In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model.

JournalofInorganicBiochemistry104(2010)79–86 ContentslistsavailableatScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model A. Bergamoa,*, A. Masia, A.F.A. Peacockb, A. Habtemariamc, P.J. Sadlerc, G. Savaa,d aCallerioFoundationOnlus,ViaA.Fleming22-31,34127Trieste,Italy bSchoolofChemistry,UniversityofEdinburgh,WestMainsRoad,EdinburghEH93JJ,UK cDepartmentofChemistry,UniversityofWarwick,GibbetHillRoad,CV47AL,UK dDepartmentofLifeSciences,UniversityofTrieste,ViaL.Giorgieri7,Trieste,Italy a r t i c l e i n f o a b s t r a c t Articlehistory: Wehavecomparedtheorganometallicarenecomplexes[(g6-biphenyl)M(ethylenediamine)Cl]+RM175 Received8May2009 (M=RuII) and its isostructural osmium(II) analogue AFAP51 (M=OsII) for their ability to induce cell Receivedinrevisedform21September detachmentresistancefromfibronectin,collagenIVandpoly-L-lysine,andcellre-adhesionaftertreat- 2009 ment, their effects on cell migration and cell viability, on matrix metalloproteinases production, and Accepted7October2009 onprimarytumourgrowthofMCamammarycarcinoma,theeffectofhumanserumalbuminontheir Availableonline14October2009 cytotoxicity.Therearedifferencesbetweenrutheniumandosmium.TheOscomplexisupto6(cid:2)more potentthanRM175towardshighly-invasivebreastMDA-MB-231,humanbreastMCF-7andhumanepi- Keywords: thelialHBL-100cancercells,butwhereasRM175wasactiveagainstMCamammarycarcinomainvivo Metastasis andcausedmetastasisreduction,AFAP51wasnot.Intriguinglythepresenceofhumanserumalbumin Invasion Mammarycancer inthegrowthmediumenhancedthecytotoxicityofbothcompounds.RM175increasedtheresistance Ruthenium ofMDA-MB-231cellstodetachmentfromsubstratesandbothcompoundsinhibitedtheproductionof Osmium MMP-2.Thesedataconfirmthekeyroleofrutheniumitselfinanti-metastaticactivity.Itwillbeinterest- Organometallic ingtoexploretheactivityofosmiumarenecomplexesinothertumourmodelsandthepossibilityof changingthenon-areneligandstotunetheanticanceractivityofosmiuminvivo. (cid:2)2009ElsevierInc.Allrightsreserved. 1.Introduction platin-resistantcolorectalcarcinomaandNAMI-A[9]aleadcom- pound for its ability to combat the development of metastasis of Despite the progress in medicine, metastases cause 90% of solidtumours. deaths from solid tumours and display a remarkably diverse set + + ofclinicalmanifestations.Researchondrugsbasedonmetalcom- poundsofferspromiseinthisfight[1,2].Inorganicchemistryoffers widescopeforthedesignofnoveldrugsbasedonthecoordination and redox properties of metal ions [3], and the exploration of Ru Os medicinalapplicationsisdrivenbythenecessitytofilltheunmet NH NH needsoftumourchemotherapy.Inparticulartheseneedsinclude Cl 2 Cl 2 theminimisationofundesirableside-effects,overcomingtheresis- H 2 N H 2 N tanceproblem,enlargingthespectrumofactivitytomoretumour typesandtometastatic(secondary)cancers. RM175 AFAP51 Rutheniumcompounds,asanalternativetoplatinum-basedtu- mour inhibitors, are receiving a great attention [4,5] and two The hypothesis [10] that ruthenium(III) complexes are pro-drugs ruthenium(III) compounds have successfully concluded a phase I which are activated by reduction suggests that RuII may be an clinical trial [6,7]. KP1019 [8], known for its activity against cis- importantcomponentofthefinalreactivedrugandthishasstim- ulatedinvestigationsoftheactivityofRuIIcomplexesthemselves. Arene ligands stabilise RuII and various classes of organometallic half-sandwichRuIIcompoundshavebeenfoundtobeactiveboth * Correspondingauthor.Tel./fax:+39040569933. invitroandinvivo[11–13].Amongsttheseisthebiphenylethy- E-mailaddress:a.bergamo@callerio.org(A.Bergamo). 0162-0134/$-seefrontmatter(cid:2)2009ElsevierInc.Allrightsreserved. doi:10.1016/j.jinorgbio.2009.10.005 80 A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 lenediamine chlorido complex RM175. RM175 exhibits in vitro berHTB-22)andmaintainedinDulbecco’smodifiedEagle’smed- cytotoxiceffectssimilarorgreaterthanthoseofcarboplatin[12], ium/F12medium1:1 v/v (EuroClone(cid:3), Devon,UK) supplemented without cross resistance with other platinum drugs, induces with 10% FBS, 2mM L-glutamine, and 100IU/mL penicillin and invitroG1andG2growtharrestandapoptosis[14],andasignif- 100lg/mLstreptomycin. icantgrowthdelayoftumoursinvivo[12,15]. The HBL-100 human non-tumorigenic epithelial cell line was The aim of the present study was to evaluate the effects of kindly supplied by Dr. G. Decorti (Department of Biomedical Sci- RM175 in an in vitro model of tumour invasion and metastasis. ences,UniversityofTrieste,Italy),and maintained inMcCoy’s5A For this purpose we investigated the role of the metal centre by medium supplemented with 10% FBS, 2mM L-glutamine, and comparing the ruthenium complex RM175 with its osmium ana- 100IU/mLpenicillinand100lg/mLstreptomycin. logue AFAP51. In general organometallic half-sandwich RuII and AllcelllineswerekeptinaCO incubatorwith5%CO and100% 2 2 OsII complexes have similar (often almost identical) structures relativehumidityat37(cid:4)C.Cellsfromaconfluentmonolayerwere butdifferintheirratesofreaction,oftenca.100(cid:2)slowerforOsII, removedfromflasksbyatrypsin–EDTAsolution.Cellviabilitywas and with an increased acidity of its aqua adducts (by ca. 1.5 pKa determinedbythetrypanbluedyeexclusiontest.Forexperimental units)[16,17].Hydrolysisofthechloridoadductsappearstoplay purposescellsweresowninmultiwellcultureclusters. a role in intracellular activation. Although isostructural with RM175,theOsIIcomplexAFAP51hydrolysesmoreslowlyandre- 2.3.Resistancetodetachmentassay actsmoreslowlywithnucleobases[17]althoughlikeRM175itis activetowardshumanlungandovariancancercellsinvitro[18]. The ability of cells to resist detachment after treatment with Interestingly,initialexperimentshaveshownthattheOsIIcomplex RM175 and AFAP51 was measured by the following procedure. can induce unwinding of plasmid DNA to a greater extent than Ninety-sixwellplasticplates(CorningCostar,Milano,Italy)were eithertheRuIIanalogueorcisplatinbutcauseslittleDNAbending coated with the following substrates: 10lg/mL poly-L-lysine, [19,20]. 20lg/mLfibronectinfromhumanplasma,and20lg/mLcollagen Metastatic progression is mimicked in vitro by opportune IV from human placenta, and left in a humidified cell-culture experiments to study cell detachment from the primary tumour, chamberat37(cid:4)Cfor4h.Beforecellseeding,plateswerewashed extracellular matrix degradation, migratory ability, invasion and with CMF-DPBS (calcium and magnesium-free Dulbecco’s phos- re-adherencetoasubstrate,usingcelllinesofthemammarygland phatebufferedsaline),then6(cid:2)103cellsin0.2mLcompletemed- with different degree of aggressiveness: MDA-MB-231, a highly- ium were sown in each well. After 2days at 37(cid:4)C, complete invasivebreastcancercellline,MCF-7atumorigenicbutnon-inva- medium was replaced with serum-starved medium, containing sive cell line, and HBL-100 a non-tumorigenic cell line of the 0.1% w/v BSA (bovine serum albumin). After 24h the medium mammary epithelium. The in vitro study is compared with the was removed and the plates washed with CMF-DPBS, before the anti-tumour and anti-metastatic effects of the same compounds treatment with 10(cid:3)4M RM175 or AFAP51, dissolved in DPBS, invivointhemousemodelofMCamammarycarcinoma. was added to the wells and incubated for 1h. At the end of the treatment,theRM175-andAFAP51-containingsolutionswerere- moved, the plates were washed twice with CMF-DPBS, and a 2.Materialsandmethods 0.008%w/vtrypsinsolutionaddedtoeachwell.Plateswerekept inagitationfor30minatroomtemperaturethenthetrypsinsolu- 2.1.Drugsandreagents tion was removed and wells washed with CMF-DPBS. Cells that werestilladherenttotheplatesweredetectedbythesulforhoda- AFAP51, [(g6-biphenyl)Os(ethylenediamine)Cl]BF , was pre- 4 mineB(SRB)test.Resistancetodetachmentisexpressedinarbi- pared as described previously [18] by refluxing the chlorido- trary units, calculated by dividing the mean absorbance of bridged dimer, [(g6-biphenyl)OsCl ] , and ethylenediamine in 22 treated cells by the mean absorbance of control cells. The resis- methanol, followed by the addition of NH BF so as to generate 4 4 tancetodetachmentofcontrolsissetequalto1. theBF(cid:3) salt.ThecomplexwaspurifiedbySoxhletextractionwith 4 dichloromethane, with purity confirmed by both 1H NMR (>99%) 2.4.Re-adhesionassay andCHNanalysis. TherutheniumcomplexRM175waspreparedbyasimilarroute The effect on the ability of the cells to re-adhere after RM175 [11,18] starting from the chlorido-bridged dimer [(g6-biphe- and AFAP51 treatment, was studied in cells maintained for 24h nyl)RuCl 2 ] 2 exceptasaPF(cid:3) 6 saltandwaspurifiedsimilarlybySoxh- in serum-starved medium, and then treated for 1h with 10(cid:3)4M letextraction.Thepurityasdeterminedby1HNMRspectroscopy RM175orAFAP51inDPBS.Attheendofthetreatmentcellswere wasca99%,andtheCHNelementalanalysisagainshowedexcel- removedfromflasksbyatrypsin–EDTAsolution,collectedbycen- lentagreementbetweenthecalculatedandexperimentally-deter- trifugation,re-suspendedinserum-starvedmediumsupplemented minedvalues. with0.1%w/vBSAandkeptfor30minatroomtemperaturetoal- AllreagentswerepurchasedfromSigma–Aldrich(St.Louis,MO) lowsurfacereceptorreconstitution.Thecellswerethenseededata unlessotherwiseindicated. density of 1(cid:2)104 cells in 0.1mL/well on 96-well plastic plates previouslycoatedasdescribedabovewithpoly-L-lysine,fibronec- 2.2.Tumourcelllinesforinvitrotests tin, collagen IV or 20lg/mL Matrigel(cid:3) (BD, Biosciences, San Josè, CA). Cells were left to adhere for 60min at 37(cid:4)C with 5% CO 2 TheMDA-MB-231humanhighly-invasivebreastcancercellline and 100% relative humidity, then the medium containing the was kindly supplied by Dr. P. Spessotto (Cro, Aviano, Italy), and non-adherent cells was removed and wells were gently washed maintained in Dulbecco’s modified Eagle’s medium (EuroClone(cid:3), with CMF-DPBS. Cells that had adhered to the substrates in Devon,UK)supplementedwith10%fetalbovineserum(FBS,Gibco, 60minweredetectedbythesulforhodamineB(SRB)test. InvitrogenTM,Paisley,Scotland,UK),2mML-glutamine(EuroClone(cid:3), Devon,UK),1%non-essentialaminoacids,and100IU/mLpenicillin 2.5.SulforhodamineBassay and100lg/mLstreptomycin(EuroClone(cid:3),Devon,UK). TheMCF-7humanbreastcancercelllinewasobtainedfromthe Adherent cells were detected with the SRB test described by AmericanTypeCultureCollection(Manassas,VA;cataloguenum- Skehanetal.[21].Briefly,adherentcellswerefixedwith10%v/v A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 81 cold trichloroacetic acid (TCA) at 4(cid:4)C for 1h. After fixation TCA medium containing 5% FBS, for 24h. Analysis was performed at was discarded and wells washed five times with distilled water the end of the incubation time by the MTT [3-(4,5-dimethylthia- andair-dried.SRBsolution(0.4%,w/v,in1%aceticacid)wasadded zol-2-yl)-2,5-diphenyltetrazolium bromide] viability test [24]. tothewellsandplateswerekeptfor30minatroomtemperature. Briefly, a solution of MTT dissolved in CMF-DPBS (5mg/mL) was UnboundSRBwasremovedbywashingthreetimeswith1%acetic added to each well (10lL per 100lL of medium) and the plates acid. Plates were air-dried, then bound stain was dissolved with wereincubatedat37(cid:4)Cwith5%CO and100%relativehumidity 2 un-buffered 10mM Tris base (Tris-hydroxymethyl-aminometh- for4h.Afterthistime,themediumwasdiscardedand200lLof ane) at pH 10.5 and the absorbance was read at 570nm with an DMSO was added to each well to dissolve the formazan crystals. automaticcomputerisedspectrophotometer (SpectraCount; Pack- The absorbance was measured at 570nm with an automatic ard,Meriden,CT). computerised spectrophotometer (SpectraCount; Packard, Meri- den,CT). 2.6.Migrationassays Moreoverthesametestwasusedalsotoevaluateiftreatment with RM175 and AFAP51, under the experimental conditions Migratoryabilityresultingfromahaptotacticorachemotactic adoptedfor the migrationassays, affectscell viability.Cells were stimuluswasmeasuredinTranswell(cid:3)cell-culturechambers(Cost- treated as described above for migration tests, except they were ar,Milano,Italy).Inthehaptotaxisassay,thelowersurfaceofthea seededon96wellplates.After24hthecellviabilitywasmeasured polyvinylpyrrolidone-free polycarbonate filter (8-lm pore size) bytheMTTassay. was coated with 10lg/mL fibronectin and left in an humidified cell-culturechamberat37(cid:4)Cfor2h,thenwashedwithCMF-DPBS 2.10.EffectofHSAoncytotoxicactivityofRM175andAFAP51 before cell seeding. In the chemotaxis assay, inserts were used without coating. Cells were treated for 1h with RM175 and This experiment was performed to evaluate if the binding of AFAP5110(cid:3)4MinDPBS.Aftertreatmentcellswereremovedwith RM175andAFAP51to HSAcould influencethecytotoxicactivity atrypsin–EDTAsolution,collectedbycentrifugation,re-suspended ofthetwometalcompoundsinthehighlyinvasiveMDA-MB-231 in serum-starved medium supplemented with 0.1% w/v BSA and cell line, in comparison to the compound alone. Cells were sown 1(cid:2)105 cells in 0.2mL were sown in the upper compartment of on96-wellplates24hbeforeincubationwithRM175andAFAP51 each chamber. The lower compartment was filled with serum- inpresenceorabsenceofHSAatscalarconcentrationsfor24h. starvedmediumsupplementedwith0.1%w/vBSA,andwithcom- RM175 and AFAP51 were tested at dose levels corresponding plete medium for the haptotaxis and the chemotaxis assays, for each compound to 1/5 of the IC calculated after a 24h cell 50 respectively. exposure.EachcompoundconcentrationwascombinedwithHSA Cellswerelefttomigratefor24h,thenthecellsontheupper concentrations in order to obtain the following ratios between surfaceofthefilterswereremovedwithacottonswabandmigrat- thecompoundandHSA:1/1,1/5,1/10.Attheendoftheincubation ingcells,presentinthelowersurface,weredetectedbythecrystal timethetreatmentsolutionwasremovedandreplacedwithcom- violetassay. plete medium containing 5% of FBS for an additional 48h, after whichthecellviabilitywasmeasuredbytheMTTassay. 2.7.Invasionassay 2.11.Zymography Invasive ability was measured in a Transwell(cid:3) cell-culture chamberaccordingtothemethodofAlbinietal.[22].Briefly,the TovisualisethedirecteffectofRM175andAFAP51ontheactiv- uppersurfaceofthepolycarbonatefilter(8lmporesize)ofTrans- ityand/orproductionofMMP-2andMMP-9enzymes,SDS–PAGE well(cid:3)cell-culturechamberswascoatedwith50lLofa600lg/mL zymography was carried out with conditioned medium of MDA- Matrigel(cid:3) solution and air-dried overnight at room temperature. MB-231andHBL-100cells.Cellsat70%confluencewereincubated Thefilterswerere-constitutedwithDMEMmediumfor90minun- for24hinserum-starvedmediumcontaining0.1%w/vBSA,before der gently shaking immediately before use. Cells were treated as beingtreatedwith10(cid:3)4MRM175orAFAP51for1h.Attheendof described for the migration assays and 0.5(cid:2)105 cells in 0.2mL the treatment, the RM175 and AFAP51 solutions were discarded weresownineachchamber.Cellswerelefttoinvadefor96h,then and complete serum-free medium containing 0.1% w/v BSA was thecellsontheuppersurfaceofthefilterswereremovedwitha addedforafurther24h.Culturemediawerethencollected,centri- cottonswabandinvadingcells,presentinthelowersurface,were fuged to remove cellular debris, then concentrated ca. 15 times detectedbythecrystalvioletassay. usingAmicon(cid:3)Ultra-1530,000nominalmolecularweightcut-off centrifugal filter devices (Millipore Corporation, Bedford, MA). 2.8.Crystalvioletassay Theconditionedmedia obtained werestoredat (cid:3)80(cid:4)Cuntil use. Equalamountsofproteinsforeachsample,asdeterminedbythe Thecrystalvioletassaywasperformedaccordingtothemethod Bradford method [25], were eluted with Laemmli non-reducing describedbyKuengetal.[23].Briefly,thecellspresentonthelow- samplebufferandanalysedbySDS–PAGEona7%polyacrylamide ersurfaceofthefilterwerefixedwitha1.1%w/vglutaraldehyde gelcontaining0.1%w/vgelatine.Attheendoftheelectrophoresis solution for 15min. After fixation, the wells were washed three in a dual-laboratory system (Protean II, Bio-Rad Laboratories, times with distilled water and air-dried. Cells were stained for Hercules,CA),thegelswerewashedtwotimesfor30minat4(cid:4)C 20minwith0.1%w/vcrystalvioletpreparedin200mMboricacid, in2.5%v/vTritonX-100toremoveSDS.Afteradditionalwashing pH9.0,thenwashedthreetimeswithdistilledwaterandair-dried in water (three times for 5min), the gels were incubated at priortodissolvingthedyewith10%aceticacidsolution.Theabsor- 37(cid:4)C overnight in collagenase buffer [200mM NaCl, 50mM bancewasreadat590nmwithanautomaticcomputerisedspec- tris(hydroxymethyl)aminomethane, 5mM CaCl , adjusted to pH 2 trophotometer(SpectraCount;Packard,Meriden,CT). 7.4]toreactivateenzymeactivity.Thegelswerethenstainedwith 0.5% w/v Coomassie brilliant blue. The gelatinolyticregions were 2.9.Cellviability observedaswhitebandsagainstabluebackground.Quantitative evaluation of the band intensity, on the basis of grey levels, was Cellsweresownon96-wellplates24hbeforebeingincubated performed using Image Master 2D version 4.01 and Magic Scan with 10(cid:3)6–10(cid:3)4M RM175 or 10(cid:3)5–10(cid:3)3M AFAP51, in culture 32version4.3software. 2.12.Invivotests RM175 MDA-MB-231 MCF-7 HBL-100 The in vivo experiments were carried out with the murine 3.5 mammarycarcinoma(MCa),originallyobtainedfromtheDepart- ** ment of Biology, Rudjer Boskovich Institute (Zagreb, Croatia), 3.0 growninCBAfemalemice,obtainedfromalocalbreedingcolony * grown according to the standard procedures for inbred strains. 2.5 The tumour line was locally maintained by serial biweekly pas- sagesof106viabletumourcells,ofacellsuspensionpreparedfrom 2.0 mincing(withscissors)theprimarytumourmassesobtainedfrom 1.5 donorssimilarlyimplanted2weeksbefore.Themincedtissuewas filteredthroughadoublelayerofsterilegauze,centrifugedat250g 1.0 for 10min, and re-suspended in an equal volume of CMF-DPBS; viable cells were counted by the trypan blue exclusion test. 106 0.5 viable tumour cells were injected i.m. into the left hind calf of experimental groups. RM175 and AFAP51 were administered as 0.0 P F C P F C P F C 10%DMSOsolutionsinsterilesaline(0.9%NaCl)andgiventomice by i.p. (intra peritoneal) administrations at two dose levels of 7.5mg/kg/dayand10mg/kg/dayforsixconsecutivedays,starting whenprimarytumourbecamepalpable,i.e.fromday8to13after tumourimplantforbothRM175andAFAP51atdoseof7.5mg/kg/ day.Theeffectsof10mg/kg/daydosesofRM175wereevaluatedin a separate experiment starting the treatment on day 6 after tu- mour implant; because RM175 at this dose level was toxic the treatmentwasstoppedafterfourconsecutiveinjections. Primary tumour growth was determined by calliper measure- ments,bymeasuringtwoorthogonalaxes,andthetumourvolume was calculated with the formula: (P/6)(cid:2)a2(cid:2)b, where a is the shorteraxisandbthelongeraxis,assumingtumourdensityequal to1g/mL.Theevaluationofthenumberandweightoflungmetas- taseswasperformedbyexaminingthesurfaceofthelungsimme- diatelyaftersacrificingtheanimalsbycervicaldislocation.Lungs weredissectedintofivelobes,washedwithCMF-DPBSandexam- ined under a low power microscope equipped with a calibrated grid. The weight of each metastasis was calculated by applying thesameformulausedforprimarytumoursandthesumofeach individualweightgavethetotalweightofmetastatictumourper animal. 2.13.Animalstudies Animal studies were carried out according to guidelines en- forced in Italy (DDL 116 of 21/2/1992 and subsequent addenda) andincompliancewiththeGuidefortheCareandUseofLabora- increases the resistance to detachment when they are grown on toryAnimals(DepartmentofHealthandHumanServicesPubl.No. poly-L-lysineandonfibronectin;theeffectoncollagenIVisquan- 86-23,Bethesda,MD,NIH,1985). titativelycomparablealthoughnotstatisticallysignificant.Nosuch effectswereobtainedwhentherutheniumcompoundwastested 2.14.Statisticalanalysis under the same experimental conditions on the non-invasive MCF-7 or on the non-tumorigenic HBL-100. By contrast, the os- Resultsweresubjectedtocomputer-assistedstatisticalanalysis miumderivativeAFAP51,showedadifferentactivityprofile,hav- using the One-WayAnalysis of Variance ANOVA, and the Tukey– ing no effect on the tumour cell lines, MDA-MB-231 and MCF-7, Kramerpost-test.Differencesofp<0.05wereconsideredtobesig- and showing changes of resistance to detachment on the non- nificantlydifferentfromthecontrols. tumorigenic HBL-100 cells: an increase when cells were grown onpoly-L-lysineand an importantdecreasewhen the samewere seededonfibronectinandcollagenIV.Alltheseeffectsarestatisti- 3.Results callydifferentfromcontrols. 3.1.Resistancetodetachment 3.2.Re-adhesionaftertreatment The resistance to detachment is an index of the propensity of tumour cells to detach from the primary site of growth with the The propensity to re-adhere to fibronectin, collagen IV and aim to disseminate. This ability was studied by seeding cells on Matrigel(cid:3), in comparison to poly-L-lysine, of MDA-MB-231, MCF- componentsoftheextracellularmatrix(ECM)suchasfibronectin 7 and HBL-100 cells, following a 1-h challenge with 10(cid:3)4M andcollagenIVand,forcomparison,onpoly-L-lysineasubstrateon RM175 and AFAP51 was studied by exposing cells to the com- whichcellssimplyadherebyelectrostaticinteractions(Fig.1).A1- pounds while they were adherent to the growth substrate hchallengeofMDA-MB-231cellswith10(cid:3)4MRM175statistically (Fig. 2). The two complexes showed a similar trend: both induce tnemhcated ot ecnatsiseR )stinu yrartibra( AFAP51 MDA-MB-231 MCF-7 HBL-100 3.5 3.0 * 2.5 2.0 1.5 1.0 0.5 *** *** 0.0 P F C P F C P F C tnemhcated ot ecnatsiseR )stinu yrartibra( 82 A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 Fig.1. EffectofRM175andAFAP51onresistancetodetachment.MDA-MB-231, MCF-7,andHBL-100cells,seededon96-wellplasticplatespreviouslycoatedwith poly-L-lysine, fibronectin and collagen IV, were exposed for 1h to RM175 and AFAP5110(cid:3)4Mandthentoadilutedtrypsinsolutionfor30min,beforedetecting cells still attached to the growth substrate by the SRB test. Arbitrary units are calculatedfromthemean±SDoftwoexperiments,eachperformedinquadrupli- cateandcontrolsaresetequalto1.P=poly-L-lysine,F=fibronectin,C=collagenIV. (cid:4)p<0.05;(cid:4)(cid:4)p<0.01;(cid:4)(cid:4)(cid:4)p<0.001versuscontrols,ANOVAandTukey–Kramerpost- test. RM175 3.4.Effectoncellviability MDA-MB-231 MCF-7 HBL-100 90 The data in Table1 showthe effect of RM175and AFAP51on cell viability after a 24-h cell exposure, as determined with the 60 30 0 -30 -60 * * -90 P F C M P F C M P F C M onlyslightmodificationsofthecellabilitytore-adhereaftertreat- ment,withonlyastatisticallysignificantreductionofadherenceof HBL-100 cells to poly-L-lysine and to collagen IV following expo- suretotherutheniumcompoundRM175. 3.3.Effectsonmigrationandinvasion TheeffectsofRM175andAFAP51oncellmigrationweredeter- mined with properly adapted Transwell(cid:3) chambers, where the cells were subjected to a chemical (chemotaxis) or a contact (haptotaxis) stimulus to promote cell movement (Fig. 3). Treat- mentwith10(cid:3)4MRM175for1hpredominantlyledtoastatisti- cally-significant inhibition of haptotaxis in MDA-MB-231 and HBL-100cells,whiletheosmiumcompoundinhibitedchemotaxis; bothRM175andAFAP51hadnoeffectonthemigrationabilityof MCF-7 cells, independently of the stimulus being applied. The invasionabilityofthesamecells,studiedonTranswell(cid:3)chambers coatedwitha3Dmatrix(Fig.4),wasnotaffectedsignificantlyby RM175 or AFAP51, despite an appreciable and similar profile of invasionmodulationinthethreecelllines. sllec tnerehdA slortnoc sv noitairav )%( AFAP51 MDA-MB-231 MCF-7 HBL-100 90 60 30 0 -30 -60 -90 P F C M P F C M P F C M sllec tnerehdA slortnoc sv noitairav )%( Chemotaxis Haptotaxis MDA-MB-231 20 MCF-7 10 HBL-100 0 -10 -20 * -30 -40 *** *** * -50 ** RM175 AFAP51 RM175 AFAP51 Fig.2. Effectoncellabilitytore-adhereafterRM175andAFAP51treatment.MDA- MB-231,MCF-7,andHBL-100cellsweretreatedfor1hwithRM175andAFAP51 10(cid:3)4M,thenthecellswereremovedfromtheflasks,collected,re-suspendedand seededon96-wellplasticplatespreviouslycoatedwithpoly-L-lysine,fibronectin, collagen IV and Matrigel(cid:3). After 60min of incubation cells that adhered to the substratesweredetectedbytheSRBtest.Dataarethepercentofvariationversus controls calculated from the mean±SD of two experiments, each performed in triplicate.P=poly-L-lysine,F=fibronectin,C=collagenIV,M=Matrigel(cid:3).(cid:4)p<0.05 versuscontrols,ANOVA,andTukey–Kramerpost-test. sllec gnitargiM slortnoc sv noitairav )%( Fig.3. EffectofRM175andAFAP51onmigrationofcellsthroughpolycarbonate filters.MDA-MB-231,MCF-7,andHBL-100cellsweretreatedfor1hwithRM175 andAFAP5110(cid:3)4M,thenthecellswereremovedfromtheflasks,collected,re- suspendedandseededontheinsertsofTranswellTMcell-culturechambers.Data representcellsthatafter24hhavemigratedandarepresentonthelowersurfaceof the filter. Data are the percent of variation versus controls calculated from the mean±SDoftwoexperimentseachperformedintriplicate.(cid:4)p<0.05;(cid:4)(cid:4)p<0.01; (cid:4)(cid:4)(cid:4)p<0.001versuscontrols,ANOVA,andTukey–Kramerpost-test. 90 MDA-MB-231 80 70 MCF-7 60 HBL-100 50 40 30 20 10 0 -10 -20 -30 RM175 AFAP51 sllec gnidavnI slortnoc sv noitairav )%( A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 83 Fig.4. EffectofRM175andAFAP51oninvasionofcellsthroughMatrigel(cid:3).MDA- MB-231,MCF-7,andHBL-100cellsweretreatedfor1hwithRM175andAFAP51 10(cid:3)4M,thenthecellswereremovedfromtheflasks,collected,re-suspendedand seededoninserts.Datarepresentcellsthatafter96hhaveinvadedandarepresent onthelowersurfaceofthefilter.Dataarethepercentofvariationversuscontrols calculatedfromthemean±SDoftwoexperiments,eachperformedintriplicate. Table1 IC50valuesofRM175andAFAP51after24incubationoftheMDA-MB-231,MCF-7and HBL-100cells. IC50(lM) RM175 AFAP51 MDA-MB-231 62 48 MCF-7 93 15 HBL-100 54 16 MDA-MB-231,MCF-7andHBL-100cells,seededon96-wellplates24hbefore,were treatedwith10(cid:3)6(cid:5)10(cid:3)4MRM175and10(cid:3)5(cid:5)10(cid:3)3MAFAP51for24h.Attheend ofincubationtimecellviabilitywasmeasuredbyMTTtestandIC50valuescalcu- latedbyGraphPadPrismversion4.00forWindows(GraphPadSoftware,SanDiego, CA). MTTtest.ThecytotoxicactivityoftheosmiumcompoundAFAP51 RM175+HSA wasgenerallymorepronounced(IC valuesof48lM,15lMand 50 16lM for MDA-MB-231, MCF-7 and HBL-100 cells, respectively) 120 than that of RM175 (IC values of 62lM, 93lM and 54lM for 50 MDA-MB-231, MCF-7 and HBL-100 cells, respectively) in all cell lines. Both compounds showed an anti-proliferative effect on 90 HBL-100cellsgreaterorcomparabletothatonthetumorigeniccell ** *** lines, suggesting lack of selectivity for these compounds towards *** theseparticularcelllines. 60 TheeffectoncellviabilityofRM175andAFAP51wasalsostud- iedunderthesameexperimentalconditionsusedtoexaminecell detachment, re-adhesion, migration and invasion, i.e. 1-h treat- mentat10(cid:3)4M(Table2).RM175andAFAP51reducedcellviability 30 ofMDA-MB-231byabout20%,hadnoeffectonMCF-7,andcaused areductionof20%and30%,respectively,ontheviabilityofHBL- 100cells.Allthesevariationsarenotstatisticallysignificant. 0 3.5. T I h n e flu d e a n ta ce in of F H ig S . A 5 o s n ho cy w to t t h o a x t ic t it h y e o p f r R e M se 1 n 7 c 5 e a o n f d H A S F A A c P a 5 n 1 influence C o ntr ols H S A 1/1 H S A 1/5 H S A 1/10 R M R 1 M 7 1 5 75/ H S A R M 1/ 1 1 75/ H S R A M 1 1 /5 75/ H S A 1/10 the effects of RM175 and AFAP51 on the viability of MDA-MB- 231 cells (Fig. 5). The MTT test was applied 48h after a 24h exposure to acombinationofthe organometalliccompound,at a concentrationof12.4lMRM175correspondingto1/5oftheIC 50 (i.e.reducedcellgrowthby20%ofcontrolswhenusedaloneTable 1),andHSAataratio1/1,1/5or1/10.ThepresenceofHSA,atall concentrationused,gaverisetoasignificantincreaseincytotoxic- ity. A similar behaviour was found when the experiment was performedwithAFAP51at9.6lM;thepresenceofHSAreinforces theanti-proliferativeefficacyofthecomplexesalsointhiscase. 3.6.EffectonMMPsproductionand/oractivity The effects of the two organometallic compounds on matrix metalloproteinase(MMP-2andMMP-9)productionand/oractiv- ity were studied by the gelatine zymography test. MDA-MB-231 cellsproducethe92kDaMMP-9inappreciableamountswhereas HBL-100cellsprevalentlyproducethe72kDaMMP-2.AFAP51re- ducedtheproduction/activityofMMP-9byapproximately25%of controls, whereas RM175 was completely inactive. Conversely, both compounds strongly inhibited the production/activity of MMP-2((cid:3)65%versusuntreatedcontrols;Fig.6). 3.7.Effectonlungmetastasesinvivo TheeffectsofRM175andAFAP51treatmentonprimarytumour growth and on lung metastases formation were studied in the model of MCa mammary carcinoma, a murine transplantable tu- mourthatspontaneouslymetastasisestothelungs,inthreesepa- rate experiments (Table 3). Dose of 7.5mg/kg/day RM175 given from day 8 to 13 after tumour implantation (experiment 1), and 10mg/kg/day RM175 from day 6 to 9 (experiment 2), reduced Table2 thegrowthoftheprimarytumour,measuredonday13,byapprox- EffectofRM175andAFAP51oncellviability. imately50%ofuntreatedcontrols,atday13.Aftertreatmentwith- drawal,theprimarytumourstartstogrowagainand,onday20an MDA-MB-231 MCF-7 HBL-100 inhibitionby20%and30%onlywasdetectedrespectivelyforthe Controls 2.284±0.066 2.631±0.055 2.308±0.424 two doses. RM175 affected also the development and growth of RM175 1.899±0.064(83%) 2.665±0.105(101%) 1.803±0.098(78%) lung metastases. Metastasis reduction was evident in both the Controls 1.027±0.078 2.631±0.055 0.266±0.051 experimentsandconsistedofareductionbyabout70%ofthemean AFAP51 0.890±0.073(87%) 2.735±0.101(103%) 0.191±0.013(71%) numberandweight,expressedinmgperanimal,with4outof10 MDA-MB-231, MCF-7, and HBL-100 cells were treated for 1h with RM175 and animals being free of macroscopically detectable metastases in AFAP51 10(cid:3)4M, then the cells were removed from theflasks, collected, re-sus- experiment1,andofthemorepronouncedreductionby85%and pendedandseededon96wellplates.After24hcellviabilitywasdeterminedbythe 95%, respectively, of numberand weight, in the experiment with MTTassay.Dataarethemeanopticaldensity±SDoftwoexperimentseachper- formed in quadruplicate. Data in parentheses represent the percentage of each the higher daily dose of 10mg/kg/day. Indeed, at this daily dose treatedgroupversustherelevantcontrols(T/C%). asignificanttoxicitywasregisteredwith6outof10treatedani- )%( ytilibaiV lleC AFAP51+HSA 120 90 ** *** *** *** 60 30 0 C o ntr ols H S A 1/1 H S A 1/5 H S A 1/10 A F A A P F 5 A 1 P51/ H S A A F A 1/ P 1 51/ H A S F A A 1 P / 5 5 1/ H S A 1/10 )%( ytilibaiV lleC 84 A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 Fig. 5. Effect of RM175 and AFAP51 on cell viability. MDA-MB-231 cells were treatedfor24hwithRM175andAFAP51atvariousconcentrationsinthepresence ornotofHSA(seetableinSection2),thenthetreatmentsolutionswereremoved from the wells and replaced by complete medium containing 5% of FBS. Data representcellsviable48haftertheendoftreatment,asmeasuredbytheMTTassay andarethepercentageofeachtreatedgroupversustherelevantcontrols(T/C%) calculatedfromthemean±SDoftwoexperiments,eachperformedintriplicate. (cid:4)(cid:4)p<0.01;(cid:4)(cid:4)(cid:4)p<0.001versuscontrols,ANOVA,andTukey–Kramerpost-test. malshavingdiedbeforetheevaluationoflungmetastases.Inthe sameexperimentalmodel,AFAP51administeredatthesamedose levelsusedforRM175ispracticallyinactive,withanegligibleinhi- bitionoftheprimarytumourgrowth(20%versuscontrols)andno detectableeffectsonsecondarylungtumours. 4.Discussion Thestudyofmetal-basedcomplexesaspotentialdrugsincan- cer chemotherapy is still largely based on the enormous knowl- edge derived from the clinical success of platinum analogues (cisplatin, carboplatin, oxaliplatin, and to a lesser extent about six other platinum drugs). All these platinum drugs are thought to have DNA as the major target site and DNA binding efficiency andinducedconformationalchangesplaycriticalroles[26].Corre- spondingly,thesearchforinnovativemetal-baseddrugshassofar mainlyinvolvedtheevaluationoftheroleofthecentralmetaland itsligandsratherthanthestudyofdifferentmodesofaction. Ruthenium,asaplatinum-groupmetal,hasalsobeenfoundto MMP-9 display some favourable properties, and it is generally believed 10 MMP-2 thatrutheniumcompoundsarelesstoxicthanothertransitionme- 0 talcompounds,perhapsbecauseofthesimilarityofrutheniumto -10 iron,anessentialmetal.Theorganometallichalf-sandwichruthe- niumcompoundRM175belongstothisclassofnewpotentialanti- -20 cancer complexes. It was synthesized because ruthenium(II) is -30 thoughttobethemoreactiveformofruthenium.Thearenestabi- -40 lizes this oxidationstate as well as providinga hydrophobicface -50 thatcanfacilitatetransportacrosscellmembranesandplaysarole -60 inbiologicalrecognitionprocesses.ChloridoRuIIarenecomplexes -70 arethoughttobeactivatedbyhydrolysis[27]inasimilarfashion tocisplatin,followedbybindingtonuclearDNA,furthermoreex- RM175 AFAP51 tended arenes such as biphenyl are capable of intercalating be- tweenDNAbase-pairs[20,28,29]. Indeed,rutheniumcomplexeshavealsobeenshowntobeen- dowedwithotherpropertiesthatmaketheminterestingforcancer chemotherapy. The use of ruthenium coordination geometry has allowed Meggers and co-workers to design compounds in which anon-specificenzymeinhibitorsuchasstaurosporineshowsasig- nificantlyincreasedselectivityforGSK-3betaandcorrespondingly inhibits selectively the growth of melanoma cells [30]. Also the class of ruthenium compounds containing the lead compound NAMI-Ahas proved to be highlyinnovative,being selectivelyac- tive in the control of the formation and growth of solid tumour metastases[9].Theeffectonmetastasesisparticularlyimportant inthatitopensupthepossibilityofobtainingdrugs,basedontran- sitionmetals,thatcopewiththeworstaspectoftumourgrowth, that are responsible for the unfavourable prognosis in almost all thesolidhumantumours.Giventhatacertaindegreeofserendip- ityallowedtheselectivityofNAMI-Aformetastasestobediscov- ered, it is now worthwhile to test if other classes of ruthenium compoundsshareanyoftheseproperties. Datareportedinthepresentstudyfortheorganometallicruthe- nium compound RM175 and its isostructural osmium derivative AFAP51showtheimportanceofthemetalcentreandoftheinter- actionwithsomeinvitro-simulatedstepsoftumourdissemination ontheinvivoeffectsofasolidmalignanttumour. RM175ratherthanAFAP51isabletoslowdownmetastasisfor- mation more effectively than the reduction of the tumour in the primarysite.Indeed,attemptstocomparetheeffectsinvivowith thebehaviourinvitroinaseriesofmodelsmimickingthedetach- mentofcellsfroma primarymass andtheirinvadingproperties, does not always produce congruencies. If on the one hand, RM175 is able to inhibit cell detachment of a malignant cell (MDA-MB-231)betterthanofa‘normal’cell(HBL-100),theeffects onchemotaxis,haptotaxisandmoregenerallyoninvasionarenot ytivitca/noitcudorp sPMM stinu yrartibra slortnoc sv noitairav )%( A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 85 MDA-MB-231 10-4M RM175 – + – 10-4M AFAP51 – – + 92 KDa → HBL-100 10-4M RM175 – + – 10-4M AFAP51 – – + 72 KDa → Fig.6. EffectofRM175andAFAP51onMMPsproductionand/oractivity.MDA-MB- 231,andHBL-100cellsweretreatedfor1hwithRM175andAFAP5110(cid:3)4M,then incubatedforadditional24hinserumstarvedcompletemediumcontaining0.1% w/v BSA. Supernatants containing MMPs were collected and concentrated and equal protein amounts (100lg) subjected to SDS–PAGE. Gelatine digestion by proteasesisdetectedaswhitebandsagainstabluebackground(upperpanel).Band digestionisquantifiedbyusingImageMaster2Dversion4.01andMagicScan32 version4.3software(lowerpanel). Table3 EffectofRM175andAFAP51onprimarytumourandonlungmetastasesinmice carryingtheMCamammarycarcinoma. Primarytumourweight(mg) Lungmetastasesa Day13 Day20 Number Weight(mg) (A) Controls 884±202 2455±376 24.4±15.8 8.99±6.47 RM175 466±125 2042±321 8.67±7.03b 3.02±5.18b 7.5mg (53%) (83%) (36%) (33%) Controls 1627±286 3068±616 29.2±8.11 18.6±12.1 RM175 768±292 2353±420 4.50±3.32 0.861±0.749 10mg (47%) (77%) (15%) (5%) (B) Controls 1004±174 2415±439 17.5±9.98 5.68±6.90 AFAP51 837±145 2066±300 19.6±13.3 5.08±5.23 7.5mg (83%) (86%) (111%) (90%) AFAP51 943±164 2078±407 19.7±14.2 7.35±8.16 10mg (94%) (86%) (112%) (129%) Groupsof10CBAmice,inoculatedi.m.with106MCatumourcellsonday0were treatedi.p.withRM175(A)andAFAP51(B)at7.5mg/kg/dayand10mg/kg/day fromday8to13aftertumourimplant,orfrom6to9asreportedinSection2.Data aremean±SD,datainparenthesesareexpressedasapercentageofthetreated versuscontrols(T/C%). a Lungmetastasesweredeterminedonday20aftertumourimplant. b Meansexcludedtheanimalsfreeofmacroscopicallydetectablemetastasis(4 outof10). 86 A.Bergamoetal./JournalofInorganicBiochemistry104(2010)79–86 socleartoallowustostatethatthereissomeselectivityforthe ject)aregratefullyacknowledgedforfinancialsupport.Thisstudy malignant cells versus the normal counterpart. It is possible to wasperformedwithintheframeofCOSTActionD39. statethattheseprocessesaresomewhatinhibited,butitiscontro- versialthatthisinhibitionissimilarinmalignantandnormalcells, AppendixA.Supplementarydata whilstbeingalmostnon-existentinthemammarytumourMCF-7. Also,theinhibitionofcelldetachmentdoesnothampertheability Supplementarydataassociatedwiththisarticlecanbefound,in ofcellstore-adheretoasubstratewhendetachmentisforcedby theonlineversion,atdoi:10.1016/j.jinorgbio.2009.10.005. scrapingcellsoutofthesubstrateandallowingthemtoattachto a new plate coated with substrates typical of the ECM such as References fibronectin,typeIVcollagenormatrigelitself. ItthusseemsthattheorganometalliccomplexRM175,besides [1] D.Wang,S.J.Lippard,Nat.Rev.DrugDiscov.4(2005)307–320. its well-described effects on DNA [29,31] and on cell apoptosis [2] P.C.A.Bruijnincx,P.J.Sadler,Curr.Opin.Chem.Biol.12(2008)197–206. [3] L.Ronconi,P.J.Sadler,Coord.Chem.Rev.251(2007)1633–1648. [14], has some degree of selectivity against tumour metastases. [4] Y.-K.Yan,M.Melchart,A.Habtemariam,P.J.Sadler,Chem.Commun.(2005) However, it is necessary to understand the role of its ligands for 4764–4776. thisprocess,providedthatthemetalatombeingrutheniumisrel- [5] S.J.Dougan,P.J.Sadler,CHIMIAInt.J.Chem.61(2007)704–715. [6] J.M. Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H. evantasshownbythecompletelackofactivityofitsisostructural Schellens,Clin.CancerRes.10(2004)3717–3727. osmiumderivative.Thediscrepancybetweeninvitrocytotoxicity [7] C.G.Hartinger,M.A.Jakupec,S.Zorbas-Seifried,M.Groessl,A.Egger,W.Berger, andinvivoactivityoftheosmiumcomplexAFAP51isnotableas H.Zorbas,P.J.Dyson,B.K.Keppler,Chem.Biodivers.5(2008)2140–2155. [8] C.G. Hartinger, S. Zorbas-Seifried, M.A. Jakupec, B. Kynast, H. Zorbas, B.K. itismuchmorecytotoxicthantherutheniumcomplexRM175in Keppler,J.Inorg.Biochem.100(2006)891–904. theassayscarriedouthere.HoweverincontrasttoRM175,AFAP51 [9] E.Alessio,G.Mestroni,A.Bergamo,G.Sava,in:A.Sigel,H.Sigel(Eds.),Metal doesnotcauseinvivoreductionofthemammarycancerMCa.The Ions in Biological Systems. Metal Complexes in Tumor Diagnosis and As AnticancerAgents,MarcelDekkerInc.,NewYork,2004,pp.323–351. subtle differences between the chemistry and biochemistry of [10] M.J.Clarke,Coord.Chem.Rev.236(2003)209–233. rutheniumandosmiummeanthatmodificationstothedesignof [11] R.E.Morris,R.EAird,P.D.S.Murdoch,H.Chen,J.Cummings,N.D.Huges,S. osmiumcomplexesareneededinordertoproduceinvivoactivity. Parsons,A.Parkin,G.Boyd,D.I.Jodrell,P.J.Sadler,J.Med.Chem.44(2001) For example mixed oxygen/ nitrogen ligands such as picolinates 3616–3621. [12] R.E.Aird,J.Cummings,A.A.Ritchie,M.Muir,R.E.Morris,H.Chen,P.J.Sadler, also give rise to highly cytotoxic osmium complexes [32,33] and D.I.Jodrell,Br.J.Cancer86(2002)1652–1657. itwillbeinterestingtoinvestigatetheiractivityinvivoagainsta [13] A. Habtemariam, M. Melchart, R. Fernández, S. Parsons, I.D.H. Oswald, A. rangeofcancermodels. Parkin, F.P.A. Fabbiani, J.E. Davidson, A. Dawson, R.E. Aird, D.I. Jodrell, P.J. Sadler,J.Med.Chem.49(2006)6858–6868. [14] R.Hayward,Q.Schornagel,R.Tente,J.Macpherson,R.Aird,S.Guichard,A. 5.Conclusions Habtemariam,P.J.Sadler,D.I.Jodrell,CancerChemother.Pharmacol.55(2005) 577–583. [15] S.M.Guichard,R.Else,E.Reid,B.Zeitlin,R.Aird,M.Muir,M.Dodds,H.Fiebig, Theclinicalsuccessofcisplatinandsubsequentgenerationsof P.J.Sadler,D.I.Jodrell,Biochem.Pharmacol.71(2006)408–415. platinum anticancer complexes has stimulated the search for [16] A.F.A.Peacock,P.J.Sadler,Chem.AsianJ.3(2008)1890–1899. [17] A.F.A.Peacock,A.Habtermariam,R.Fernández,V.Walland,F.P.Fabbiani,S. active complexes amongst the other platinum-group metals. Parsons,R.E.Aird,D.I.Jodrell,P.J.Sadler,J.Am.Chem.Soc.128(2006)1739– Ruthenium complexes appear to possess several novel features. 1748. FirstbothRuIIandRuIIIcomplexesadopt6-coordinateoctahedral [18] A.F.A.Peacock,A.Habtermariam,S.Moggach,A.Prescimone,S.Parsons,P.J. geometryincontrasttothesquare-planarconfigurationofPtIIcom- Sadler,Inorg.Chem.44(2007)4049–4059. [19] H.Kostrhunova,J.Florian,O.Novakova,A.F.A.Peacock,P.J.Sadler,V.Brabec,J. plexessuchascisplatin.SecondlyRuIIIcomplexessuchasNAMI-A Med.Chem.51(2008)3635–3643. canexhibitanti-metastaticactivityandpreventthespreadofcan- [20] H.Chen,J.A.Parkinson,S.Parsons,R.A.Coxall,R.O.Gould,P.J.Sadler,J.Am. cer which is a major clinical problem. The experiments reported Chem.Soc.124(2002)3064–3082. [21] P.Skehan,R.Soreng,D.Scudiero,A.Monks,J.McMahon,D.Vistica,J.T.Warren, here showthat rutheniumitself isimportantinthis activity.The H.Bokesch,S.Kennet,M.R.Boyd,J.Natl.CancerInst.82(1990)1107–1112. organometallicRuIIarenecomplexRM175alsoexhibitsanti-meta- [22] A.Albini,Y.Iwamoto,K.Kleinman,G.R.Martin,S.A.Aaronson,J.M.Kozlowski, staticactivitywhereasitsisostructuralcongenorOsIIdoesnot.Itis R.N.McEwan,CancerRes.47(1987)3239–3245. [23] W.Kueng,E.Silber,U.Eppenberger,Anal.Biochem.182(1989)16–19. curiousthatalthoughtheosmiumcomplexismorepotenttowards [24] M.C.Alley,D.A.Scudiero,A.Monks,M.L.Hursey,M.J.Czerwinski,D.L.Fine,B.J. thecellsinvestigatedhereinvitroitisnotactiveagainstMCamam- Abbott,J.G.Mayo,R.H.Shoemaker,CancerRes.48(1988)589–601. marycarcinomainvivo,unlikeRM175.Thereasonsforthisinactiv- [25] M.M.Bradford,Anal.Biochem.72(1976)248–254. [26] E.R.Jamieson,S.J.Lippard,Chem.Rev.99(1999)2467–2498. ity require further investigation. Interestingly the presence of [27] F.Wang,A.Habtemariam,E.P.L.vanderGeer,R.Fernández,M.Melchart,R.J. human serum albumin enhanced the potency of both the ruthe- Deeth,R.Aird,S.Guichard,F.P.A.Fabbiani,P.Lozano-Casal,I.D.H.Oswald,D.I. nium and osmium complexes in vitro. It will be interesting now Jodrell,S.Parsons,P.J.Sadler,Proc.Natl.Acad.Sci.USA102(2005)18269– 18274. toinvestigatetheinvivoactivityofosmiumarenecomplexesina [28] H.Chen,J.A.Parkinson,R.E.Morris,P.J.Sadler,J.Am.Chem.Soc.125(2003) wider range of cancer models in vivo and to examine the effects 173–186. ofthenon-areneligandsonactivity.Invitrocytotoxicitytestshave [29] H.-K.Liu,S.J.Berners-Price,F.Wang,J.A.Parkinson,J.Xu,J.Bella,P.J.Sadler, suggestedthatthepresenceofN,O-chelatingligandssuchaspico- Angew.Chem.Int.Ed.45(2006)8153–8156. [30] E.Meggers,Chem.Commun.(2009),doi:10.1039/b813568a. linatesinosmiumarenecomplexescanalsogiverisetocytotoxic [31] O.Novakova,H.Chen,O.Vrana,A.Rodger,P.J.Sadler,V.Brabec,Biochemistry potencyashighascisplatinandcarboplatin[33]. 42(2003)11544–11554. [32] A.F.A.Peacock,S.Parsons,P.J.Sadler,J.Am.Chem.Soc.129(2007)3348–3357. [33] S.H. van Rijt, A.F.A. Peacock, R.D.L. Johnstone, S. Parsons, P.J. Sadler, Inorg. Acknowledgements Chem.48(2009)1753–1762. FondazioneCRTrieste(‘‘MADE”Project)andRegioneAutonoma FriuliVeneziaGiulia(‘‘NuoveTerapieeFarmaciAntitumorali”Pro-