RevistaBrasileiradeFarmacognosia27(2017)776–779
w ww.e l s e v i e r . c o m / l o c a t e / b j p
Short
communication
Impact
of
chrysosplenetin,
per
se
or
in
combination
with
artemisinin,
on
breast
cancer
resistance
protein
(Bcrp)/ABCG2
mRNA
expression
levels
in
mice
small
intestine
Wei
Ma
a,1,
Yuanyuan
Zhang
a,1,
Yanli
Zhang
b,1,
Chenxu
Zhang
a,
Jianhuan
Wang
a,
Liping
Ma
a,
Bei
Yang
a,
Xiuli
Wu
a,c,d,∗,
Jing
Chen
a,c,d,∗aSchoolofPharmacy,NingxiaMedicalUniversity,Yinchuan,China
bDepartmentofPathogenBiologyandImmunology,SchoolofBasicMedialScience,NingxiaMedicalUniversity,Yinchuan,China
cNingxiaEngineeringandTechnologyResearchCenterforModernizationofHuiMedicine,Yinchuan,China
dKeyLaboratoryofHuiEthnicMedicineModernization,MinistryofEducation(NingxiaMedicalUniversity),Yinchuan,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received22February2017 Accepted14June2017
Availableonline16November2017
Keywords: Chrysosplenetin Artemisinin
Bcrp/ABCG2mRNAexpression Westernblot
RT-qPCR
a
b
s
t
r
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Ourpreviousworkrevealedthatchrysosplenetinincombinationwithartemisinininhibitedinvivo
P-glycoprotein(P-gp,oneofclassicmulti-drugresistanceproteins)mediateddigoxintransportation
activitybyreversingtheupregulatedP-gp/Mdr1mRNAexpressionlevelsbyartemisinin.Therefore,
chrysosplenetinmightbeapotentialartemisinin-resistancereversalagentasaP-gpinhibitor.Butit
stillremainsunknownifchrysosplenetinhasanimpactonanotherpivotalmulti-drugresistance
pro-tein,breastcancerresistanceprotein(Bcrp),whichisco-expressedwithP-gpinapicalmembraneof
intestinalepithelialcellandoverlapssomeofthesubstratesandinhibitors.Thisstudy,therefore,further
addressedtheimpactofchrysosplenetin,perseorincombinationwithartemisin,onBcrp/ABCG2mRNA
expressionlevelsinmicesmallintestinedeterminedbywesternblotandrealtime-quantitative
poly-merasechainreaction(RT-qPCR)assay.Thedrugswereintragastricallyadministratedonceperdayfor7
days.Novobiocin,aknownBcrpinhibitor,wasobservedtohavenoimpactonBcrp/ABCG2levelswithor
withoutartemisininversusvehicle.Interestingly,artemisininaloneattenuatedBcrplevelwhile
chrysos-plenetinaloneincreasedit(p<0.05).RelativemRNAlevelwassignificantlydecreasedwhenco-used
withartemisininandchrysosplenetininratioof1:2(p<0.05).Thediscrepantresultsforchrysosplenetin
onBcrp/ABCG2mRNAexpressionsmightbecloselyrelatedtothetranscriptionalorposttranscriptional
regulation.
©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeFarmacognosia.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Introduction
ThehumanATP-binding cassette(ABC) proteinsbelongto a largeproteinsuperfamily;thusfar,48ABCtransportershavebeen identifiedinhumansand twelveofthemhavebeenrecognized asputativedrugtransporters.Amongthem,P-glycoprotein(P-gp, genesymbol ABCB1orMdr1), themultidrugresistanceprotein 1(MRP1,genesymbolABCC1),andthebreastcancerresistance protein (BCRP, gene symbol ABCG2)are the bestdistinguished andmostparamountdrugtransportersofmultidrugresistance (MDR)(GilletandGottesman,2011).Thereismountingevidence
∗ Correspondingauthors.
E-mails:[email protected](X.Wu),[email protected](J.Chen).
1Theseauthorscontributedequallytothiswork.
tosupportthathumanBCRP/ratBcrpplaysapivotalroleindrug disposition by expelling a broad range of structurallydifferent metabolitesoutofcells.Bcrpsubstantiallyoverlapswithsubstrates andinhibitorsofP-gporMRP1andresemblesP-gpintissue distri-butionandexpression.Inthisregard,averyactiveBcrptransporter couldprobablydiminishdrugdeliverytothetargetorganswhich leadstoMDR,despiteperipheraldrugconcentrationsbeingwithin theirtherapeuticextent.
Bcrp is composed of 655 amino acids (72kDa) and orga-nized into six transmembrane ␣-helices, containing only one nucleotide-binding domain (NBD) near its N-terminal and one membrane-spanningdomain(MSD)(Lecerf-Schmidtetal.,2013; Noguchiet al., 2014; Maoand Unadkat, 2015).Therefore, Bcrp perseisahalftransporterthattransformstoafunctionalefflux pump when a disulfide bridge at Cys 603 of two proteins is
https://doi.org/10.1016/j.bjp.2017.06.005
W.Maetal./RevistaBrasileiradeFarmacognosia27(2017)776–779 777
homodimerized(Lecerf-Schmidtetal.,2013;Noguchietal.,2014; MaoandUnadkat,2015)andconfersanatypicalMDRphenotype (Lagasetal.,2009;Nietal.,2010).
Todate,artemisininantimalarialdrugsarestilloftheutmost importance in theworldwide combination therapy of resistant falciparummalaria(Tripathietal.,2013).Artemisininresistance, defined as a delayed clearance of parasites after clinical ther-apy,hasbeenreported(Meshnicketal.,1996;White,2004).The mechanismofartemisininresistanceremainsunclearand proba-blydominatedbymultiplemechanisms,whichinvolvednumerous multidrugresistanceproteinssuchasseveralmembersoftheABC transportersuper-family(Alcantaraetal.,2013).Thiscausesalow bioavailabilityandbloodconcentrationforterminaldrugs(Meng etal.,2014).
Numerous polymethoxylated flavonoids are discovered to modulate the activity of drug metabolizing enzymes and ABC transporters,whichraisesthepotentialforalterationsinthe phar-macokineticsofsubstratedrugs(Wesolowska,2011;Yuanetal., 2012).Chrysosplenetin is one of theknown polymethoxylated flavonoidsinArtemisiaannuaL.andotherseveralChineseherbs (Numonov et al., 2015). In our previouswork, chrysosplenetin wasobservedtoimprovethebioavailabilityandanti-malarial effi-ciencyofartemisininincombinationratioof1:2partiallyrelying on its strong inhibition on rat CYP3A metabolic activity in an un-competitivemanner (Wei et al., 2015)and onP-gp in vivo
effluxefficacy(Yangetal.,2016)viareversingtheup-regulated P-gp/Mdr1mRNAexpressionlevelsbyartemisinin(Maetal.,2017). Hence,chrysosplenetinmightbea dualinhibitoronCYP3Aand P-gp. However,the function ofchrysosplenetin on Bcrp/ABCG2 expressionisstill unknown,which isanobstacle forusto sys-tematicallyevaluatethepossibilityofchrysosplenetinbeingasan inhibitorofartemisininresistance.
Therefore,wehereaimedtofurtherinvestigatetheimpactof chrysosplenetin in theabsence and presence of artemisinin on Bcrp/ABCG2expressionlevelsusingbywesternblotandreal time-quantitativepolymerasechainreaction(RT-qPCR)methods.
Materialsandmethods
Artemisinin (white crystal) was purchased from Chongqing Huali KongguCo., Ltd. (purity ≥99.0%, Chongqing,China). CHR (purity≥98.0%)waspurifiedinourlabfromanacetonelayerof artemisininindustrialwastematerialsusingmultiplecolumn chro-matographymethods as described in theliterature (Wei et al., 2015).TheindustrialwasteswerekindlyprovidedbyChongqing HualiKongguCo.,Ltd.andthevoucherspecimen(20100102)has beendepositedwithCollegeofPharmacy,NingxiaMedical Uni-versity, for furtherreferences. Novobiocin waspurchased from HefeiBomeiBiotechnologyCo.,Ltd.(CAS:1476-53-5,purity≥90%, China).
HealthymaleICRmice,weighing18–22g,werepurchasedfrom ananimalcentreofNingxiaMedicalUniversity(Ningxia,China). Allanimalswerehousedinpolycarbonatecagesandacclimated inanenvironmentallycontrolledroom(23±2◦C,withadequate ventilationanda12-hlight/darkcycle)priortouseandwere pro-videdwithstandardlaboratoryfoodandwaterbeforeandduring theexperiments.Theexperimentalprotocolwasapprovedbythe UniversityEthicsCommittee(NingxiaMedicalUniversity,China; ethicapproval:2014-029).Allproceduresinvolvinganimalswere inaccordance withtheRegulations oftheExperimentalAnimal Administration,StateCommitteeofScienceandTechnology. Ani-malswererandomlydividedintoninegroups(n=6foreachgroup) includingnegativecontrol(0.5%sodiumcarboxymethylcellulose, CMC-Na), artemisinin alone (40mg/kg), novobiocin (positive control,100mg/kg),novobiocin–artemisinin(positivecontrol in
combination, 1:1, 100:100mg/kg), artemisinin–chrysosplenetin (1:0.1, 40:4mg/kg), artemisinin–chrysosplenetin (1:1, 40:40mg/kg), artemisinin–chrysosplenetin (1:2, 40:80mg/kg), artemisinin–chrysosplenetin (1:4, 40:160mg/kg), and chrysos-plenetin alone (80mg/kg). The drugs were intragastrically administrated once per day for consecutive seven days. Then the mice were euthanized and sacrificed by cervical vertebra dislocation. Small intestines wereharvested and cleaned using normalsalineatleastthreetimes.TotalRNAwasextractedfrom themousesmallintestineusingE.Z.N.A.TMTotalRNAKit(OMEGA
bio-tek, Norcross, GA, USA), in accordance with the manufac-turer’s instructions. RNA concentrations weremeasured witha microplate spectrophotometer (Bio-RAD,USA) at a wavelength of 260nm.RNA quality was evaluatedusing electrophoresis in 1% agarosegels. TotalRNA (3g) was reverse transcribedinto first-strandcomplementaryDNA(cDNA)usingThermoScientific RevertAidFirstStrandcDNASynthesisKit(ThermoScientific).Each cDNAsample(1l)wasamplifiedwith12lofThermoScientific MaximaSYBRGreenqPCRMasterMix(2X),ROXSolution(Thermo Scientific)and1Mofeachprimer.Amplificationwasperformed inaRT-qPCRIQ5System(AppliedBiosystems,FosterCity,USA) withthe following parameters:denaturationat 94◦C for2min followedby35cyclesofdenaturationat94◦Cfor30s,annealing at 61◦C for 30sand extension at72◦C for 45s. Thesequences of theoligonucleotideprimersused forthis studywere5′-GCA TTCGCTGTGGTTGAGT-3′(sense)and5′-TATCCGTGGCATCTC TGGA-3′(antisense)forABCG2(productsize,123bpfromSangon Biotech(Shanghai)Co.,Ltd.);and5′-GGTGAAGGTCGGTGTGAA CG-3′(sense)and5′-CTCGCTCCTGGAAGATGGTG-3′(antisense) forGAPDH(productsize,233bp,fromInvitrogen Biotechnology Co.,Ltd.).TherelativeexpressionlevelsofABCG2ineachsample (normalized tothat of GAPDH) were determinedusing 2−Ct
method.AllRT-qPCRexperimentswererepeatedthreetimes. The total proteins were harvested by KenGEN Whole Cell LysisAssayKit (KenGENBioTECH,Nanjing,China),andthe pro-teinconcentrationsweredeterminedusingKeyGENBCAProtein QuantitationAssaykit(NanjingKeyGENBiotech,China).Anequal quantityofprotein(80g)fromtotalproteinwasresolvedusing 7.5%SDS-PAGEgelandsubsequentlytransferredonto nitrocellu-losemembranes(Bio-Trace).Afterblockingthemembranewith5% non-fatmilkinTris-bufferedsaline(Biotopped)atroom tempera-turefor1h,themembranewasincubatedat4◦Cfor12hwithrabbit polyclonalantibodyagainstABCG2(1:100;sc-25822,SantaCruz) andmousemonoclonalantibodyagainst-actin(1:150;sc-47778, Santa Cruz). The membranes were incubated with horseradish Peroxidase-conjugatedAffiniPureGoatAnti-RabbitIgGand Goat Anti-Mouse IgG (ZSGB-BIO, Beijing, China) for 2h and signals wereobservedusingSuperSignalWestPico(ThermoScientific). Westernblottingbandsintensitywasquantifiedbydensitometric analysisusingImageJversion2x(NIHImagesoftware,Bethesda, MA,USA).
DatawasanalyzedusingtheSPSS18.0software(IBM,USA)and submittedtoaone-wayanalysisofvariance (ANOVA)todetect significantdifferences betweenstudygroups. Turkey’s test was appliedtoidentifyanydifferencebetweenmeansusinga signif-icancelevelofp<0.05.
Resultsanddiscussion
778 W.Maetal./RevistaBrasileiradeFarmacognosia27(2017)776–779
A
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β-actin p
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xpression le
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Negativ e contro
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Novo biocin
Novobiocin-artemisinin (1:1)
Artemisinin-chr
ysosplenetin (1:0.1 )
Artemisinin-chr
ysosplenetin (1:1 )
Artemisinin-chr
ysosplenetin (1:2 )
Artemisinin-chr
ysosplenetin (1:4 )
Chr
ysosplenetin alone
Fig.1.EffectofchrysosplenetinperseorincombinationwithartemisininonBcrpexpressionlevelinmicesmallintestine.Datawereexpressedasmeanvalues±SD(n=6) foreachgroup.(A)Westernblottingbands.(B)QuantificationofBcrpassessedbywesternblottinganalysiswasnormalizedtotheexpressionlevelof-actinantibody. Differentlower-caselettersindicatesignificantdifferencesbetweendifferentgroupswithasignificancelevelofp<0.05.
Negativ e contro
l
Artemisinin alon
e
Novo biocin
Novobiocin-artemisinin (1:1)
Artemisinin-chr
ysosplenetin (1:0.1 )
Artemisinin-chr
ysosplenetin (1:1 )
Artemisinin-chr
ysosplenetin (1:2 )
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ysosplenetin (1:4 )
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ysosplenetin alone
ab
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bc bc
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Relative mRNA levels
Fig.2. ImpactofchrysosplenetinperseorincombinationwithartemisininonABCG2mRNAexpressionlevelinmicesmallintestine.Dataweredescribedasmeanvalues±SD (n=6)foreachgroup.Differentlower-caselettersindicatesignificantdifferencesbetweendifferentgroupswithasignificancelevelofp<0.05.
inaccordance withtheliterature(Shiozawaet al.,2004)which reportedthatnovobiocinisacompetitiveinhibitorofBcrp. There-fore,theexpressionofBcrpdoesnotchange.Whencomparedwith artemisinin alone, however, novobiocin alone, chrysosplenetin
alone and artemisinin–chrysosplenetin combination groups in ratioof1:2and1:4remarkablyelevatedthelevels(p<0.05).
W.Maetal./RevistaBrasileiradeFarmacognosia27(2017)776–779 779
Fig.2.ContrarytotheresultsofBcrpexpression,artemisininalone increasedABCG2mRNAlevelwhileartemisinin–chrysosplenetin (1:2)significantlyattenuateditversusnegativecontrol(p<0.05). Moreover,allstudygroupsreversedtheupregulatedrelativemRNA levelsbyartemisinin(p<0.05).
Inthispaper,contradictingdataforchrysosplenetininthe pres-enceorabsenceofartemisininontheregulationofBcrp/ABCG2 mRNAexpressionareobserved.Artemisininwasfoundto down-regulate Bcrp protein expression but upregulate ABCG2 mRNA level.Itmightbebecausetranscriptionalorposttranscriptional reg-ulationforBcrpproteinplaysapredominantroleaccordingtothe literature(MaoandUnadkat,2015).Artemisinin,therefore,might promoteitsresistanceafteramulti-doseoraladministrationfrom theviewofP-gpbasedonourpreviouswork.It indicatesarisk ofartemisininresistanceinitsclinicaltherapy.Butthe possibil-ityofartemisininresistanceseemstobeweakenedatBcrpprotein expressionlevel.Sothisworkwillbeofbenefittoaccurately eval-uatetheventureof artemisininonitsresistancefromdifferent aspects.
Secondly,chrysosplenetinshowedaregulatingfunctionon P-gp/Mdr1andBcrp/ABCG2, totallyopposite toartemisininbased on our previous and this work. The reason might be closely relatedtothecompetitionofartemisininandchrysosplenetinon theidenticalbindingsitesinP-gpor Bcrpprotein.It needs fur-ther researchto certify thepotential mechanism in our future study.
Ingeneral,artemisininandchrysosplenetinhavetheopposite effectonBcrp/ABCG2mRNAexpressionlevels.Whentheyare co-usedin ratio of1:2,ABCG2 mRNAlevel wasremarkably down regulated.Theresultswillbehelpfultosomeextentunderstandthe mechanismofchrysosplenetinasapotentinhibitoronartemisinin resistanceatP-gp/Mdr1mRNAandABCG2mRNA(encodinggene forBcrp)levels.However,itistooearlytoreachaneventual con-clusionwhetherchrysosplenetincouldbeclinicallyco-usedwith artemisinintodelayorreverseartemisininresistance.Thedecisive factorsinformationofartemisininresistanceandtheregulationof chrysosplenetinonthesefactorsdeservetostudybeforethearrival ofafinalconclusion.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Author’scontributions
WM,YZandYZcontributedinrunningthelaboratorywork.CZ andJWanalyzedthedata.LMandBYrevisedthemanuscript crit-ically.XWand JCdesignedthestudy,supervisedthelaboratory workandcontributedtomodifythemanuscript.Alltheauthors havereadthefinalmanuscriptandapproveditssubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
ThisworkwasfinanciallysupportedbytheNationalNatural ScienceFoundationofChina(No.81560580).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.06.005.
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