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Picolinate ruthenium(II)-arene complex with in vitro antiproliferative and antimetastatic properties: comparison to a series of ruthenium(II)-arene complexes with similar structure.
JournalofInorganicBiochemistry108(2012)53–61
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Picolinate ruthenium(II)–arene complex with in vitro antiproliferative and
antimetastatic properties: Comparison to a series of ruthenium(II)–arene
complexes with similar structure
Nevenka Gligorijevića, Sandra Aranđelovića, Lana Filipović a, Ksenija Jakovljević a, Radmila Janković a,
Sanja Grgurić-Šipka b, Ivanka Ivanović b, Siniša Radulovića,⁎ , Živoslav Lj. Tešić b
aInstituteforOncologyandRadiologyofSerbia,Pasterova14,11000Belgrade,Serbia
bFacultyofChemistry,UniversityofBelgrade,Studentskitrg12-16,11000Belgrade,Serbia
a r t i c l e i n f o a b s t r a c t
Articlehistory: Inourpreviousstudy,ruthenium(II)-p-cymenecomplexesofgeneralformula[(η6-p-cymene)Ru(L)Cl2],L:3-
Received18July2011 acetylpyridine (1), 2-amino-5-chloropyridine (2); and [(η6-p-cymene)Ru(HL)Cl], HL: 2,3-pyridinedicar-
Receivedinrevisedform8December2011 boxylicacid(3),2,4-pyridinedicarboxylicacid(4),revealedlowantiproliferativeactivity,exceptcomplex
Accepted9December2011 [(η6-p-cymene)RuCl(picolinicacid)]·H O(5)whichexhibitedIC around80μM.Inthisstudywefurtherin-
Availableonline16December2011 2 50
vestigatedinvitropotentialofantimetastaticactionofrutheniumcomplexesonHeLaandtwoendothelial
celllines.Comparisonofstructureandactivityoffivecomplexes indicatedheterogenicmode ofactivity,
Keywords:
Ruthenium(II)–arene withregardtothepotentialofantimetastaticandantiproliferativeeffect.Replacementofsubstitutedpyri-
Antiproliferativeactivity dineligandwithpicolinicacid(complex5)aroundRu(II)centercontributedtocomplexcytotoxicityandru-
Antimetastatic theniumDNAbindingaffinity.Analysisofruthenium(II)accumulationinDNAandproteinfractionsofHeLa
ERCC1 cells,usingICP-OESrevealedsignificantlyhighercontentofcomplex5inDNAfractionincomparisontothe
PARP othertestedcompounds.Italsoalteredcellcycleprogression,affectedexpressionofDNArepairenzymes
ERCC1andMSH2,andshowedenhancedactivityincombinationwith3-aminobenzamide.
Regardlessoftheireffectoncellgrowth,Ru(II)complexesexertedantimetastaticeffectonseveraltumorcell
linesinvitro,achievedmostlybytheeffectoncelladhesion,migrationandangiogenesis,whilepicolinate
ruthenium(II)–areneadditionallyexertedinhibitoryeffectonextracellularmatrixdegradation.
©2011ElsevierInc.Allrightsreserved.
1.Introduction activity [1]. Interestingly, some ruthenium complexes demonstrate
greater efficacy against cancer metastasis than against primary tu-
Cisplatinandstructurallyrelatedplatinumbaseddrugsrepresent mors. Examples of such complexes include the two complexes
unique and important class of antitumor agents. Among the great NAMI-AandKP1019whichunderwentphaseIIclinicaltrials[1].
number of non-platinum metal complexes examined for anticancer Themostnumerousgroupofcytotoxicrutheniumcompoundsis
characteristicsuptodate,recentattentionisfocusedonruthenium Ru(II)–arenecomplexes,whichweredevelopedprimarilybyDyson
complexes.Rutheniumcomplexesarereportedtoshowpatterns of andco-workersandSadlerandco-workers,althoughnoneofthese
antitumor activity and clinical toxicity that are distinct from those compoundshasyetenteredclinicaltrials[5].
ofplatinumcomplexes[1–4]. Inourpreviousstudiesseriesofruthenium(II)complexescontaining
The most likely interactions of ruthenium complexes are with: thep-cymeneligandandapyridinederivativecoordinatedinamono-
plasma proteins, collagens of the extracellular matrix (ECM),actins dentate or bidentate manner were synthesized and characterized by
on the cell surface, regulatory enzymes within the cell membrane standardanalyticaltechniques[6,7].
and/orinthecytoplasmandDNAinthecellnucleus,whichcangive Analysis of cell growth inhibition caused by Ru complexes of
them diversity of modes of action involving both extra- and intra- structural formula [(η6-p-cymene)Ru(L)Cl ], (L: 3-acetylpyridine
2
cellularprocesses[5]. (1), 2-amino-5-chloropyridine (2)); and [(η6-p-cymene)Ru(HL)Cl],
In the last 30years, a large number of ruthenium containing (HL:2,3-pyridinedicarboxylicacid(3),2,4-pyridinedicarboxylicacid
agents have been synthesized and tested for potential anticancer (4)), and [(η6-p-cymene)RuCl(picolinic acid)]·H O (5) (Fig. 1) on
2
panel of tumor cell lines (HeLa, B16, MDA-MB-361, MDA-MB-453
and LS-174) revealed low antiproliferative activity (IC >200μM),
50
⁎ Correspondingauthor.Fax:+381112067294. exceptforcompound5withIC 50 81.97μMasalreadyreported[6,7].
E-mailaddress:sinisar@ncrc.ac.rs(S.Radulović). KnowingthatfewRucomplexeswhichfailedtraditionalanticancer
0162-0134/$–seefrontmatter©2011ElsevierInc.Allrightsreserved.
doi:10.1016/j.jinorgbio.2011.12.002
54 N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61
Fig.1.Structuresofinvestigatedruthenium-p-cymenecomplexes.
drugscreeningprogramsbasedontheirlevelofgenotoxicityandcy- DNA-repair-dependent signaling which includes components of
totoxicity in vitro, have demonstrated antimetastatic activity [1–3], NERorMMRisutilizedincellresponsetoruthenium(II)-p-cymene
wefurtherinvestigatedmechanismofactionofourRu(II)-p-cymene complexes, by following expression of ERCC1 (mRNA and protein
complexes, and extended our research on endothelial cells lines: level)andMSH2(proteinlevel)usingQuantitativeReal-TimePCR
EA.hy926(transformedhumanumbilicalveinendothelialcells)and (RQ-PCR) and Western blot. PARP is another nuclear enzyme re-
MS1(murineendothelialcells),asmodelsystemsforinvitroanalysis sponsible for recognition and repair of single-strand DNA-breaks,
ofantiangiogenicactivity[8,9]. which might be induced by metal based drugs or oxidative stress
PotentialofourRu(II)-p-cymenecomplexestomodulateseveral [15–17]. PARP also takes role in cell cycle progression and cell
steps of metastatic progression like adhesion, migration, invasion, deathsignaling,andispotentialmoleculartargetfordevelopment
proteoliticdegradationofextracellularmatrixandformationofnew of PARP inhibitors as chemo-sensitizer to DNA-damaging agents
bloodvesselshasbeenstudiedinvitro. [18].InordertodeterminecontributionofPARPincellresponseto
Intracellular distribution of ruthenium among protein and DNA ruthenium-basedcomplexesweinvestigatedwhetherPARPinhibi-
cellularfractionsinHelacellswasanalyzedbyICP-OES,whileeffect tion by 3-aminobezamide may potentiate action of complex 5 on
oncellcycleprogressionwasdeterminedbyFlowcytometry. HeLacells.
Though thepharmacological target of rutheniumcomplexeshas
notbeenunequivocallyidentified,itisgenerallyacceptedthattheir 2.Resultsanddiscussion
cytotoxicityisrelatedtotheirabilitytobindDNA,comparablytoplat-
inumbaseddrugs[10].CellDNA-damageresponsedependsuponthe 2.1.ResultsofMTTassay
natureofthelesionandrecruitscomplexsystemofDNA-damagerec-
ognition/repairproteins. AntiproliferativeactionofaseriesofRu(II)-arenecomplexesof
Major mechanisms involved into repair of cisplatin induced structural formula [(η6-p-cymene)Ru(L1,2)Cl ], where L1,2 is 3-
2
DNA-lesions are reported to be Nucleotide Excision Repair (NER) acetylpyridine(1)and2-amino-5-chloropyridine(2),correspond-
andMismatchRepair(MMR)mechanisms,androleofexcisionre- ingly, and [(η6-p-cymene)Ru(H L3,4)Cl], where H L3,4 represent
2 2
pair cross-complementing 1 (ERCC1) protein and MutS homolog 2,3-pyridinedicarboxylicacid(3),2,4-pyridinedicarboxylicacid(4)
(MSH)proteinsincellresponsetoplatinumdrugshasbeenexten- havebeenreportedpreviouslyonsixtumorcelllinesshowingno
sively investigated [11–14]. In this study we evaluated whether cytotoxicactivityupto200μM[6].Inthisstudyadditionalstructure
N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61 55
Table1 marginallythemostsensitivetothiscomplex.Prolongationofincu-
ResultsofMTTassayforcomplexes1−5arepresentedasIC50values,obtainedafter48 bationtime(from48hto72h)didnotimprovetheantiproliferative
or72htreatment.
activity.
Complex IC50(μM) Asalreadymentioned,thelowcytotoxicityinvitroisverycommon
EA.hy926 MS1 HeLaa featureforruthenium-basedanticanceragentswhichwereshownina
numberofcasestopossessgoodantitumor,andaboveall,antimetastatic
1 48h >300 >300 275.71±1.57
properties[20,21].
72h >300 270.04±3.71 244.00±1.91
2 48h >300 220.82±15.98 >300
72h 258.46±2.79 218.91±6.21 283.20±3.12 2.2.Cellcycle
3 48h >300 >300 >300
72h 259.42±9.83 >300 >300
Theeffectofinvestigatedruthenium(II)-p-cymenecomplexeson
4 48h >300 >300 >300
72h >300 >300 >300 cellcycleprogressionofHeLaandEA.hy926cellswasexaminedby
5 48h 95.47±7.36 78.3±4.09 81.97±14.17 flow cytometry after continual treatment for 24h, using staining
72h 88.36±8.10 108.54±9.45 149.35±12.06 withPI[22].Examinationofthehistogramsindicatedthatonlycyto-
a ResultsofactivityonHeLacellsarealreadypublished[6,7]. toxiccomplex5inducedperturbationsofcellcycleofHeLacells,and
resultsarepresentedinFig.2.Othercomplexeshadnoeffectoncell
cycle phase progression in both HeLa and EA.hy 926 (data not
presented).
activity comparison has been performed on endothelial cell lines Obtainedresultsshowthatcomplex5induceddecreaseofpercent
(modelsystemsforinvitroangiogenesis)byMTTassay[19],includ- ofcellsinG1andslightarrestintheSphaseofcellcycle,withnoap-
ingcompoundofstructuralformulae[(η6-p-cymene)RuCl(picolinic optotic effect (evaluated as Sub-G1 fraction). Slower progression
acid)]·H O (5) [7]. Results are shown in Table 1. in terms of IC through replication phase indicates its preferential binding to DNA
2 50
values,determinedfrom3to4independentexperiments. andcorrelateswiththeresultsobtainedbyICP-OES(seebelow).
Antiproliferativeactivityofinvestigatedcomplexesonendothelial
cellswerelow,indicatedbyhighIC 50 values(>200μM),exceptfor 2.3.Intracellulardistributionofruthenium(II)inproteinvs.DNAfraction
complex 5. Ruthenium compound 5 exhibited cytotoxic activity on
allinvestigatedcelllinesafterboth48hand72hofcontinualincuba- Whileinvestigatingmechanismofactionofpotentialdrugcandi-
tion,withIC 50 valuesfallinginthe80–150μMrange.Nosignificant datesitisimperativetolocatedruginteractionswithkeytargetsin
tumor cell type selectivity was observed, although HeLa are the cell [20]. We separately analyzed ruthenium(II) distribution
Fig.2.Effectofthecomplex5(IC50)oncellcycleprogressionofHeLacellsfollowing24hincubation.Histogramspresentedarerepresentativeofthreeindependentexperiments.
amongtheDNAandproteinfractionsinHeLacellstreatedwithinves- Other rutheniumcomplexes have not shown any inhibitory effect in
tigated ruthenium(II)-p-cymene complexes, using ICP-OES analysis, thisassay.
after24htreatmentwith100μMofinvestigatedcomplexes.Eachin-
vestigatedRu(II)compoundwasfoundintracellular,althoughexhi- 2.4.2.Invasionassay
biting different levels of intracellular accumulation and different Inordertoinvestigateinvitropotentialofrutheniumcomplexes
affinitiesforproteinorDNAbinding(Fig.3).Complex5waspreferen- toinhibitinvasionofHeLacells,weusedBDBioCoatMatrigelinva-
tiallyaccumulatedinDNAfraction,showingdrasticallyhigheraffinity sion chambers as a system with the reconstituted basement mem-
forDNA-bindingincomparisontootherinvestigatedRucomplexes, brane matrix-matrigel as an invasive substrate coated on a
which may be a reason for its cytotoxic action. Level of ruthenium microporousfiltermembrane[26].Allinvestigatedrutheniumcom-
bound to protein fraction was comparable among the tested com- plexes exhibited the potential of inhibiting HeLa cells invasion
plexes.Complexes1,3,4exhibitednoDNAbinding,whichindicates (Fig.4A).Amongthenontoxicrutheniumcomplexeswhichwereap-
thatligandstructuraldifferencesoftestedRu(II)compoundssignifi- pliedatequimolarconcentrations(100μM),complex2reducedinva-
cantlyinfluencedaffinityorstabilityofDNAinteractions.Complexes sion with 33.6% of inhibition compared to the control (100%
1and2aswellas3and4havesomecharacteristicswhichprobably invasion).Cytotoxiccomplex5exhibitedinhibitoryactivityonHeLa
obstructDNAinteractions,especiallydrugintercalationthroughη6- cell invasion up to 20%, although at lower concentration being
bonded arene (such as rotation of the organic ligand about the 41μM(whichishalfofitsIC value).
50
Ru\N bond in complexes 1 and 2; and \COOH residue in meta-
andpara-positionwithrespecttotheNatomofthearomaticringat 2.4.3.Adhesionassay
complexes3and4). Pro-adhesive properties of investigated ruthenium(II)-p-cymene
In contrast, complex 5 has a relatively planar surface, free from complexesonadhesionofHeLacellsonplasticsubstrate(uncoated
substituents, which enables interactions with DNA thus inducing bottomoftheplates)wereevaluatedasresistancetotrypsintreat-
theobservedantiproliferativeactivity. ment,whichisagoodmeasureofadhesivestrength[27].Wedeter-
mined that after 90min treatment with 100μM concentration,
2.4.Invitroantimetastaticpotential complexes 2 and 3 contributed 3 times more to the adhesion of
HeLacells,onplasticsurface,comparedtothatinthesametestper-
Tumor metastasis is one of the major obstacles to cancer treat- formedwithcontrolcells(Fig.4B).
mentandisacomplexmultistepprocessthatinvolvesinvasion,ad- Resultsoftheadhesionassayseemtobecompatiblewiththere-
hesion, direct migration, and proteolitic activity to degraded ECM sults of the matrigel invasion assay (Fig. 4A). The same ruthenium
barriers[23,24].Ru-basedcomplexesarereportedtoexhibitunique complexes2and3,whichamongthetestedcomplexescontributed
antimetastaticpropertiesregardlessoftheircytotoxiceffect[20,25]. themosttothecelladhesion,alsoexhibitedthegreatestinhibitory
Thus far, we investigated potential of our ruthenium(II)-p-cymene effect on invasion behavior of the cell, through Boyden chamber
complexestoinhibitseveralstepsoftumorcellinvasionandangio- membranes.
genesis, starting with analysis of their inhibitory activity on matrix
metalloproteinases(MMPs),inHeLacells. 2.4.4.Inhibitionofcellmigration(Scratchassay)
Woundmigrationassaywasusedasinvitroassaytodetermine
2.4.1.Gelatinzymography atfirstglancewhetherinvestigatedrutheniumcomplexeshavedi-
MMPsare the family of tissue degrading enzymesthat facilitate rectinfluenceonmigrationofHeLacells[28].Forthisassayconflu-
invasionandmetastasisthroughdissolutionofthebasementmem- entculturesofHeLacellswerewoundedandthenincubatedinfresh
brane and degradation of the ECM. Among the MMPs, MMP-9 and completemedium.Migrationofcellswasobservedperiodicallyover
MMP-2 (capable of degrading gelatin) are thought particularly im- timeunderthemicroscopeandrepresentativepicturesweretaken
portantintheinvasionofthecancercells[24]. at 24h and 48h. Complex 5 induced inhibition of migration of
TheactivitiesofsecretedMMP-2andMMP-9after24htreatmentof HeLa cells which was very visible after 48h incubation (Fig. 4D
HeLaandEA.hy926cellswereanalyzedbygelatinzymography.Results shows results of wound assay for complex 5 only). Other tested
show that only complex 5 exhibited inhibitory activity on secreted complexes1,2,3and4inducedmildinhibitioneffects(resultsnot
MMP-2andMMP-9inHeLaandEA.hy926cells,respectively(Fig.4C). shown).
18
16
14
12
10
8
6
4
2
0
1 2 3 4 5
AND
ro
snietorp
gµ/uR
gp
56 N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61
Proteins DNA
Complexes
Fig.3.Ruthenium(II)contentinHeLacells(DNAvs.proteinfractionofcells)after24htreatment,measuredbyICP-OES.Representativeexperiment.
N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61 57
Fig.4.Invitroantimetastaticpropertiesofthecomplexes.A.TheeffectofinvestigatedcomplexesonHeLacellinvasion,after24htreatmentand48hinvasiondeterminedbyMatrigel
invasionassay.Dataarepresentedasthepercentagerelativetothecontrol(untreatedcells)andarerepresentativeofoneexperimentinduplicates.B.Theeffectofcompoundsonadhesion
abilityofHeLacellstoplasticsurface(atadoseof100μMfor90min)determinebySRBassay.Dataarerepresentativeofoneoutofthreeseparateexperimentswithstandarddeviations,
comparedtonontreatedcontrols.C.GelatinzymographyofHeLaandEA.hy926celltreatedfor24hwith100μMofinvestigatedcomplexes.D.Typicalresultsofmigrationassay(scratch
assay)forHeLacellseitherintheabsenceorpresenceof0.5×IC50complex5.Centerofthegapisindicatedwiththespotline.
2.4.5.Tubeformationassay ruthenium complexes on angiogenesis in vitro, the tube formation
Angiogenesisinvolvesendothelialcellproliferation,migrationand assaywasperformed.Whenseededontomatrigelinthepresenceofap-
capillary tube formation. To examine the effect of our investigated propriatemedia MS1 (endothelial)cellsstarttoreorganize,showing
Fig.5.Tubeformationassay(antiangiogenicpotentialinvitro).RepresentativemicrographsoftubeformationofMS1cellstreatedfor24hwithcomplex5(0.5×IC50)versus
control.
Fig.6.ExpressionlevelofERCC1mRNAdeterminedbyReal-Time-PCR(A)andproteinexpressionleveldeterminedbyWesternblot(B),inHeLacellsafter24hexposurewithinvestigated
complexes2,3,4,5(100μM)andCDDP(7μM).
projectionsandformingcell–cellcontactsandpolygonstructures[8].In DNAreplicationandhasbeenimplicatedintriggeringofapoptosisin-
ourexperimentMS1endothelialcellsweretreatedwithsub-toxiccon- ducedbyanumberofcytotoxicanticanceragents[8].Basedonthe
centrationsofinvestigatedcomplexestodeterminetheirpotentialto idea that MSH components mediate sensitivity to DNA damaging
decreaseabilityofthesecellstoextendanddifferentiateintotube-like agents,weinvestigatedwhetherMSH2mightbeincludedincellre-
structures.Ourcomplexes1,4,and5showedsomepotentialtoinhibit sponse to Ru(II)-p-cymene complexes, especially complex 5 which
characteristicsofangiogenicbehaviorofendothelialcells.Complex5in- showed preferential affinity for DNA binding. We have analyzed
ducedthehighestinhibitoryeffectamongthetestedcomplexes,andre- MSH2proteinexpressionlevelinHeLaafter24htreatmentwithin-
sultsarepresentedinFig.5. vestigatedcomplexes,comparingtocisplatinandnontreatedcontrol.
Data obtained indicated that protein level of MSH2 decreased after
2.5.EffectonexpressionofERCC1mRNAlevelbyQuantitativeReal-Time treatmentwithcomplex5,whilerestofthecomplexes,includingcis-
PCR(RQ-PCR)andproteinlevel(Westernblot) platinasareference,hadnoeffect(Fig.7).
Thereisconsiderableinterestintheinteractionsofruthenium(II)
2.7.Combinationaldrugstudy
complexeswithDNAaswellasthebiologicalimpactoftheinteractions
[10,29,30].Inordertoevaluatewhetherinvestigatedrutheniumcom-
PARPisanuclearenzymeinvolvedinsinglestrand-breakrepair
plexesinduceDNA-repair-dependentcellresponseastheresultofcyto-
andmaybeinhibitedtoincreasechemosensitivitytowidespectrum
toxicDNAlesions,weinvestigatedmRNAexpressionlevelofERCC1,
of DNA-damaging agents including alkylating agents (cisplatin),
andproteinexpressionlevelofERCC1andMSH2,whichmightbecoop-
topoisomeraseIinhibitors(etoposide)etc.[17].Assumingthatruthe-
erativelyinvolvedin cell responsetoDNA-bindinganticanceragents
nium complex 5 bind to DNA, inducing lesions, we investigated
[31].ERCC1iscriticalcomponentofNERwhichisprimarilyinducedin
whether combination study of complex 5 with PARP-1 inhibitor 3-
therepairofbulkyplatinum-DNAadducts,anditisknownthatitsre-
aminobezamideonHeLacells,mayinterferewithcomplex5induced
pairfunctionextendsbeyondNERandmayplayroleincellresponse
cytotoxicity.Resultsofcombinationaldrugstudy(Fig.8)showedthat
to the other type of DNA lesions [31]. Data obtained on HeLa cells
3-ABatconcentrationof2mMpotentiatedcytotoxicityofcomplex5
after24hcontinualtreatmentshowedthatcytotoxiccomplex5signif-
which indicates that Ru complexes may induce DNA-single-strand
icantlyincreasedexpressionofERCC1onmRNAlevel(Fig.6A).Other
breaks,eitherastheresultofdirectbindingtoDNAorasanindirect
rutheniumcompoundsshowednoeffectornegativelymodulatedrela-
cytotoxiceffect[16].
tive expressionlevelsof ERCC1mRNA, comparedto thenon treated
control, as determined by RQ-PCR. This data are in accordance with
theresultsofWesternblot(Fig.6B),whichshowedincreasedprotein 3.Conclusion
expressionlevelinducedbycomplex5,incomparisontothecontrol.
ERCC1has been investigatedasa markerof cisplatin resistance, and Based on their in vitro anticancer properties, investigated com-
ERCC1mRNAexpressioncorrelatestosensitivitytocisplatintosome plexes have one complicated mechanism of action, involving both
extend[12],thusfarcisplatinhasbeenusedasreferencecompound
in this study. After 24h cisplatin treatment in HeLa cells, ERCC1 120
mRNAexpressionwasslightlydownregulated,whichwasoppositeto
theeffectofRucomplex5.Anadditionalstudyisnecessarytoaddress 100
the role of ERCC1 upregulation in response to complex 5 induced
cytotoxicity. 80
2.6.EffectonexpressionofMSH2proteinlevel(Westernblot) 60
MSH2isacomponentofmismatchrepairsystem(MMR)which 40
has central role in correcting mismatches in DNA occurring during
20
0
0 0.5xIC50 IC50 1.5xIC50
Fig.7.MSH2proteinexpressionlevelinHeLacellsafter24htreatmentwithcomplexes
1–5(100μM)andCDDP(7μM)determinedbywesternblot.
)lortnoc
fo
%(
htworg
lleC
58 N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61
Complex 5
Complex 5+1mM 3-AB
Complex 5+2mM 3-AB
Fig.8.CellgrowthinhibitiongraphofHeLacellsafter48hcontinualactionofcombined
treatmentofcomplex5(atconcentrationscorrespondingto0.5×IC50,IC50and1.5xIC50)
and3-AB(atconcentrations1mMand2mM),determinedbySRBassay.Dataarerepre-
sentativeforoneoutofthreeseparateexperimentswithstandarddeviations.
N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61 59
extraandintra-cellularprocesses,quitedistinctfromthatofclassical weremaintainedasmonolayercultureinnutrientmedium,Dulbecco's
platinum-basedcompounds. ModifiedEagleMedium(DMEM).PowderedDMEMmediumwerepur-
In accordance with the previously published results [6,7] where chasedfromSigma-AldrichCo.NutrientmediumDMEMwaspreparedin
replacement of pyridine ligands in the molecule of the ineffective sterileionizedwater,supplementedwithpenicillin(192IU/ml),strepto-
ruthenium(II)-arene (complexes 1–4), with picolinic acid, resulted mycin(200µg/ml),4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid
in an enhancement of antiproliferative activity, additional study on (HEPES) (25mM), L-glutamine (3mM) and 10% of heat-inactivated
twoendothelialcelllines(EA.hy926,MS1)showedagainthatpicoli- fetalcalfserum(FCS)(pH7.2)andD-glucose(4.5g/l).
nate ruthenium(II)–arene (complex 5) exhibited enhanced activity, The cells were grown at 37°C in 5% CO and humidified air
2
comparedtotheothercomplexes.Complex5exerteditscytotoxicef- atmosphere.
fect through S phase cell cycle arrest and significant preference for
DNAbinding.Ourinvestigationofintracellulardistributionofruthe- 4.2.2.MTTassay
niumcomplexesallowedustoconcludethatDNA,ratherthanpro- Antiproliferativeactivityofcomplexes1–5wasdeterminedusing
teins,maypresenttargetforcomplex5cytotoxicaction.Moreover, 3-(4,5-dymethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT,
it upregulated expression of DNA repair enzyme ERCC1 on both Sigma-AldrichCo.)assay[19].Cellswereseededin96-wellcellcul-
mRNAandproteinlevel,andaffectedproteinlevelofMSH2,which tureplates(NUNC)EA.hy926(4000c/w)andMS1(7000c/w)incul-
indicated involvement of DNA-repair-dependent cell response. Po- ture medium and grown for 24h. Compounds were dissolved in
tentiationofactionofcomplex5incombinationwithPARP-1inhibi- DMSOandfurtherdilutedwithnutrientmediumtodesiredconcen-
tor3-AB,suggestsimportanceofunderstandingcellularpathwaysor trations(upto300μM).FinalconcentrationofDMSOneverexceeded
targetsutilizedbyrutheniumcomplexeswhichshowenhancedsen- 1%.Solutionsofvariousconcentrationsofexaminedcompoundwere
sitizationoftumorcellsincombinationwithPARPinhibition. addedtothewells,exceptthecontrolwellswhereonlynutrientme-
Complexes1–4whichpreferentiallyaccumulatedinproteinfrac- diumwasadded.Alltestswerecarriedoutintriplicate.Afterincuba-
tionhadminorgrowthinhibitoryeffectandmodestanti-invasivepo- tion periods of 48 and 72h 20μL of MTT solution, 5mg/mL in
tential.Complex5showedinhibitionofMMP-sactivity,inhibitionof phosphatebuffersolution,pH7.2wereaddedtoeachwell.Samples
migrationofHeLacells,andinhibitionoftubeformationofMS1endo- were incubated for 4h at 37°C, with 5% CO in humidified atmo-
2
thelialcells(antiangiogenicpotentialinvitro).ComparablytoNAMI- sphere. Formazan crystals were dissolved in 100µl of 10% sodium
Alikerutheniumcompounds(3),Ru(II)complex5showedcapacity dodecyl sulfate (SDS). Absorbances were recorded on an enzyme-
tomodifyimportantparametersofmetastasis.However,thenature linked immunosorbent assay (ELISA) reader (ThermoLabsystems
of the target(s) responsible for the antimetastatic activity remains Multiskan EX 200–240V) after 24h at the wavelength of 570nm.
tobeelucidated. IC (μM)valuesweredeterminedfromthecellsurvivaldiagrams.
50
Structure–activity comparison revealed heterogenic mode of ac-
tivity of tested ruthenium(II) complexes with regard to antimeta- 4.3.Flow-cytometricanalysisofcellcyclephasedistribution
static effect and cytotoxic potential. Variations of the nature of
monodentateorbidentateligandsignificantlyinfluencedpharmaco- Quantitative analysis of cell cycle phase distribution was per-
logicalpropertiesofthecomplexes.Moreover,areneidentitymayaf- formed by flow-cytometric analysis of the DNA content in fixed
fect drug-uptake and interactions with the potential intracellular HeLaandEA.hy926cells,afterstainingwithpropidiumiodide(PI)
targetssuchasDNA,contributingtononcovalent,hydrophobicinter- [22].
actionswithDNAbases,suchasintercalation(29). Cellswereseededat densityof2×105cells/wellat6-wellplate
Basedonthedataobtainedinthisresearch,picolinaterutheniu- andgrowthinnutritionmedium.After24hcellswerecontinuallyex-
m(II)–arene(complex5)distinguishedamongthetestedruthenium posedtoinvestigatedcompounds1–4(300μM)and5(82μM/IC ).
50
complexes,according toitshighestantiproliferativeandantimetas- After24hofcontinualtreatmentcellswerecollectedbytrypsiniza-
taicpotentialinvitro.Replacementofsubstitutedpyridineligand(in tion, washed twice with ice-cold PBS, and fixed for 30min in 70%
complexes1,2,3,4)withligandofplanarsurface,freefromsubstit- EtOH.AfterfixationcellswerewashedagainwithPBS,andincubated
uentssuchaspicolinicacid(complex5)allowedforbetterDNAbind- with RNaseA (1mg/mL) for 30min at 37°C. Just before flow-
ingaffinityandcytotoxicity. cytometric analysis cells were stained with PI at concentration of
DuetotheinteractionwithDNAandinvitroantimetastaticchar- 400μg/mL.Cellcyclephasedistributionwasanalyzedusingafluores-
acteristicswebelievethatcomplex5ispromisingcandidateforfur- cence activated sorting cells (FASC) Calibur Becton Dickinson flow
therinvivoantitumorstudy. cytometerandCellQuestcomputersoftware.
4.Experimentalprotocols 4.4.Intracellulardistributionofinvestigatedcomplexes(proteinsvs.DNA
fraction)
4.1.Synthesis
Ruthenium level in proteins vs. DNA fraction after treatment of
The complexes of structural formula [(η6-p-cymene)Ru(L1,2)Cl ], HeLa cells with investigated complexes was determined using ICP-
2
whereL1,2is3-acetylpyridine(1)and2-amino-5-chloropyridine(2), OES. 6×106cells/75cm2 falcon dish (NUNC) were treated with in-
correspondingly,and[(η6-p-cymene)Ru(H L3,4)Cl],whereH L3,4rep- vestigatedcomplexesin100μMconcentrationsfor24h.Aftertreat-
2 2
resent2,3-pyridinedicarboxylicacid(3),2,4-pyridinedicarboxylicacid mentcellswerewashedbyicecoldPBSandharvestedbyscraping.
(4),and[(η6-p-cymene)RuCl(picolinicacid)]∙H O(5)weresynthesized Aftercentrifugationat2000rpm,10minat4°Ccellpelletwasfrozen
2
andcharacterizedaspreviouslydescribed[6,7]. at−70°C.
DNA and proteins were isolated using TRI Reagent® (Sigma-
4.2.AnalysisofcellgrowthinhibitionbyRu(II)-complexes AldrichCo.)accordingtomanufacturer'sprocedureandconcentrations
determined spectrofotometrically by measuring absorbance at A260
4.2.1.Cellculture andA280nmrespectively(EppendorfBioPhotometer6131).Rutheni-
TheendothelialcelllinesEA.hy926(permanenthumancelllinede- umlevelsweredeterminedinisolatedDNAandproteinfraction,after
rived from a fusion of human umbilical vein endothelial cells-HUVEC sample preparation for ICP-OES according to standard procedure,
andhumanlungadenocarcinomacells-A549)andMS1(murineendo- usingThermoScientificiCAP6500DuoICP(ThermoFisherScientific,
thelialcellsimmortalizedbyinfectionencodingSV40largeTantigen) Cambridge,UK)
60 N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61
4.5.Invitroantimetastaticpotential andabsorbancesrecordedonanenzyme-linkedimmunosorbentassay
(ELISA) reader (ThermoLabsystems Multiskan EX 200–240V) at the
4.5.1.Gelatinzymography wavelengthof570nm.Eachexperimentwasdoneintriplicate.
Gelatinolitic activity of secreted MMP-2 and MMP-9 in drug-
treated HeLa and EA.hy 926 cells was analyzed by zymography in 4.5.4.Cellmigrationassay(Scratchassay)
10%SDS-polyacrylamidegelsimpregnatedwith0.1%gelatin. Togainfirstinsightsinthepotentialofinvestigatedrutheniumcom-
Cellswereseededin6-wellplate(NUNC)induplicate(6×105c/w) plexestoinhibitmigrationofHeLacells,invitroscratchassaywasdone
in nutrition mediumbeing RPMIfor HeLaand DMEM forEA.hy 926 [22].4×105cells/wellwereseededinto6-wellcellcultureplateandcul-
cells.At70%ofconfluencecellswereexposedtoinvestigatedcomplexes turedinRPMI1640with10%FCSintissueculture5%CO incubatorto
2
(100μM)inserum-freemedium,for24h.Equalamountsofsuperna- confluence. A scrape was made in the center of the cell monolayer
tantproteinsforeachsample,asdeterminedbyBradfordassay[32], withasterilepipettetip(p200)tocreateagapofconstantwidth.Cells
weremixedwithloadingbuffer(loadingbuffer:0.125MTris–HCl,pH detachedbyscrapingwereremovedbygentlywashingwithPBS.The
6.8;20%glycerol;10%SDSand0.25%bromophenolblue)beforeloading initialimagesofthewoundswerecapturedwithOlympusdigitalcam-
to the SDS-polyacrylamide gels. Electrophoresis was performed at eraconnectedtotheinvertedmicroscope(CarlZeiss,Jena,Germany,ob-
125V, gels were washed 3 times for 20min in renaturing solution jective6.3/0.20)andcellswerefurtherincubatedfor24hand48hin
(2.5% Triton X-100) followed by a 48h incubation at 37°C in the fresh medium for control or with investigated complexes in desired
enzyme assay buffer (50mM Tris–HCl, pH 7.5; 0.2M NaCl; 5mM concentrations1–4(100μM)and5(0.5×IC ).Picturesweretakento
50
CaCl ; 0.05% NaN ) for the development of enzyme activity bands. qualitativelydeterminetheinfluenceofinvestigatedcomplexesonmi-
2 3
Afterincubation,thegelswerestainedwith0.05%Coomassiebrilliant gration.Photographsoftheinitialwoundedmonolayerswerecompared
blueG-250inamixtureofMeOH:AcOH:H O(2.5:1:6.5)anddestained withthecorrespondingimagesoftreatedcells.Artificiallinetraversing
2
in 4% MeOH with 8% AcOH. In order to increase the sensitivity, the throughthemiddleofgapwasdrawnonpicturesoforiginalwounds
gelswerefurtherdestainedinasolutionof1%TritonX-100(1–2h) andoverlaidontheimagesofculturesafterincubation.
[24,33].Thegelatinolyticactivitieswerevisualizedascleartransparent
bandsagainstthebluebackgroundofCoomassiebrilliantblue-stained 4.5.5.Tubeformationassay(invitroangiogenesisassay)
gelatin. Potentialofinvestigatedcomplexestoinhibitangiogenesisinvitro
was analyzed by tube formation assay in mouse endothelial cells
4.5.2.Matrigelinvasionassay (MS1). MS1 cells plated into gel of basement membrane proteins,
Thepotentialofourruthenium(II)-p-cymenecomplexes,toinhib- rapidlyorganizeintomulticellulartube-likestructures,whileantian-
itinvasionofHeLacellswecarriedoutbymatrigelinvasionassayin giogeniceffectoftestedcompoundsisobservedasreductionoftube
24-well BD BioCoat Matrigel invasion chambers (BD Biosciences) formation[8].Briefly,24-wellplateswerecoatedwithcollagenand
[26].2×105cells/wellofHeLacellswereseededin6-wellcellculture allowed to solidify at 37°C for 1h. MS1 (mouse endothelial cells)
plate(NUNC)innutritionmedium.After24hcellsweretreatedwith wereseeded1×105cells/wellinmedium.Treatmentwasadded2h
investigated ruthenium complexes 1–4 (100μM) and 5 (0.5×IC ) after cells settled (for complexes 1–4 concentration was 100μM,
50
for24h.Controlwerecellsinthenutritionmediumonly.Aftertreat- and for complex 5 concentration was 0.5×IC ). Tube formation
50
mentcellswereleftfor4hinserum-freeRPMI1640mediumwith wasobservedperiodicallyovertimeundermicroscopeandrepresen-
0.2%BSA.Afterstarvationcellswereharvestedbytrypsinizationand tativepicturesweretakenafter24hincubationwithOlympusdigital
resuspended in serum-free RPMI 1640 medium with 0.2% BSA. cameraconnectedtotheinvertedmicroscope(CarlZeiss,Jena,Germany,
4×104cells/0.5mL/wellwereseededontothematrigel-coatedfilters objective6.3/0.20).
(with8μm poresize)inBioCoatMatrigelinvasionchambers.RPMI
1640 medium with 10% FCS was added to the lower compartment 4.6.QuantitativeReal-TimePCR(RQ-PCR)
aschemoattractant.Afterincubationinahumidifiedtissueculturein-
cubator,at37°C,5%CO atmospherefor48h,thenon-invadingcells 4.6.1.RNAisolationandcDNAsynthesis
2
areremovedfromtheuppersurfaceofthemembraneby“scrubbing”. RNAwasisolatedfrom6×106HeLacells,aftertreatmentwithin-
Cellsthatinvadedthelowersurfaceofthefiltersweredetachedand vestigated complexes 2–5 (100μM) for 24h, using TRI Reagent®
stainedwithdetachmentbufferTrypsine/EDTA(0.05%/0.02%)inPBS (Sigma-Aldrich) according to the manufacturer's procedure. Total
with5μMCalcein-AM.200μLoftheTrypsine/EDTAsolutioncontain- RNAofeachsamplewasquantifiedbyaspectrophotometer(Eppen-
ingthedislodgedcellswastransferredfromeachwellof24wellplate dorfBioPhotometer)measuringtheabsorbanceat260nm.Theratio
toa96-wellplateinduplicate.Invadedcellswerequantifiedin96- A /A wasusedtoassessthepurityofisolatedRNA.TheRNAcon-
260 280
well plate with the fluorescence microplate reader (Fluoroskan As- centrationwascalculatedinμg/μL.ToanalyzetheRNAbandingpat-
cent FL, Thermo Electron Corporation) at an excitation wavelength tern, gel electrophoresis (Pharmacia Biotech) in 2% agarose gels
of485nmandanemissionwavelengthof520nm.Assaywasdone with ethidium bromide was performed. The RNA samples were
induplicate. mixedwithxylenecyanolcolor(0.25%xylenecyanolin30%glycerol
inwater)andrunin0.5×TBEbufferfor50minat25mA.TheRNA
4.5.3.Celladhesionassay(resistancetodetachmentassay) bandswerevisualizedonaUVtransilluminator(Hoefer).Toperform
To evaluate potential influence of investigated ruthenium(II)-p- RT-PCRwithrandomprimers,2μgoftotalRNAwasusedastemplate
cymenecomplexesonadhesionoftumorcells,theadhesionofHeLa for MultiScribe™ Reverse Transcriptase (50U/μL) in High-Capacity
cellsonplasticsubstrate(uncoatedbottomoftheplates)wasevalu- cDNA Reverse Transcription kit (Applied Biosystems), according to
atedaccordingtomethodreportedby[27]. themanufacturer'sinstructions.Thereactionmixtureswereincubat-
HeLacellsweregrownatadensityof3×104cells/mL,in0.2mL/ edinMastercyclergradient(Eppendorf).
wellnutritionmediumina96-wellplasticplate(NUNC)andincubated
at37°C.At70%confluencecellsweretreatedwithinvestigatedcom- 4.6.2.QuantitativeReal-TimePCR(RQ-PCR)
plexes at 100μM concentrations, for 90min.Briefly,30μL of(0.05%, TheanalysisofgeneexpressionlevelofERCC1andGAPDH(endoge-
w/v) trypsin solution per well was used to detach cell at 37°C for nous control) was done by using TaqMan® Gene Expression Assay
30min.Nondetachedcells were fixed with 200μl of10% (w/v) cold (ERCC1probe,AppliedBiosystem,ID:Hs_01012155_g1,andTaqMan®
trichloroacetic acid (TCA) at 4°C for 1h. Number of adherent cells MGBGAPDHprobe,AppliedBiosystem,ID:Hs_00355782_m1),onABI
was determined using sulforhodamine B (SRB; Sigma) staining [30], PRISM® 7500 PCR instrument (Applied Biosystems Inc., Foster City,
N.Gligorijevićetal./JournalofInorganicBiochemistry108(2012)53–61 61
CA).PCRreactionswerepreparedinduplicateforeverysamplein96- HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid
well optical plates. Each well contained 11μL of TaqMan® Universal FCS Fetalcalfserum
PCR Master Mix (Applied Biosystems), 1μL of cDNA (100ng/μL)and MTT 3-(4,5-dymethylthiazol-yl)-2,5-diphenyltetrazolium
8μLH O.Notemplatecontrolinduplicateaswell,wasusedasaPCR bromide
2
negativecontrol(11μLTaqMan®UniversalPCRMasterMix(Applied ELISA Enzyme-LinkedImmunosorbentAssay
Biosystems),9μLH O).PCRprofileconsistedofthefollowingsteps:en- FASC FluorescenceActivatedSortingCells
2
zymeactivation50°C2min;initialdenaturation95°C10min;40cy- PBS PhosphateBufferedSaline
cles of denaturation at 95°C 15s, and annealing/extension at 60°C TCA Trichloroaceticacid
1min. SRB SulforhodamineB
CDDP Cis-diamminedichloroplatinum(II)
4.7.Westernblot ECL EnhancedChemiluminescence
GAPDH Glyceraldehyde3-phosphatedehydrogenase
HeLa cells were treated with investigated ruthenium complexes
1–5 in concentration 100μM and CDDP in concentration 7μM for
24h. Control were untreated cells, maintained in medium only.
After treatment cells were washed with phosphate buffered saline Acknowledgments
(PSB)(Sigma-AldrichCo.)andwholecelllysateswereharvestedin
100μLofLysisbuffercontaining:37mMTris–HCl,75mMNaCl,25% ThisworkwassupportedbytheMinistryofScience,Republicof
TritonX-100,and10μlproteaseinhibitorcocktail(Sigma).Samples Serbia,Grant,No.III41026andGrant,No172017.
werestoredat−20°Cuntilfurtheranalysis.
Proteinconcentrationsoftheresultinglysatesweredetermined
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