Studies on the structures, cytotoxicity and apoptosis mechanism of 8-hydroxylquinoline rhodium(iii) complexes in T-24 cells

NJC PAPER Studies on the structures, cytotoxicity and apoptosis mechanism of 8-hydroxylquinoline Citethis:DOI:10.1039/c6nj00182c rhodium( ) complexes in T-24 cells† III Hai-Rong Zhang,*abc Yan-Cheng Liu,b Zhen-Feng Chen,*b Ting Meng,b Bi-Qun Zou,b You-Nian Liua and Hong Liang*ab Two rhodium(III) complexes (Rh(OQ) 3 (1) and Rh(BrQ) 2 (CH 3 OH)Cl (2), HOQ = 8-hydroxyquinoline, HBrQ = 5-bromo-8-hydroxyquinoline) of 8-hydroxylquinoline were synthesized and characterized. By MTTassay,theinvitrocytotoxicityofcomplexes1and2,comparedwithHOQ,HBrQandcisplatin,was evaluatedtowardsaseriesoftumorcelllinesaswellasthenormallivercelllineHL-7702.Complexes1 and 2 showed higher cytotoxicity against the tested tumor cell lines than the corresponding ligands, among which T-24 was the most sensitive cell line for complexes 1 and 2 (IC = 13.42 mM for 1, 50 18.91mMfor2).Comparedwithcisplatin,complex1exhibitedhighercytotoxicityagainstT-24cellsbut lower cytotoxicity against HL-7702(IC = 15.93 mM). Considering the better cytotoxicity of complex 1 50 than complex 2 against T-24 cells, the underlying anticancer molecular mechanisms were also investigated. DNA interaction studies revealed that complex 1 interacted with ct-DNA mainly via an Received(inVictoria,Australia) 19thJanuary2016, intercalative binding mode. Further investigation of intracellular mechanisms revealed that complex 1 Accepted27thApril2016 causedG2phasecellcyclearrestandinducedT-24cellapoptosisinadose-dependentmode.Targeting DOI:10.1039/c6nj00182c the mitochondrial pathway, the apoptotic mechanism in T-24 cells treated with 1 was studied by ROS detection, intracellular Ca2+ concentration measurements and caspase-9/3 activity assay, which suggested www.rsc.org/njc thatcomplex1inducedT-24cellapoptosisbythedisruptionofmitochondrial-relatedmechanisms. 1. Introduction andnephrotoxicity.4,5Thosedrawbacksprovideusthemotivation tofindnovelnon-platinummetal-basedcomplexeswithmaximal The serendipitous discovery of the cytotoxic properties of beneficialantitumorpropertiesandminimalsideeffects. cisplatin in the early 1960s has led to research into platinum- Rhodium complexes belong to one of the most promising baseddrugsinthefieldofmedicinalinorganicchemistry,1and classesofantitumoragents.RhCl 3 (cid:2)3H 2 Owasthefirstrhodium(III) a series of platinum-based anticancer drugs such as cisplatin, complexwithantitumorpropertiesreportedin1953,preceding carboplatin, and oxaliplatin are currently usedin the clinic or the discovery of cisplatin activity by more than a decade.6 in the combined chemotherapy for the treatment of various Subsequently,somerhodiumcomplexeswithsignificantantitumor malignancies such as bladder, ovarian, testicular, lung, head activitysuchasmer-[RhCl(NH)]andmer,cis-[RhCl(DMSO)(NH)] 3 33 3 2 3 andneckcancers.2,3However,theclinicaluseofplatinumdrugs werereportedinthenexthalfcentury.7,8Butthecellulareffectsof suffers from some major drawbacks such as drug resistance cytotoxic Rh(III) complexes have been systematically investigated and severe side effects including neurotoxicity, hepatotoxicity, only in recent years. Now, rhodium(III) complexes are the subject of current research on the anticancer activities of metal-based aCollegeofChemistryandChemicalEngineering,CentralSouthUniversity, complexes.Anda seriesofpapershave beenpublishedonthe Changsha,Hunan,410083,P.R.China.E-mail:zhr328@163.com synthesis and cytotoxicity of rhodium(III) complexes as anti- bStateKeyLaboratoryfortheChemistryandMolecularEngineeringofMedicinal cancer agents.9–11 They have been shown to bind nucleobases, Resources,SchoolofChemistry&PharmaceuticalSciences,GuangxiNormal dinucleotides,andDNAdodecamersinglestrands.12–14Thishas University,Guilin,Guangxi,541004,P.R.China. E-mail:chenzf@mailbox.gxnu.edu.cn,hliang@gxnu.edu.cn; arousedgreatinterestindiscoveringtheantitumoractivitiesof Fax:+867732120958;Tel:+867732120958 rhodium(III)complexes. cCollegeofMaterialsandEnvironmentalEngineering,HunanUniversityof Ontheotherhand,inthepastdecade,8-hydroxyquinolineand Humanities,ScienceandTechnology,Loudi,Hunan,417000,P.R.China itsderivativeshaveattractedgreatinterestfrommedicinalchemists †Electronicsupplementaryinformation(ESI)available.CCDC1447746(1)and and have been investigated for various medical applications, 1447747(2).ForESIandcrystallographicdatainCIForotherelectronicformat seeDOI:10.1039/c6nj00182c including as potential antitumor/antineoplastic agents.15,16 Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 NewJ.Chem. .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online View Journal Paper NJC A series of investigations of the cytotoxicity of metal-based Infraredspectra(inKBrpellets)weremeasuredonaPerkinElmer complexes of 8-hydroxyquinoline and its derivatives against FT-IR spectrometer. NMR spectra were recorded on a 600 MHz human cancer cell lines were reported along with the rise Fourier NMR spectrometer. C, H, N elemental analyses were of medicinal inorganic chemistry. For instance, Tardito et al. performed on a PerkinElmer Series II CHNS/O 2400 elemental studied the structure–activity relationship of a series of analyzer.Highperformanceliquidchromatography-massspectro- 8-hydroxoquinolinederivativesandfocusedonthecytotoxicity metry (HPLC-MS) was performed on a Thermo Fisher Exactive of the copper complex of 5-Cl-7-I-8-hydroxyquinoline, which massspectrometer.UV-Visible(UV-Vis)absorptionspectraand showedpotentialanticanceractivitieswithminoradverseside fluorescence measurements were collected on a PerkinElmer effects.17 Barilli et al. reported the antiproliferative/cytotoxic Lambda 45 UV-Visible spectrophotometer and a Shimadzu propertiesofthetransitionmetal(Cu2+,Fe3+,Co2+,Ni2+)complexes RF-5301/PCspectrofluorophotometer,respectively.Thecircular of8-hydroxyquinolinederivativesagainstHeLacells.18Yanetal. dichroism (CD) spectra of ct-DNA were recorded on a JASCO synthesized a series of endoperoxide–iron chelator conjugates J-810 automatic recording spectropolarimeter. Flow cytometry and found that 5-amino-8-hydroxyquinoline conjugates exhibited (FCM) was performed usinga FACS Aria II flow cytometer (BD highcytotoxicityandgoodselectivityagainsthumancancercells.19 Biosciences, USA). Fluorescence images were photographed Correiaetal.studiedthecytotoxicactivitiesofvanadium(IVandV) usingaNikonTE2000microscopysystem(Japan). and copper(II) complexes as potential anti-tuberculosis and anti-tumor agents.20 Inspired by these studies, weweregreatly 2.3. Synthesisofcomplexes1and2 interestedinexploringmoremetal-basedcomplexesof8-hydroxy- 2.3.1. Synthesis of 1. Complex 1 was prepared by treating quinolineligandswithotherrationalsubstitutivegroups. HOQ(0.1mmol,0.015g)withRhCl (cid:2)3H O(0.1mmol,0.021g) Although quite a few rhodium(III) complexes of 8-hydroxy- 3 2 inmethanol/chloroform(10:1)undersolvothermalconditions quinoline anditsderivativeshavebeenreported,theirbiological at801Cforthreedays.Brownblockcrystalsof1wereobtained activities, especially their anticancer activities, have still not been fully explored.21–25 In an effort to develop metal-based andsuitablecrystalswereselectedforX-raydiffractionanalysis. Yield (0.016 g, 88%). ESI-MS (in DMSO containing aqueous drugs with a better combination of anticancer activities of solution): m/z 574.0 [Rh(OQ) + K]+, 558.0 [Rh(OQ) + Na]+. 8-hydroxyquinoline and rhodium complexes, two rhodium(III) 3 3 Selected IR (KBr, cm(cid:3)1): 3402 (m, n(OH)), 3050 (w, (C–H)), complexes of 8-hydroxyquinoline (HOQ) and its derivative 1574 (s, n(CQN)), 1459 (s, n(CQC)), 1319 (s, n(C–N)), 1218 5-bromo-8-hydroxyquinoline (HBrQ) were synthesized and (s,n(C–O)),526(s,n(Rh–N)),415(s,n(Rh–O))(asshowninFig.S7, structurally characterized in this work. In an effort to explore ESI†).1HNMR (600MHz,DMSO-d )d 8.42(d, J=11.8Hz,3H), newantitumoragents,theirinvitrocytotoxicityagainstaseries 6 8.39 (s, 3H), 7.52 (ddd, J = 27.7, 16.6, 4.7 Hz, 3H), 7.41 (dt, ofhumancancercelllineswasscreenedandtheDNAbinding J=23.3,5.4Hz,3H),7.14–7.02(m,3H),7.00–6.85(m,3H)(see properties of the better cytotoxic complex (1) were primarily Fig.S10,ESI†).Anal.calcdforC H RhN O :C59.27;H3.91; studied.FocusingonthemostsensitivetumorcelllineT-24,the 28 22 3 4 N 7.41%. Found: C 59.10; H 3.98; N 7.38% (for the synthetic intracellular apoptotic pathway under the treatment of the route,seeScheme1). rhodium(III) complex 1 was further studied and discussed for 2.3.2. Synthesisof2.Complex2waspreparedbyreplacing betterunderstandingitspossibleanticancermechanism. HOQ with HBrQ using the same processing step for 1. Brown crystalsof2wereharvestedafterthreedays,andsuitablesingle 2. Experimental crystals were selected for X-ray diffraction analysis. Yield (0.029g,92%).ESI-MS(inDMSOcontainingaqueoussolution): 2.1. Chemicals (cid:3)m/z632.7[Rh(BrQ) (CH OH)Cl+OH](cid:3).SelectedIR(KBr,cm(cid:3)1): 2 3 All chemicals including rhodium(III) salts were of analytical 3427 (m, n(OH)), 2969 (w, (C–H)), 1580 (s, n(CQN)), 1448 grade and used without purification. HOQ and HBrQ were (s, n(CQC)), 1359 (s, n(C–N)), 1306 (s, n(C–O)), 769 (s, n(C–Br)), purchasedfromAlfa-Aesar.RPMI1640andfetalbovineserum 678 (s, n(Rh–N)), 544 (s, n(Rh–O)) (as shown in Fig. S8, ESI†). (FBS)werepurchasedfromHyclone(USA).3-(4,5-Dimethylthiazol- 1HNMR(600MHz,DMSO-d )d9.04(d,J=2.1Hz,2H),8.92(d,J= 6 2-yl)-2,5-diphenyltetrazoliumbromide(MTT),RNaseA,propidium 5.1 Hz, 1H), 8.79 (d, J = 8.3 Hz, 1H), 7.96 (dd, J = 23.7, iodide (PI), Hoechst 33258, acridine orange/ethidium bromide 10.9 Hz, 4H), 7.23 (s, 1H), 7.22 (s, 1H), 3.43 (d, J = 7.0 Hz, 3H), (AO/EB), rhodamine-123 (Rh123), DCFH-DA, Fluo-3/AM were obtained from Sigma Chemicals Co. (USA). The CasPGLOWt FluoresceinActiveCaspase-9/3Stainingkitwaspurchasedfrom BioVision. Calf thymus DNA (ct-DNA) was purchased from Sigma-Aldrich. Tris(hydroxymethyl)aminomethane (Tris buffer) waspreparedusingtwicedistilledwater. 2.2. Instrumentation Electrosprayionizationmassspectra(ESI-MS)wererecordedon a Bruker HCT electrospray ionization mass spectrometer. Scheme1 Syntheticrouteoftherhodium(III)complex1. NewJ.Chem. Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online NJC Paper immunosorbent assay (ELISA) enzyme labelling instrument with570/630nmdoublewavelengthmeasurement.Cytotoxicity wasevaluatedfromtheIC values,whichwerecalculatedusing 50 theBlissmethod(n=5). 2.6. DNAbindingexperiments All the spectral analyses for DNA binding experiments were Scheme2 Syntheticrouteoftherhodium(III)complex2. performed in Tris buffer saline (0.1 M, pH = 7.43). The stock solution of ct-DNA (2.0 mM) was stored at 4 1C for not more 1.90(s,1H)(seeFig.S12,ESI†).Anal.calcdforC H Br ClN O Rh: than5daysbeforeuse.Thestocksolutionofcomplex1(2.0mM) 19 13 2 2 3 C37.08;H2.13;N4.55%,found:C37.03;H2.34;4.50%(forthe waspreparedbydissolvingitinDMSOanddilutedsuitablywith syntheticroute,seeScheme2). Trisbuffertorequiredconcentrationsfornextspectralexperiments. ForUV-Visspectralanalysis,afixedsolution(3.0mL)with2.0(cid:4) 2.4. Crystallography 10(cid:3)5Mofcomplex1waspreparedandthenthestocksolution of ct-DNAwas gradually added. The UV-Vis absorption spectra ThesinglecrystalX-raydiffraction dataforcomplexes1and2 were recorded after the mixture solution had reacted enough. wereobtainedusingaBrukerSmartApexIICCDdiffractometer Theelectrostaticinteractionexperimentforcomplex1wasalso equipped with graphite monochromated Mo Ka radiation performedusingUV-Visabsorptionspectroscopy.3.0mLsolution (l = 0.71073 Å). CrysAlis RED was used for cell refinement, containing2.0(cid:4) 10(cid:3)5Mofcomplex1wastitratedbysuccessive data reduction and absorption correction. The structures additionsofsodiumdodecylsulfonate(SDS)solution.Thecorres- were solved by direct methods using SHELXL-97 programs.26 ponding absorption intensity was then recorded. The DNA Non-hydrogen atoms were refined by full-matrix least squares competitive binding studies between EB and complex 1 were methods. All ofthe hydrogenatoms were addedgeometrically carriedoutusingfluorescenceemissionspectroscopybymaintaining andrefinedusingaridingmodel.DIAMONDwasusedforthe aconstantconcentrationofct-DNA(2.0(cid:4)10(cid:3)5M)andEB(2.0(cid:4) drawingrepresentationsofthecomplexes. 10(cid:3)6M)andonlyvaryingthecomplexconcentration.CDspectra wererecordedin0.1MTrisbuffer(pH7.43).A3.0mLsolution 2.5. CellcultureandMTTassay ofct-DNA(1.0(cid:4)10(cid:3)4M)wasaddedtocomplex1(0,0.5,1.5,2.5, ThetumorcelllinesT-24,BEL-7404,Hep-G2,MGC-803,SK-OV-3 3.5(cid:4) 10(cid:3)4 M) andincubated for 5 min. Each sample solution andthehuman normalliver cell line HL-7702 were purchased was then tested in the range of 200–400 nm. The sample from the Shanghai Cell Bank of the Chinese Academy of containingonlyTrisbufferwasemployedastheblanksample. Sciences. Those cell lines were cultured in DMEM medium 2.7. Cellcycleexperiment (Gibco) supplemented with penicillin (100 units per mL), streptomycin (100 mg mL(cid:3)1) and fetal bovine serum (10%) at T-24cellswereseededatadensityof1(cid:4)105cellspermLina 371Cinahumidifiedatmosphereof5%CO /95%air.Thestock 75mLculturedishandincubatedfor24h.Theculturemedium 2 solutions of complexes 1 and 2 (2.0 mM, dissolved in DMSO) wasthenreplacedwithmediacontainingdifferentconcentrations weredilutedwithPBStotherequiredconcentrationimmediately ofcomplex1(0,7.0,14.0,28.0mM)andincubatedfor24h.After before use. Cisplatin (a commonly used anticancer drug) was 24 h treatment, T-24 cells (106–107 cells) were collected, washed chosenasareferencemetallodrugforevaluatingthepotencies with PBS (pH 7.40), and fixed with ice-cold 70% ethanol (no of complexes 1 and 2.27 The stock solution of cisplatin was cryoprotectant was used) at (cid:3)20 1C overnight. Before testing, prepared by dissolving cisplatin in 0.154 M NaCl to get a ethanolwasremovedbycentrifugingthecellsthefixedT-24cells concentrationof1mM.28 wereresuspendedin500mLofPBS(containing50mgmL(cid:3)1PIand The in vitro cytotoxicity evaluation was carried out using 100mgmL(cid:3)1RNaseA)andstainedfor45mininthedarkat371C. MTTassay.Around1(cid:4)104cellswell(cid:3)1wereseededin180mLof The cell cycle distribution was recorded on a FACS AriaII flow supplemented culture medium in 96-well micro plates, and cytometer(BD)andanalyzedusingMFLT32LTsoftware. incubatedfor24hat371Cinahumidifiedatmosphereof5% 2.8. Cellapoptosisexperiment CO . Then, appropriate concentrations (2.5, 5, 10, 20, 40 mM, 2 respectively) of HOQ, HBrQ, complexes 1 and 2, and cisplatin T-24cellswereincubatedin6-wellplates(1(cid:4)105cellspermL) wereadded(thevolumepercentageofDMSOwasnomorethan in 2 mL of culture medium; 24 h later, the cells were further 2%).Andthecellsweretreatedfor48h.Thecellsculturedwith incubated with complete medium only (control) and medium the culture medium not containing the compounds served as containing7.0,14.0,and28.0mMconcentrationsofcomplex1 the control. Upon completion of the treatment, 10 mL of MTT for8h,respectively.Then,thecellswereharvestedandwashed (5mgmL(cid:3)1)inPBS(pH7.40)wasaddedtoeachmicrowelland withPBS(pH7.40)and100mLof1(cid:4)bindingbufferwasadded. the plates were incubated for another 4 h in a cell culture After that, the cells were labelled with 5 mL of annexin V-FITC incubator. After that, the medium was removed and replaced and5mLofPI(50mgmL(cid:3)1)for20mininthedarkat41C,and with100mLofDMSOtodissolvetheformedformazancrystals. finally,thecellswereresuspendedin400mL1(cid:4)bindingbuffer Finally, the absorbance was read using an enzyme-linked beforebeinganalyzedbyflowcytometry. Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 NewJ.Chem. .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online Paper NJC 2.9. Hoechst33258andAO/EBexperiments cytometer. The analysis results were described as the percent changeintheactivitycomparedwiththeuntreatedcontrol. The morphological changes of T-24 cells were detected by Hoechst33258andAO/EBdoublestainingforcellularapoptosis. For the Hoechst 33258 experiment, T-24 cells adhered on a 3. Results and discussion coverslipweretreatedwith7.0,14.0,and28.0mMofcomplex1 for 8 h, respectively. Upon completing the treatment, 0.5 mL 3.1. Synthesisandstructuralcharacterization of 4% para formaldehyde was added to fix the cells on the coverslipfor10min,rinsedtwicewithPBS,stainedwith0.5mL Hydroxyquinolinato rhodium(III) complexes 1 and 2 were ofHoechst33258fluorescentdyefor5minat371Cinthedark. prepared by the reaction of the corresponding ligand with RhCl (cid:2)3H Oinequimolaramountsundersolvothermalconditions TheexcessdyewasremovedbywashingtwicewithPBS.Then, 3 2 at801Cforthreedays.Amixtureofmethanolandchloroform a drib of an anti-fluorescence quenching liquid was dropped withavolumeratioof10:1wasusedasasolvent(asshownin on a slide, which was then covered by a coverslip. The images Schemes1and2). of stained nuclei were captured using a Carl Zeiss LSM710 TheUV-Visabsorptionspectraofcomplexes1and2in0.1M confocal microscope (Ex/Em = 346 nm/460 nm). For the Tris buffer (pH 7. 43) are shown in Fig. S1 and S2 (ESI†). As AO/EB double staining experiment, the T-24 cells were treated shown in Fig. S1 (ESI†), two characteristic absorption bands with7.0,14.0,and28.0mMofcomplex1for8h.Aftertreatment, were found. One of the intensive bands at ca. 269 nm was the T-24 cells were collected and suspended in PBS and the attributed to the p–p* electron transition of the aromatic suspended cells were stained with AO-EB working solution (containing 100 mg mL(cid:3)1 AO and 100 mg mL(cid:3)1 EB) for 5 min structureanditshowedanobviousblue-shiftof38nmcompared at 37 1C. The stained cells were then observed and photo- with the HOQ ligand, which suggested the existence of a strong coordinationeffectbetweenthemetalionandHOQ.Theotherless graphed immediately (Ex/Em = 488 nm/510–550 nm) using a intense band at ca. 399 nm is typical of metal to ligand charge fluorescencemicroscope(NikonTE2000,Japan). transfer (MLCT). As shown in Fig. S2 (ESI†), three characteristic 2.10. Measurementofmitochondrialmembranepotential(Dw) absorption bands were found. The bands at ca. 275 nm and 343 nm were attributed to the p–p* electron transition of the T-24 cells were seeded in 6 well plates and allowed to adhere aromaticstructureandn–p*electrontransitionofthehalogenated for 24 h and then treated with 14.0 mM of complex 1. After structure of the HBrQ ligand, respectively.29 Both the bands had treatment,thecellswereharvestedandstainedwith10mgmL(cid:3)1 obvious blue-shifts of 27 nm and 18 nm, respectively compared Rh123for30minat371Cinthedark,washedtwiceandmixed withtheHBrQligand.Thebandatca.423nmwasattributedto with300mLofserum-freeculturemediumbeforebeingmonitored the MLCT. These results indicated that a coordination reaction byflowcytometry. happenedbetweentheRh(III)metalionandHBrQ. The FT-IR spectra of complexes 1 and 2 and the corres- 2.11. Measurementofreactiveoxygenspecies(ROS) pondingligandsareshowninFig.S3–S6(ESI†).Comparedwith productionandcytoplasmiccalciumconcentration([Ca2+] ) c theIRspectrumofHOQ,obviousabsorptionchangescouldbe The intracellular ROS production level and the intracellular foundintheIRspectrumofcomplex1.Thestrongabsorption Ca2+concentrationweretestedbyflowcytometryanalysisusing bandatca.3050cm(cid:3)1wasweakened,indicatingthedeprotonated DCFH-DA staining and Fluo-3/AM staining, respectively. T-24 8-hydroxylmoietyofHOQduetoitscoordinationtothemetalion. cells were exposed to 14.0 mM of complex 1 for 8 h at 37 1C. Theringstretchingfrequenciesofn(CQN)andn(CQC)shiftedto Then, the treated cells were loaded for 30 min with DCFH-DA 1574 cm(cid:3)1 (Dn = (cid:3)2 cm(cid:3)1) and 1498 cm(cid:3)1 (Dn = (cid:3)4 cm(cid:3)1), (100 mM) or Fluo-3/AM (5 mM) at 37 1C. After that, the loaded respectively.TheC–OstretchandC–Nstretchshiftedto1218cm(cid:3)1 T-24 cells were washed twice with serum-free cell culture (Dn=13cm(cid:3)1)and(Dn=(cid:3)60cm(cid:3)1),respectively.Themagnitudeof medium, and then maintained in 500 mL serum-free culture theshiftDnobviouslyindicatedthatOandNcoordinatedwiththe medium. Finally, the cells were examined by flow cytometry Rh(III)metalion.30Thiswasfurthersupportedbybandsinthefar analysisimmediately. infraredregion,526cm(cid:3)1correspondingtoRh–Nand415cm(cid:3)1to Rh–Obondstretches.31Similarly,comparedtheIRspectraofHBrQ 2.12. Determinationofcaspase-9/3activitybyflowcytometric with complex 2, the broad band of at 3317 cm(cid:3)1 disappeared, analysis indicatingthedeprotonated8-hydroxylmoietyofHBrQowingtoits Thecaspase-9/3activitywasevaluatedbyflowcytometryusing coordination to the metal ion. There were also some shifts. The FITC-LEHD-FMK (for caspase-9) or FITC-DEVD-FMK (for bands of n(CQN), n(CQC), n(C–O) and n(C–N) shifted to caspase-3) staining. The control T-24 tumour cells and the 1580cm(cid:3)1(Dn=6cm(cid:3)1),1448cm(cid:3)1(Dn=53cm(cid:3)1),1306cm(cid:3)1 T-24 tumour cells after exposure to 14.0 mM of complex 1 (Dn=158cm(cid:3)1)and1359cm(cid:3)1(Dn=81cm(cid:3)1),respectively.The for 8 h were harvested, washed twice with PBS, and then appearance of new bands of 678 and 544 cm(cid:3)1 was supported maintained in 300 mL buffer, followed by addition of 1 mL of the formation of Rh–N and Rh–O bonds. These results strongly FITC-LEHD-FMK (for caspase-9) or FITC-DEVD-FMK (for indicatedthattheBrQ(cid:3)coordinatedwiththeRh(III)metalion. caspase-3), and incubation for 1.0 h at 37 1C in the dark. Themolecularstructuresofthetworhodium(III)complexes Finally, the cells were monitored using a FACS AriaII flow 1 and 2 were further authenticated by single crystal X-ray NewJ.Chem. Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online NJC Paper diffractionanalysesandaredepictedinFig.1and2.Thedetails another chelated 8-hydroxyquinoline form a tight equatorial ofcrystallographicdataandstructurerefinementparametersof plane, while one N(3) atom and one O(1) atom from other complexes1and2aresummarizedinTableS1(ESI†).Complex1 different chelated 8-hydroxyquinoline derivatives are located crystallizes in a monoclinic crystal system with space group in apical positions. The bond lengths and angles are shown P2 /c, while complex 2 crystallizes in a triclinic crystal system in Fig. 1. The NMR data for complex 1 also supported the 1 withspacegroupP1 % .Boththecomplexespresentmononuclear structurecomparedwithHOQ(seeFig.S9andS10,ESI†)and structure, but include different numbers of corresponding agreed well with the reported results.32 Different from quinoline ligands. Complex 1 possesses one metal centre and complex 1, the Rh(III) atom of complex 2 is chelated by three three8-hydroxyquinolineligands.However,complex2hasone oxygenatoms(twoofthemfrom5-bromo-8-hydroxyquinoline Rh(III)metalcentre,two5-bromo-8-hydroxyquinolinatoligands, andthe other Oatomfromthemethanol molecule), twonitro- onechloridoanionligandandonemethanolmoleculeligand. gen atoms from 5-bromo-8-hydroxyquinoline and one chloride The coordination geometries of the metal Rh(III) centre of anion. The Rh(1) atom, one chelated 5-bromo-8-hydroxy- complexes 1 and 2 can be described as a distorted octahedral quinoline, one O(1) atom from another chelated 5-bromo-8- geometry.TheRh(III)atomofcomplex1ischelatedbythreeoxygen hydroxyquinolineandonechlorideanionformatightequatorial atomsandthreenitrogenatomsfromthree8-hydroxyquinolines. plane, while one N(1) atom from another chelated 5-bromo-8- TheRh(1)atom,onechelated8-hydroxyquinoline,oneN(1)atom hydroxyquinolineandoneO(3)atomfromthemethanolmole- from chelated 8-hydroxyquinoline and one O(3) atom from cule are located in apical positions. The bond lengths and angles are shown in Fig. 2. The NMR data for complex 2 also agreed with the structure compared with HBrQ (see Fig. S11 andS12,ESI†). Moreover, to analyze the exact species of the two metal complexes existing in solution state, the existing species of complexes 1 and 2 in aqueous solution were investigated by ESI-MSanalysis,inordertobetterunderstandtheirantitumor activities and their potential structure–activity relationships (SAR). As demonstrated in Fig. S7 (ESI†), it was found that themajorabundancesofESI-MSfor1peakedat574.0and558.0 inthepositiveionmode,correspondingto574.0[Rh(OQ) +K]+and 3 558.0 [Rh(OQ) + Na]+, respectively. The SAR of 1 was also 3 monitoredbyHPLC-MSusingamobilephaseofmethanol–H O 2 (70/30), as shown in Fig. S13 (ESI†). The major abundance of HPLC-MSfor1peakedat558.0[Rh(OQ) +H]+inthepositiveion 3 Fig.1 Theball-stickpresentationofthecrystalstructureofcomplex1. mode,whichalsosuggestedthatitscoordinatedstate(Rh(OQ) ) 3 Selectedbondlengths(Å)andangles(1),Rh(1)–O(1)2.0166(17),Rh(1)–O(2) of1wasretainedforover72hinanaqueoussolution.Whilethe 2.0223(16), Rh(1)–O(3) 2.0172(15), Rh(1)–N(1) 2.026(2), Rh(1)–N(2) majorabundanceofESI-MSfor2peakedat632.7inthenegative 2.0122(19), Rh(1)–N(3) 2.0125(19), O(1)–Rh(1)–O(2) 91.46(7), O(1)–Rh(1)– N(1)82.96(7),O(2)–Rh(1)–N(1)92.46(7),O(1)–Rh(1)–N(3)174.57(7),O(3)–Rh(1)– ion mode, corresponding to [Rh(BrQ) 2 (CH 3 OH)Cl + OH](cid:3) (as N(2)94.09(7). shown in Fig. S8, ESI†). The HPLC-MS analysis for 2 was performed using a mobile phase of methanol–H O (60/40) (see 2 Fig. S14, ESI†). The major abundance of HPLC-MS for 2 also peakedat632.8[Rh(BrQ) (CH OH)Cl+OH](cid:3)inthenegativeion 2 3 mode,suggestingthatRh(BrQ) (CH OH)Clof2wasretainedafter 2 3 72hinanaqueoussolution.TheESI-MSanalysisandHPLC-MS results strongly suggested that complexes 1 and 2 tended to maintain a mononuclear coordination structure in aqueous solution, with an OQ/Rh(III) ratio of 3:1 for 1 and a BrQ/Rh(III) ratioof2:1for2. 3.2. InvitroantitumoractivityevaluationbyMTTassay TheinvitrocytotoxicityoftheligandsHOQ,HBrQandcomplexes1 and2wasevaluatedbyMTTassayagainstT-24,BEL-7404,Hep-G2, Fig.2 Theball-stickpresentationofthecrystalstructureofcomplex2. MGC-803,SK-OV-3tumorcelllines.Thehumannormallivercell Selected bond lengths (Å) and angles (1), Rh(1)–O(1) 2.027(3), Rh(1)–O(2) lineHL-7702wasalsotestedfortheselectivitystudy.Cisplatinwas 2.025(4),Rh(1)–O(3)2.075(4),Rh(1)–N(1)1.988(4),Rh(1)–N(2)2.007(4),Rh(1)– usedasthepositivecontrol.AsshowninFig.3andTableS2(ESI†), Cl(1)2.3284(15),O(1)–Rh(1)–O(2)91.17(15),O(1)–Rh(1)–Cl(1)91.03(11),N(1)– complexes 1 and 2 were more sensitive to the T-24 cell line Rh(1)–O(1) 82.81(16), N(1)–Rh(1)–O(2) 87.85(17), N(1)–Rh(1)–Cl(1) 91.09(14), N(1)–Rh(1)–N(2) 97.74(17), N(1)–Rh(1)–O(3) 174.97(16), O(3)–Rh(1)–N(2) than the other four tested cancer cells with the IC 50 value of 87.29(16),O(3)–Rh(1)–O(1)92.19(14),O(3)–Rh(1)–O(2)92.78(16). 13.42–18.91 mM. Compared with the corresponding ligands Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 NewJ.Chem. .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online Paper NJC (HOQ: IC = 76.24 (cid:5) 0.11 mM and HBrQ: IC 4 200 mM), absorptionspectrumofmetalcomplex1exhibitedanmaximum 50 50 complexes 1 and 2 showed enhanced cytotoxicity against the absorption band centered at 265 nm attributable to the p–p* T-24 cell line. It should be noted that both metal complexes electron transition of the aromatic structure of HOQ. The displayed lower cytotoxicity than cisplatin against the normal addition of increasing amounts of SDS did not cause obvious human liver HL-7702 cells and the IC values of complexes 1 hypochromicity at all at the maximum absorbance, which 50 and2againstHL-7702cellswerealsohigherthanthatagainst suggested that the electrostatic interaction between complex 1 the T-24 cancer cells, thereby, complexes 1 and 2 exhibited andDNAcanbeexcluded. lower side effects than cisplatin and slight selectivity to T-24 Moreover, to ascertain the possible binding mode between bladder cancer cells. Between the two complexes, complex 1 complex1andDNA,theinteractionofcomplex1withct-DNA exhibitedhighercytotoxicitythancomplex2againstT-24tumor was also investigated here. As shown in Fig. 4B, a significant cells with an IC value of 13.42 (cid:5) 0.04 mM. From their hypochromic ratio of 83% was observed at 265 nm with an 50 structures,itcouldbeenvisionedthatthedifferentsubstituent obvious red-shift of 19 nm when the [DNA]/[1] ratio reached groupsandthenumberof8-hydroxyquinolinederivativeligands 7:1. The hypochromic effect and red shift were regarded as may play a key role in determining their cytotoxicity. Since typical phenomena of intercalation between small molecules complex1exhibitedhighercytotoxicitythancomplex2toward andDNA.35Theresultsstronglyrevealedthatmetalcomplex1 the T-24 tumor cells, it was chosen to study DNA binding and interactedwithct-DNAinanintercalativebindingmode. theintracellularactionmechanisminT-24cells. 3.3.2. Fluorescence spectral analysis. The DNA binding modeofcomplex1withct-DNAwasalsodiscussedbycompeting 3.3. DNAbindingstudies with ethidium bromide (EB) as an intercalative probe. The fluorescence emission intensity of the EB–ct-DNA system will The anticancer activity of many drugs is achieved by blocking be significantly quenched if EB was replaced by other small DNAreplicationofcancercells.Intumorcells,DNAreplication molecules, which can be used to distinguish the intercalative can be blocked by the intercalation of small molecule drugs and non-intercalative compounds.36 As shown in Fig. 5, in into the base pairs of DNA.33 Thus, the interactions between the absence of complex 1, the EB–ct-DNA system gave strong small molecule drugs and DNA are believed to be one of the fluorescence emission with the maximum emission intensity primary action mechanisms for the antitumor activity. To at 581.4 nm, indicating that the intercalated EB molecules better understand the antitumor activity of metal complexes, were sufficiently protected by the neighboring base pairs in theDNAbindingofmetalcomplex1wasinvestigatedbyUV-Vis the ct-DNA from being quenched by polar solvent molecules. absorption,fluorescenceemissionandCDspectroscopy. Underthegradientadditionof1,thefluorescenceintensitywas 3.3.1. UV-Visabsorptionspectralanalysis.UV-Visabsorption gradually quenched, which indicated that the intercalation spectroscopy is one of the most useful techniques to examine bindingmodeof1withDNAexisted.Thefluorescencequenching the binding mode of a metal complex with DNA. Thus, on constant(K )wascalculatedusingtheStern–Volmerequation:37 addition of SDS, the electrostatic interaction of 1 with ct-DNA q wasprimarilyexaminedbyUV-Visspectrophotometry.SDSisa I /I=1+K (cid:4)[Q] (1) 0 q kindofprobeusedforunderstandingwhethertheelectrostatic interactionexistedduetotheaggregatedSDSanionsactingas whereI isthefluorescenceintensityoftheEB–DNAsystemin 0 an appropriate substitute for the DNA polyanionic backbone, theabsenceofaquencher,Iisthefluorescenceintensityofthe whichledtospectralchanges.34AsshowninFig.4A,theelectronic EB–DNA system in the presence of a quencher.38,39 [Q] is the equilibrium concentration of the quencher, which is usually replaced by the total concentration of the complex.40–42 As displayed by the inset plot of Fig. 5, K was calculated to be q Fig.4 (A)UV-Visabsorptionspectraofcomplex1(2.0(cid:4)10(cid:3)5M)inthe absence(dashedline)andpresence(solidline)ofincreasingamountsof Fig.3 IC 50 (mM) values of ligands and complexes 1 and 2 against five SDSfrom1:1to5:1.(B)UV-Visabsorptionspectraofcomplex1(2.0(cid:4) selected tumor cell lines and the normal human liver cell line HL-7702 10(cid:3)5M)intheabsence(dashedline)andpresence(solidline)ofincreasing aftertreatmentfor48h. amountsofct-DNAfrom1:1to7:1. NewJ.Chem. Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online NJC Paper comparedtothecontrolcells(7.28%).Inlinewiththisfinding, thecellpopulationsinG1andSphasesdecreasedaccompanied bytheincreaseofG2phasearrestedcells.Theseresultsdemon- strated that, in T-24 cells, cell cycle arrest in the G2/M phase contributedtothecytotoxicityofcomplex1.SincetheG2/Mcell cyclecheckpointisoneofthecrucialimportantresponsestothe DNAdamage,theseresultsfurthersuggestedthatDNAwasan importantintracellulartargetforthiscomplex. 3.5. Cellapoptosisassay Fig.5 (A)FluorescenceemissionspectraofEB–ct-DNAsystems([DNA]= Consideringtheessentialroleofcellcyclearrestintumorcells 2.0 (cid:4) 10(cid:3)5 M, [EB] = 2.0 (cid:4) 10(cid:3)6 M) in the absence and presence of apoptosis,45 the annexin V-FITC/PI assay was performed to complex1with[complex1]/[EB–ct-DNA]ratiosrangefrom1:1to10:1. determine whether the metal complex-induced cell growth (B)Fluorescencequenchingconstant(K )byI /Iversus[Q]. q 0 inhibition of T-24 cells was the result of apoptosis. As shown inFig.8,thepercentagesofT-24apoptoticcells,includingearly 1.56 (cid:4) 104 M(cid:3)1. The results further exhibited considerably apoptotic cells (see Q3 zone) and late apoptotic cells (see Q2 strongintercalationofcomplex1withct-DNA.Theyareconsistent zone) after treatment 1, increased from 4.7% to 8.5%, 12.5%, withtheaboveresultsoftheUV-Visspectralanalysis. and 17.1%, respectively. These results suggest that complex 1 3.3.3. Circular dichroism spectral analysis. CD spectro- caninducetheapoptosisofT-24cellsandtheapoptoticeffectis scopyisoneofusefultechniquesformonitoringtheconformational concentrationdependent. variationsofDNAinthepresenceofametalcomplexinsolution. To further elucidate the T-24 cell apoptosis induced by The ct-DNA exhibited a positive band at 278 nm due to base treatment with complex 1, the nuclear morphology was also stackingandanegativebandat246nmduetotheright-handed monitored by Hoechst 33258 staining and AO/EB double helicityoftheB-DNAform.Thesetwobandswerequitesensitive stainingassays.Generally,cellapoptosisisusuallyaccompanied to the mode of DNA interactions with small molecules.43 As by morphological changes such as cell shrinkage, nuclear showninFig.6,withgradualadditionofcomplex1toct-DNA, fragmentation, chromatin condensation and the formation of the intensities of both positive and negative bands underwent apoptotic bodies.46 As shown in Fig. 9A, after treatment with great changes, the decrease percentages in the maximal DNA complex 1 at gradient concentrations, an increasing number positive and negative bands by 1 were 34.04% and 53.12%, of cells with apoptotic features (cell shrinkage and nucleus respectively. These results further confirmed the intercalation fragmentation)wereobserved,comparedwiththecontrolcells. binging mode of complex 1 with DNA via the planar aromatic AsshowninFig.9B,underthesameconditions,typicalapoptotic structureofquinolinol.44 changesinnuclearchromatin(brightgreenandorangefluores- cence indicated early-stage apoptotic, orange-red fluorescence 3.4. Cellcyclearrest indicated late-stage apoptotic) were also shown in T-24 cells To explore the intracellular mechanism of complex 1 for an treatedwithcomplex1,comparingwiththelivingcontrolcells anti-proliferative effect on T-24 tumor cells, the cell cycle (greenfluorescenceindicated).Andtheapoptoticfeatureswere progressionoftheT-24cellstreatedby1wasprimarilyexamined muchmoreobviouswiththeincreaseinthedoseofcomplex1. byPI-flowcytometricanalysis.AsshowninFig.7,treatmentof Thus, the results of Hoechst 33258 and AO/EB staining assays T-24cellswithincreasingconcentrationofcomplex1increased furtherconfirmedthatcomplex1couldeffectivelyinduceT-24 cell cycle arrest at the G2 phase, leading to an increase in cell cellapoptosisinadose-dependentmanner.47,48 population in the G2 phase (13.39%, 22.17%, and 24.44%) Fig.6 CDspectraofct-DNAintheabsenceandpresenceofcomplex1, Fig.7 Cell cycle analysis by flow cytometry for T-24 cells treated with [DNA]=1.0(cid:4)10(cid:3)4M,[complex1]=0to3.5(cid:4)10(cid:3)4M. complex1. Thisjournalis©TheRoyalSocietyofChemistryandtheCentreNationaldelaRechercheScientifique2016 NewJ.Chem. .73:73:40 6102/50/32 no ygolonhceT fo etutitsnI ainrofilaC yb dedaolnwoD .6102 lirpA 82 no dehsilbuP View Article Online Paper NJC Fig.10 The T-24 cells treated with 14.0 mM of complex 1 for 8 h and stained with Rh123 indicated the loss of the mitochondrial membrane potential(Dc)byflowcytometricanalysis. thedisruptionofmitochondrial-relatedmechanisms,theupstream regulatory mechanisms leading to complex 1-induced mito- chondrialdysfunctionwereinvestigated.Theeffectsofcomplex Fig.8 TheinductionofapoptosisofT-24cellsmeasuredbyflowcyto- metry using PI and FITC-Annexin V assay after treatment with different 1onintracellularROSlevelsandintracellularconcentrationof concentrationsofcomplex1for8h. Ca2+wereexamined,respectively.ForROSgenerationdetection assay,T-24cellstreatedwithcomplex1wereexaminedbyflow cytometryanalysisusingDCFH-DAasaprobedye.Asshownin 3.6. Themitochondrialmembranepotentialassayforcell Fig.11A,theROSgenerationlevel(88.0%)incomplex1treated apoptosis T-24cellswassignificantlyenhancedcomparedwiththecontrol It is well known that mitochondria play a critical role in the (52.7%). The result suggested complex 1-induced T-24 cells regulation of apoptosis.49,50 Many of apoptotic stimuli trigger a apoptosiswascloselyrelatedtotheROS-mediatedmitochondrial changeinthemitochondrialmembranepermeability.Thelossof dysfunction pathway. For intracellular concentration of Ca2+ mitochondrial membrane potential (Dc) is widely regarded as a detection assay (as shown in Fig. 11B), compared with the characteristic of cell apoptosis in the early stage. Hence, the control, the intracellular Ca2+ concentration in T-24 cells alterationofDcT-24cellswastestedbyflowcytometricanalysis exposed by complex 1 increased from 48.9% to 64.6%. These using rhodamine 123 (Rh123) staining. As shown in Fig. 10, the results proposed that the complex 1-induced mitochondrial leftmarkerwasconsideredasmitochondriawithalowmembrane apoptoticpathwaywasachievedviathemitochondriadysfunction potential,i.e.depolarizedmitochondria,andtherightmarkerwas triggered by ROS generation, which could be proven by the considered as mitochondria with a high membrane potential, enhancementoftheintracellularCa2+concentration. i.e. polarized mitochondria.51 The mitochondrial polarization decreasedfrom51.1%to37.3%aftertreatmentofT-24cellswith 3.8. Assessmentonthecaspase-9/3activationforcell 14.0mMcomplex1for8h,comparedtocontrolcells.Theresult apoptosis intimatedthatcomplex1mayinducetheT-24cellapoptosisbythe disruptionofmitochondrial-relatedmechanisms. Itisgenerallyacceptedthatcaspase-9and-3playessentialroles as executors of cell apoptosis.56,57 To further confirm the 3.7. DetectionofROSgenerationandintracellular disruption of mitochondrial-related mechanisms induced by concentrationofCa2+ complex 1, the activation of caspase-9 and -3 was assessed by IntracellularROSgenerationisaneventupstreamoflossofthe mitochondrial membrane potential.52–55 To further authenticate Fig.11 (A)ThegenerationofROSinT-24cellstreatedwith14.0mMof Fig.9 Cell morphological observation on the T-24 cells for apoptosis complex 1 for 8 h. (B) The detection on the intracellular level of Ca2+ induced by complex 1 for 8 h. Hoechst 33258 staining (A) and AO/EB concentrationintheT-24cellstreatedwith14.0mMofcomplex1for8h doublestaining(B)areshown,respectively. byflowcytometryusingFluo-3/AMasafluorescentprobe. NewJ.Chem. 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