhibitionofapoptosis
induction. Butmore investigations are needed toelucidate the
probableunderlyingmechanismsofthesetherapeuticeffects.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
thatnoexperimentswereperformedonhumansoranimalsfor
thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
ThisworkwassupportedbyToxicologyMedicalResearch
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