Inorganica Chimica Acta 423 (2014) 156–167

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Inorganica Chimica Acta

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Characterization of the antiproliferative potential and biological targetsof a trans ketoimine platinum complex

http://dx.doi.org/10.1016/j.ica.2014.07.0670020-1693/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Departamento Ciências da Vida, Faculdade Ciências eTecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Tel./fax:+351 21 294 85 30.

E-mail address: ma.fernandes@fct.unl.pt (A.R. Fernandes).

Joana Silva a, António Sebastião Rodrigues b, Paula A. Videira c, Jamal Lasri d, Adília Januário Charmier d,e,Armando J.L. Pombeiro d, Alexandra R. Fernandes a,d,⇑a Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugalb Human Molecular Genetics Research Centre (CIGMH), Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, Lisbon, Portugalc CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon, Portugald Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugale Faculdade de Engenharia, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal

a r t i c l e i n f o

Article history:Received 30 April 2014Received in revised form 29 July 2014Accepted 30 July 2014Available online 13 August 2014SI: Antitumor Active Organotin Compounds

Keywords:CancerCisplatinCytotoxicityDNASelectivitytrans-Pt

a b s t r a c t

The characterization of the antiproliferative potential of the ketoimine platinum complex trans-[PtCl2{RC(@O)N@CN–(H)C(Me)2–CH2CH2}2] (R = CH2CO2Me) is reported. It showed a higher cytotoxicityagainst HCT116 and HepG2 cancer cells (IC50 values of 22.74 ± 0.04 lM and 22.08 ± 0.08 lM, respec-tively) compared to fibroblasts and a non-tumorigenic cell line. It was also observed a moderate abilityof the complex to induce apoptosis in HCT116 cells, as observed by fluorescence microscopy and flowcytometry. The observed antiproliferative activities of the complex are mostly due to delay in the cellcycle progression. In vitro DNA interaction studies revealed a DNA affinity constant of 6.67 � 105 M�1,suggesting a high affinity to DNA, by comparison to the value obtained for doxorubicin. A decrease inthe electrophoretic mobility of the supercoiled plasmid DNA (pDNA) suggested the formation of com-plex-DNA adducts. However, the complex did not exhibit relevant genotoxicity in V79 cells. Proteomicassays demonstrated that the ketoimine Pt(II) complex promotes an overexpression of two negative cellcycle regulators, PA2G4 and 14-3-3r, and PHB, and a decrease in expression of VDAC1 and HSP90B, prob-ably associated with the antiproliferative potential. The ketoimine Pt(II) complex is able to trigger anoverexpression of cytoskeleton-associated proteins in agreement with its ability to maintain cell struc-ture, and an overexpression of oxidative stress enzymes, coping with the induction of ROS formation,observed by in vitro EMSA assays. In conclusion, the ketoimine Pt(II) complex is an antiproliferative agentwith potential to be used against cancer cells.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

The chemotherapeutic agents applied today in the clinic presenttwo major drawbacks: the development of high toxicity towardshealthy tissues and the emergence of intrinsic and acquired drugresistance. These problems trigger the search for novel compoundswith greater cytotoxicity against tumor cells and reduced sideeffects. N-platinum complexes containing heterocyclic groups arewidely used in medicine due to their biological activities. The threeapproved platinum based complexes, cisplatin, oxaliplatin, andcarboplatin, play a major role in cancer chemotherapy [1–3]. Thefirst complex of the class, cis-[PtCl2(NH3)]2 (cisplatin), exerts its

antitumor activity by interacting with DNA [4,5]. Cisplatin directlybinds to the DNA molecule of tumor cells, forming a cross-link thatleads to the arrest of DNA synthesis and replication and also itstranscription [6,7]. Bi-functional adducts between two adjacentbases in the same DNA strand are the main type of adducts inducedby cisplatin, accounting for more than 90% of all adducts [6,8,9]. Inrapidly dividing cells, i.e. cancer cells, cisplatin can also induceDNA damage, which ultimately leads to irreversible cellular injuryand death by apoptosis [6,7]. Apoptosis can be induced by the acti-vation of various signal transduction pathways that includes cal-cium signaling, death receptor signaling, and the activation ofmitochondrial pathways [10]. However, despite its broader appli-cation there are several drawbacks of cisplatin such as undesirableside effects, mutagenic effects, and the occurrence of drugresistance [1,6,11–14]. These problems triggered the developmentof a high number of cisplatin’s analogues, in order to find new

J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167 157

molecules that are more effective against cisplatin-resistanttumors, minimize cisplatin resistance and that produced a lessertoxicity to non-tumor cells [6]. Carboplatin, oxaliplatin and nedapl-atin are less toxic than cisplatin to the gastrointestinal tract andless nephrotoxic. They are structural congeners of cisplatin andhence their toxicity towards healthy cells still cause serious sideeffects [15,16].

Transition platinum complexes play a crucial role in antitumortherapy. For this reason, a growing interest has been triggeredregarding the investigation of Pt(II) complexes of trans configura-tion, due to their cytotoxic activity (equal or even higher than ofcisplatin) [17–27] and their ability to revert tumor cells’ resistanceto cisplatin [28–31]. The emergence of this type of platinum com-plexes is based in the established substitution of the amine ligandsin the trans platin molecule by bulky ligands. This process leads toimprove the efficiency of the drug molecule, enhancing its anti-tumor potential in vitro and in vivo [30]. Moreover, heterocyclicmoieties as five-membered heterocyclic aromatic compounds asoxadiazolines represent an important class of heterocycles whichhave drawn greater attention due to their promising biologicalactivities, appear as interesting moieties in drug molecules [32]and are thought to improve the interaction between the compoundand the DNA molecule and these ligands can also produce someDNA damage enhancing the toxic effects of the compound [14].The mechanism underlying to the capacity of these complexes inovercoming the resistance problems to the cis configuration plati-num complexes is thought to be by the production of DNA adductsquantitatively and qualitatively different from those induced bycisplatin. These are mainly monofunctional adducts or interstrandcrosslinks [8,30,31].

Previously we have found that [2 + 3] cycloaddition reaction of acyclic nitrone with the trans-[PtCl2(RCN)2] (R = CH2CO2Me) formed,under mild conditions, a bicyclic oxadiazoline complex. The sponta-neous ring opening on the N–O bond of the obtained bicyclicoxadiazoline complex gave a new ketoimine Pt(II) complex via anunprecedent single-pot reaction [34]. In this regard, the majoraim of this study was the characterization of the antiproliferativepotential and the biological targets of the ketoimine Pt(II) complextrans-[PtCl2{RC(@O)N@CN(H)C–(Me)2CH2CH2}2] (R = CH2CO2Me),hoping that this trans heterocyclic platinum complex could reducedside effects in non-tumor cells compared to cisplatin. We show thatthe complex is cytotoxic for two tumor cell lines, HCT116 andHepG2, with higher cytotoxicity towards tumor cells comparingto non-tumor cells. The induction of apoptosis and the interferencein cell cycle progression are the two mechanisms underlying itsantiproliferative potential. The Pt(II) complex interacts in vitro withDNA, but does not show genotoxicity in a cellular environment. Pro-teomics provided a detailed comparison of proteins expressed inthe presence or absence of the Pt(II) complex, allowing to clarifythe mechanisms underlying the antiproliferative activity of thiscompound [33].

2. Materials and methods

2.1. Compounds

The ketoimine Pt(II) complex trans-[PtCl2{RC(@O)N@CN(H)C–(Me)2CH2CH2}2] (R = CH2CO2Me) was synthesized in Centro deQuímica Estrutural of Instituto Superior Técnico, by the processdescribed by Charmier et al. [34]. The fused bicyclic D4-1,2,4-oxadiazoline Pt(II) complex trans-[PtCl2{N@C(CH2CO2Me)ONC(H)CH2CH2CMe2}2] has been synthesized, in excellent yield 90%,by treatment of trans-[PtCl2(NCCH2CO2Me)2] with two equivalentsof the cyclic nitrone (pyrrolin N-oxide)�O+N@CHCH2CH2CMe2,under reflux or using microwave irradiation, the instable obtained

D4-1,2,4-oxadiazoline Pt(II) complex in CH2Cl2 affords, in 89%isolated yield, the ketoimine Pt(II) complex trans-[PtCl2

{N(C(@O)(CH2CO2Me))@CCH2CH2C(Me2)NH}2] containing twomethyl-2-(5,5-dimethylpyrrolidin-2-ylidenecarbamoyl)acetateligands. A stock solution of 23.2 mM was prepared in dimethyl sulf-oxide (DMSO) (Sigma, Spain) and stored at�20 �C. A 0.9% (w/v) NaClstock solution of 1 mg/mL cisplatin (Teva Parenteral Medicines, Inc.,Teva Pharmaceuticals; courtesy of Dr. Joaquim Henriques (Faculty ofVeterinary Medicine, Universidade Lusófona de Humanidades eTecnologias, Portugal) was used and stored at room temperature.A stock solution of 38 mM of doxorubicin (DOX) (TRC – TorontoResearch Chemicals; Canada) was prepared in DMSO and stored at�20 �C.

2.2. Nuclear magnetic resonance spectroscopy (NMR)

NMR spectra of the Pt(II) complex (in CDCl3 and CDCl3/DMSO)were recorded on a Bruker Avance 400 MHz (Ultra Shield TM Mag-net) spectrometer. 1H and 13C chemical shifts (d) are in ppm rela-tive to Si(CH3)4 and J values are in Hertz (Hz).

2.3. Cell lines and culture conditions

HCT116 human colorectal carcinoma and HepG2 human hepa-tocellular carcinoma cell lines were grown as previously described[33,35]. Non-tumorigenic human mammary epithelial cells (MCF-10A) were grown in DMEM/F12 (Gibco, Life Technologies, Spain)supplemented with 0.5 mg/mL hydrocortisone (Sigma), 5% (v/v)horse serum (Invitrogen, Life Technologies, Spain), 20 ng/mL epi-dermal growth factor (Sigma), 100 ng/mL cholera toxin (Sigma)and 10 lg/mL insulin (Sigma). Normal human fibroblasts weregrown in the same conditions as HepG2 cell line and were kindlyprovided by Isabel Carreira (Laboratory of Cytogenetics andGenomics, Faculty of Medicine, University of Coimbra, Portugal).

2.4. In vitro viability assays

Cells were plated at 7500 cells per well in 96-well plates. Mediawas removed 24 h after platting and replaced with fresh mediacontaining the working concentrations of Pt(II) complex or 0.2%(v/v) DMSO (vehicle control). Increasing concentrations of cisplatinor 0.2% (v/v) of a solution of 0.9% (w/v) NaCl were also studied(vehicle control). Pt(II) complex solutions were prepared from1000-times concentrated stock solutions to assure a maximumvolume of DMSO in culture medium of 0.2% (v/v). After 48 h of cellincubation in the presence or absence of each compound, cell via-bility was evaluated using the CellTiter 96� AQueous Non-Radioac-tive Cell Proliferation Assay (Promega, Madison, WI, USA), asdescribed in Silva et al. [33,35].

2.5. Apoptotic potential

2.5.1. Hoechst 33258 labelingHCT116 and HepG2 cells were seeded in 35 mm dishes at

1.5 � 105 cells per plate. Culture medium was removed 24 h afterplatting and replaced with 2 mL of fresh medium containing either0.2% (v/v) DMSO (vehicle control) or 20, 35 and 50 lM of the Pt(II)complex for 48 h. Hoechst staining assay was performed asdescribed in Silva et al. [33,35].

2.5.2. Flow cytometryHCT116 cells were plated into 35 mm dishes at 1.5 � 105 cells

per plate. Culture medium was removed 24 h after platting andreplaced with 2 mL of fresh medium containing either 0.2% (v/v)DMSO (vehicle control) or 35 and 45 lM of the Pt(II) complex. Cellswere incubated in the presence of each compound for 48 h and

158 J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167

then stained with fluorescein isothiocyanate (FITC) labeledannexin V and propidium iodide (PI) as described in Silva et al.[33]. The analysis and quantification of apoptosis was performedon an Attune� Acoustic Focusing Flow Cytometer (Life Technolo-gies, Carlsbad, Califórnia) using an Attune� Cytometric Software(Life Technologies).

2.5.3. Caspase-3/-7 activitiesHCT116 cells were seeded at 7500 cells/well in a black opaque

96-well microplate (Corning, USA). Culture medium was removed24 h after platting and the cells were treated with fresh mediumcontaining increasing concentrations of the complex (25, 35 and45 lM) or 0.2% (v/v) DMSO (vehicle control). A blank control wasprepared just with culture medium without cells. Cells were incu-bated for 48 h in these conditions and caspase-3/-7 combinedactivity was quantified by the Apo-ONE� Homogeneous Caspase-3/7 Assay (Promega) according to manufacturer’s instructions.Briefly, 100 lL of the mixture with the profluorescent substratewas applied to each well and after 2 h at 37 �C, the fluorescencewas measured in an Anthos Zenyth 3100 (Anthos Labtec Instru-ments) plate reader with excitation an emission wavelengths of485 and 535 nm, respectively.

2.6. Cell cycle analysis

HCT116 cells were seeded in 25 cm2 culture flasks at225000 cells/flask and were synchronized in early S-phase by adouble thymidine block, as previously described [33,36]. Briefly,cells were first blocked with medium containing 2 mM of thymidine(Sigma, St Louis, MO, USA) 8 h after plating, and release from theblockage with fresh medium. The cells were release from the secondblockage by substituting the medium with 2 mM thymidine forfresh medium without thymidine and either with 0.2% (v/v) DMSO(vehicle control) or 35 lM of the Pt(II) complex. After incubationperiods of 4 and 8 h at 37 �C and 5% CO2 the media was removedand the cells collected by trypsinization and centrifugation (1000gfor 5 min) and washed with PBS 1�. Cell pellet was resuspendedin 1 mL of cold PBS 1� and 1 mL of cold 80% (v/v) ethanol was addeddrop by drop to each tube while gently vortexing. Cell suspensionwas kept in ice for 30 min and stored at 4 �C at least 14 h. Cells wereharvested by centrifugation, resuspended in 50 lg/mL RNase A inPBS 1� and incubated at 37 �C for 30 min. PI was added to a finalconcentration of 2.5 lg/mL and DNA content was analyzed on aAttune Flow Cytometer (Life Technologies).

2.7. DNA interaction

2.7.1. UV titrationsThe interaction of the Pt(II) complex with Calf Thymus (CT) DNA

(Invitrogen) was studied by UV spectroscopy as described in Silvaet al. [35]. The absorbance spectra were acquired in a ShimadzuUV-2010PC double beam spectrophotometer. The dilution effectresulting from the addiction of DNA was first corrected and theaffinity binding constant (Kb) was determined using the UV titra-tion data at 265 nm, by the application of the Eq. (1):

½DNA�ðea � ef Þ

¼ ½DNA�ðeb � ef Þ

þ 1Kbðeb � ef Þ

ð1Þ

where [DNA] is the concentration of calf-thymus DNA (CT-DNA)(per nucleotide phosphate) and ea, eb and ef corresponds to theapparent, bond and free metal complex extinction coefficients,respectively [37]. The DNA concentration (expressed as molarityof phosphate groups) was determined using the NanoDrop2000spectrophotometer and e260 = 6600 M�1 cm�1 [37]. The ef valuewas obtained by a calibration curve of several solutions of the Pt(II)

complex (11.6–46.4 lM) in 5 mM Tris–HCl, 50 mM NaCl, pH 7, at265 nm, following the Beer’s law. In the other hand, the ea valuewas calculated by Aobs/[complex].

2.7.2. Electrophoretic assays to analyze DNA-complex interactionsThe interaction of the Pt(II) complex with pBluescript II

SK(+)(pBSKII) DNA (Agilent Technologies, California, USA) andpUC18 (Ferments, USA) were performed as described in Silvaet al. [35]. Plasmids were obtained from Escherichia coli trans-formed cells as described in Luís et al. [38] and plasmid extractionwas carried out by the Invisorb� Spin Plasmid Mini Two Kit (Invi-tek, Berlin, Germany). DNA was quantified by spectrophotometryusing the NanoDrop 2000 (Thermo Scientific, Massachusetts,USA). Concentration-dependent studies were performed withpBSKII plasmid DNA (200 ng) incubated in the presence of increas-ing concentrations of the Pt(II) complex (ranging from 60 to350 lM) or in the absence (0% or 1.5% DMSO; vehicle control), in50 mM Tris–HCl, 10 mM NaCl, pH 7.25, to a final reaction volumeof 20 lL. These solutions were incubated for 24 h at 37 �C. Specificbase pair interactions with DNA by the complex was determinedusing 30 lM (per nucleotide phosphate) of pUC18 incubated with3 lM (r = 0.1) and 9 lM (r = 0.3) of the Pt(II) complex (final volumeof 20 lL) or DMSO and digested with the restriction enzymes DraIand SmaI as described in Silva et al. [35]. After the incubation per-iod, 4 lL of loading buffer (25 mM Tris–HCl, 25 mM EDTA (pH 8.0),50% glycerol, 0.1% of bromophenol blue) were added to the sam-ples and then loaded on a 0.8% (w/v) agarose gel (Agarose Sea-Kem�LE, Maine, USA) dissolved in 1� TAE buffer (4.84 g Tris-Base (Merck), EDTA (Riedel-de Haën) 0.5 M, 1.142 mL acetic acid(Panreac), pH 8.0). Electrophoresis was performed for 2 h in 1�TAE buffer at 80 V as constant voltage. Subsequently, DNA wasstaining by immersing the agarose gel in an ethidium bromidesolution (0.5 mg/mL in distilled water) for 20 min and the gelwas washed in distilled water for 10 min. The results were ana-lyzed and photographed using a UVI TEC transilluminator (Cam-bridge, UK) with a Kodak Alpha-DigiDoc camera (Alpha Innotech,California, USA) coupled, and using the AlphEaseFC software(AlphaDigiDoc 1000, Alpha Innotech) for image acquisition.

2.7.3. Chromosomal aberrationsChinese hamster pulmonary fibroblasts (V79) cells were seeded

in 25 cm2 culture flasks at 100000 cells/flask. Cells were incubatedwith 5, 10 and 20 lM Pt(II) complex, 0.1% (v/v) DMSO (vehicle con-trol) and 1.5 lM Mitomycin C (MMC; positive control) for 16 h in37 �C and 5% (v/v) CO2. After 14 h incubation, 10 lL of a colchicinesolution (300 lg/mL; Sigma) pre-warmed at 37 �C were added toeach sample. Cells were washed with 1 mL of a versene solution sup-plemented with sodium bicarbonate, harvested with a ver-sene:trypsine solution 3:1 (v/v) and by centrifugation at 1500 rpmfor 5 min, and each pellet were resuspended in 6 mL of a pre-warmed (37 �C) 75 lM KCl solution for 5 min. Samples were thawedin a water bath at 37 �C for 4 min, centrifuged (1500 rpm for 5 min)and incubated for 15 min at�20 �C with 5 mL of pre-chilled (�20 �C)solution of methanol:acetic acid 3:1 (v/v) (fixing solution). Cellswere centrifuged (1500 rpm for 5 min), washed twice with 5 mL offixing solution and resuspended in 2 mL of this same solution. Eachcell suspension was mounted on to a glass slide and chromosomalaberrations were evaluated under a bright-field microscope by usinga 4% (w/v) Giemsa staining.

2.8. Inhibition of topoisomerase II decatenation activity

The assessment of the inhibition of the decatenation activity ofhuman topoisomerase II (TopoII) was performed by the Kit HumanTopo II Decatenation Assay (Inspiralis, Norwich, United Kingdom)following the manufacturer’s instructions. Briefly, catenated DNA

Fig. 1. Ketoimine Pt(II) complex trans-[PtCl2{RC(@O)N@CN(H)C–(Me)2CH2CH2}2](R = CH2CO2Me) used in this study.

J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167 159

(kDNA) was incubated with 75 lM Pt(II) complex and 1 lM DOX(positive control) and 0.5% (v/v) DMSO (negative control) at 37 �Cfor one hour and a half. A second negative control was preparedwithout adding topoisomerase II to the kDNA. The samples wereprepared by adding each component in the followed order: com-plex, topoisomerase II and kDNA. After the incubation period,4 lL of loading buffer were added to the samples and those wereloaded on a 1% (w/v) agarose gel dissolved in 1� TAE buffer andstained with 2% (v/v) RedGel (Biotium). Electrophoresis was per-formed in 1� TAE buffer for 1 h at 90 V as constant voltage. Theresults were analyzed and photographed using a UVI TEC transillu-minator coupled with a Kodak Alpha-DigiDoc camera, and usingthe AlphEaseFC software for image acquisition.

2.9. Proteomic studies

2.9.1. Protein sample preparationHCT116 cells were seeded into 25 cm2 culture flasks at 6 � 105

cells per flask. Culture medium was removed 24 h after plattingand replaced with fresh medium containing 35 lM of the ketoim-ine Pt(II) complex or its solvent DMSO at 0.2% (v/v) (vehicle con-trol). Cells were also exposed to 25 lM of cisplatin or 0.75% (v/v)of a NaCl solution at 0.9% (w/v) (vehicle control). Cells were incu-bated in the presence of the complex for 24 h and collected forprotein extraction. In order to minimize sample degradation, allsolutions for sample preparation contained 1 � phosphatase inhib-itor (PhosStop, Roche, Basel, Switzerland), 1 � protease inhibitor(complete Mini, Roche�, Basel, Switzerland), 1 mM PMSF and0.1% (w/v) dithiothreitol. Collected cells were concentrated in lysisbuffer (150 mM NaCl; 50 mMTris, pH 8.0; 5 mM EDTA, 2% (w/v)NP-40) by centrifugation at 14000 g for 30 min at 4 �C. Superna-tants were collected and samples stored at �80 �C.

2.9.2. Two-dimensional gel electrophoresis (2-DE)2-DE was performed as described in Conde et al. and Silva et al.

[33,39]. Briefly, prior to 2-DE, protein concentration was deter-mined via the 2-D Quant kit (GE Healthcare, Little Chalfont, UK).Isoelectric focusing (IEF) was performed using Immobilized pHgradient (IPG) strips (7 cm long IPG strips covering pH range from3 – 10; GE Healthcare�) in an EttanIPGphor 3 focusing unit (GEHealthcare�). All 2-DE gel images were digitalized (PIXMA M250;Canon, Uxbridge, UK) and analyzed with the Melanie 7.0 Software(GeneBio, Geneva, Switzerland). Each one of the conditions studiedwas evaluated in triplicate. Spots with significantly altered intensi-ties between conditions (up or down-regulated peptides in com-parison to control samples) were selected and picked from gelstowards identification through MALDI-TOF mass spectrometry.

2.9.3. In-gel digestion and MALDI-TOF mass spectrometry analysisSelected protein spots were manually excised from 2-DE gels

and sent for peptide mass fingerprinting analysis at the Mass Spec-trometry Laboratory in ITQB/iBET (paid service, Oeiras, Portugal).

2.10. Statistical analysis

All data were expressed as mean ± SEM from at least three inde-pendent experiments. Statistical significance was evaluated usingthe Student’s t-test; p < 0.05 was considered statistically significant.

3. Results and discussion

Previously our group reported the synthesis and characteriza-tion of nitrile-derived ketoimine Pt(II) complexes by ring openingof D4-1,2,4-oxadiazolines [34]. The synthesis of this new Pt(II)complex has been performed by EtCN displacement in

trans-[PtCl2(EtCN)2] by the appropriate nitrile, in refluxing CH2Cl2,for a few hours or, for a shorter period (30 min), under focusedmicrowave irradiation [34].

The major aim of this study is the characterization of the anti-proliferative potential and the biological targets of the ketoiminePt(II) complex trans-[PtCl2{RC(@O)N@CN(H)C–(Me)2CH2CH2}2](R = CH2CO2Me) (Fig. 1).

Dimethyl sulfoxide (DMSO) is one of the solvents of choice forpharmaceutical compounds due to its excellent solubility proper-ties. However, DMSO can also be used as a ligand in platinum com-plexes, as described for cisplatin by Fischer and collaborators [40].Cisplatin and DMSO react to form an adducted compound withreduced cytotoxicity and neurotoxicity compared to cisplatin in a0.9% (v/v) NaCl solution [40]. In this regard, we performed the 1HNMR spectroscopy of the ketoimine Pt(II) complex in CDCl3 andCDCl3/DMSO to confirm that no substitution of the complexligands occurred during the biological assays using DMSO. The 1HNMR spectrum obtained in the presence of DMSO as a solvent isvery similar to that obtained in CDCl3 (Supplementary Fig. S1).The 1H NMR (CDCl3/DMSO) spectrum exhibits the chemical shiftat 2.6 ppm attributed to the SCH3 methyl protons characteristicof the free DMSO which is shifted 1 ppm downfield relative tocoordinated DMSO [41,42]. The absence of the characteristic 1Hchemical shift at 3.6 ppm attributed to the SCH3 methyl protonsof the coordinated DMSO confirms that no substitution occurred(Supplementary Fig. S1).

3.1. Cytotoxic potential

The in vitro antiproliferative activity of the ketoimine Pt(II)complex was analyzed in two human tumor cell lines HCT116and HepG2 by the application of the MTS colorimetric assay. Thismethodology relies in the MTS reduction into a brownish formazanproduct by mitochondrial dehydrogenases in metabolically active,viable cells [43]. A decrease of the cell viability in a dose-depen-dent manner was observed for both tumor cell lines after 48 hexposure to the ketoimine Pt(II) complex (Fig. 2).

The relative IC50 (concentration that inhibits the proliferation of50% of the cell population) values of 22.74 ± 0.04 lM and22.08 ± 0.08 lM were determined for HCT116 and HepG2 cells,respectively. These values revealed an identical cytotoxic effectof the complex in both tumor cell lines. Furthermore, the Pt(II)complex does not exhibit significant antiproliferative activityagainst the two non-tumor cell lines, fibroblasts and MCF-10A, atthe concentrations tested (Fig. 2B and C), demonstrating a highercytotoxicity towards the tumor cells (IC50 higher than 100 lM forboth normal cell lines).

In order to compare the cytotoxic potential of this complex tothe current anti-tumor drugs, the antiproliferative activity of cis-platin was also determined in HCT116 and HepG2 tumor cell linesand in the non-tumor cell line MCF-10A (Fig. 2D).

The IC50 values for cisplatin in HCT116 and HepG2 cells were18.18 ± 0.06 lM and 5.29 ± 0.08 lM, respectively, and 87.89 ±

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Fig. 2. Cytotoxicity of the ketoimine Pt(II) complex and cisplatin in tumor and non-tumor cell lines. Cytotoxic effect of the Pt(II) complex in A. tumor cell lines (HCT116 (blackbars) and HepG2 (grey bars)) and in normal cell lines B. MCF-10A and C. fibroblasts. D. Cytotoxicity of cisplatin in HepG2 (black bars), HCT116 (grey bars) and MCF-10A (whitebars). Tumor and non-tumor cells were treated with increasing concentrations of the Pt(II) complex or cisplatin for 48 h and cell viability was determined by MTS assay. Thedata were normalized against the control (0.2% (v/v) DMSO or 0.2% (v/v) of a solution of 0.9% (w/v) NaCl, respectively for the Pt(II) complex or cisplatin). The results showedare expressed as mean ± SEM of three independent assays.

160 J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167

6.01 lM for MCF-10A. Although, cisplatin demonstrated to be,respectively, 1.25 and 4.2 times more effective against HCT116and HepG2 cells compared to the ketoimine Pt(II) complex,100 lM of this complex induces a lower reduction of cell viabilityin MCF-10A epithelial cells compared with cisplatin (Fig. 2). There-fore, the complex seems to demonstrate a cytotoxic effect towardstumor cells and to induce reduced side effects in non-tumor cellscompared to cisplatin.

3.2. Apoptotic potential

3.2.1. Hoechst 33258 stainingThe reduction of cell viability promoted by the ketoimine Pt(II)

complex (Fig. 2) prompted us to evaluated the underlined mecha-nisms of cell death. In fact, platinum complexes promote their cel-lular toxicity by inducing apoptosis [44,45]. A preliminary analysiswas performed by staining with Hoechst 33258 dye, due to its highaffinity for DNA allowing the detection of nuclear alterations likechromatin condensation and nuclear fragmentation, typicalfeatures of apoptotic cells [46–48]. Hoechst 33258 staining ofHCT116 and HepG2 cells after 48 h of exposure to 20, 35 and

50 lM of ketoimine Pt(II) complex allowed us to observe a reduc-tion in the number of stained cells (in a dose dependent manner)compared to control cells (DMSO treated cells) and also the nuclearcondensation and fragmentation characteristics of apoptosis(Fig. 3).

In DMSO treated cells, the nuclei have blue staining and thelevel of fluorescence is uniformly distributed [46] opposite to whatis observed in Pt(II) complex exposed cells (Fig. 3A and B: b, c and dcompared to a). These preliminary results show that the Pt(II) com-plex is able to trigger apoptosis in both tumor cells lines, as it waspreviously observed for cisplatin [49,50].

3.2.2. Annexin V/PI double-staining assayThe double-staining with annexin V-FITC and PI is capable of

identifying apoptotic cells with high specificity and sensitivity[51]. Annexin V proteins bind with high affinity to phosphatidyl-serine (PS) that is translocated to the outer membrane leaflet inan early stage of the apoptotic process (early apoptotic cells)[47,51,52]. Once the integrity of the cell membrane is lost, PI canenter the cells and stain the nucleus [53]. Therefore, viable cellsare FITC�/PI�, early apoptotic cells are FITC+/PI�, late apoptotic

Fig. 3. Apoptotic morphological changes in HCT116 (A) and HepG2 (B) cells exposed to the ketoimime Pt(II) complex. Cells were treated with 0.2% (v/v) DMSO (vehiclecontrol) (a) and with 20 lM (b), 35 lM (c) and 50 lM (d) of the complex for 48 h and stained with Hoechst 33258. Typical morphologic features of apoptosis like chromatincondensation (arrows) and nuclear fragmentation (circles) were identified.

J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167 161

cells are FITC+/PI+ and necrotic cells are FITC�/PI+ [54,55]. The incu-bation of HCT116 cells in the presence of the ketoimine Pt(II) com-plex reveals an increase in the number of apoptotic cells (late andearly stages) in a dose dependent manner, with 32.5% and 37.8% for35 and 45 lM, respectively, compared to the control that exhibits20.1% of apoptotic cells (Fig. 4A).

As observed in Fig. 4A apoptotic cells are predominantly in thelate stage of apoptosis and the number of necrotic cells is not sig-nificant. Tseng et al. observed that the exposure of HCT116 cells to33 lM of cisplatin for 24 h was able of induce 25.8% of apoptoticcells [56]. In this regard, compared to cisplatin, this complex ismore effective in the induction of apoptosis.

3.2.3. Caspase-3/7 activityQuantification of the effectors caspase-3/7 activity was deter-

mined in order to further confirm the role of apoptosis as a poten-tial mechanism of cytotoxicity of the Pt(II) complex. It wasobserved a maximum of 1.25-fold change in the activity of cas-pase-3/7 at 35 lM of the Pt(II) complex relatively to the control(0.2% (v/v) DMSO) (Fig. 4B).

Results from Zhang and collaborators show that cisplatin is ableto induce a variation of approximately 1.5 times higher than thecontrol [57]. As observed in Fig. 4B the ketoimine Pt(II) complexis able to induce approximately the same levels of caspase-3/7activity as cisplatin which corroborate with the results observedin the Annexin V/PI double-staining assay and the ability of thecomplex to induce some level of apoptosis.

3.3. Cytostatic potential

3.3.1. Cell cycle progressionIn order to characterize the cytostatic potential of the ketoimine

Pt(II) complex in HCT116 cells, flow cytometric analysis of thosecells stained with PI was accessed (Fig. 4C).

After 4 h of incubation in the presence of 35 lM of ketoiminePt(II) complex an increased number of cells in the S phase (approx-imately 70.6%) was observed (Fig. 4C A). Nevertheless, cells treatedwith DMSO (vehicle control) showed a high% of cells in G2/Mphase (Fig. 4C A) After 8 h of incubation in the presence of 35 lMof ketoimine Pt(II) complex a progress in the cell cycle wasobserved being the majority of cells in G2/M phase (approximately63.6%) while cells treated with DMSO (vehicle control) continue toprogress and the majority of cells at 8 h was distributed betweenG2/M and G0/G1 phases. These results indicate that the complex

is able to delay cell cycle progression (Fig. 4C). Cisplatin was alsocapable of interfering in the cell cycle progression by promotingan arrest in G2/M phase [56].

These results prompt us to analyze the in vitro and ex vivo inter-action of the complex with the DNA molecule.

3.4. Interaction with DNA

3.4.1. UV titrationsAbsorption spectroscopy is a useful approach for the study of a

possible interaction between a complex and the DNA molecule[58,59]. UV titrations regarding the incubation of the ketoiminePt(II) complex (23.2 lM) in the presence or absence (dark line) ofincreased concentrations of CT-DNA (0–22.7 lM) allowed us toobserved a hypochromic effect and an absence of a bathochromicshift (Fig. 5).

The co-occurrence of these two spectral alterations, hypochro-mism and bathochromism, is interpreted as the existence of anintercalation mechanism of binding for the complex [58,60]. Inthe other hand, a hypochromic effect can be indicative of a groovebinding interaction [59]. A comparison with the spectral variationof cisplatin was not possible because its ligands (two amines andtwo chlorines) have no ability to absorb in the UV region [61]. Inthe other hand, DOX, an intercalator and minor groove bindingagent [59,62], was evaluated by Luís et al. at the same conditionstested for the Pt(II) complex and it was also showed a hypochromiceffect and absence of a red shift [38]. Thus, it can be inferred thatthe ketoimine Pt(II) complex may interact with DNA by a grovebinding mechanism. The affinity binding constant (Kb) for the Pt(II)complex was determined as 6.67 (±0.42) � 105 M�1 (Fig. 5). Thevalues of Kb described in the literature for classical intercalators(ethidium-DNA 7 � 107 M�1 [63]) are at least 100 order of magni-tude higher than that calculated for the trans-ketoimine Pt(II) com-plex. Nevertheless, the ketoimine Pt(II) complex demonstrate tohave 1.6–2x more affinity to DNA compared to proflavin (Kb of4.1 � 105 M�1 [64]) or DOX (Kb equals to 3.48 (±0.03) � 105 M�1

[38]). These results confirm the in vitro interaction of the ketoiminePt(II) complex with CT-DNA, being the Kb value obtained for thiscomplex higher of that determined in similar conditions for otherPt(II) complex (2.0 (±0.2) � 104 M�1 [58], Fig. 5).

3.4.2. Electrophoretic assays to analyze DNA-complex interactionsIn order to additionally confirm the observed interaction

between the ketoimine Pt(II) complex with DNA molecule

Fig. 4. Evaluation of the apoptotic potential and cell cycle progression of the ketoimine Pt(II) complex in HCT116 cells. (A) Apoptosis was evaluated and quantified by flowcytometry analysis with annexin V-FITC and PI double staining. Cells were treated with 0.2% (v/v) DMSO (vehicle control) and the complex at 35 and 45 lM for 48 h. Viablecells (FITC�/PI�) (grey bars); early apoptotic cells (FITC+/PI�) (green bars); late apoptotic cells (FITC+/PI+) (yellow bars); necrotic cells (FITC-/PI+) (red bars). (B) Changes incaspase-3/7 activity after incubation the cells with 0.2% (v/v) DMSO (vehicle control) and the complex at 25, 35 and 45 lM for 48 h. (C) Cell cycle distribution was evaluatedafter 4 h (A) and 8 h (B) in the presence of 0.2% (v/v) DMSO (NA, vehicle control) and 35 lM of the complex (JL) by flow cytometry analysis after PI staining. The resultsshowed are expressed as mean ± SEM of three independent assays. (For interpretation of the references to colour in this figure legend, the reader is referred to the web versionof this article.)

Fig. 5. UV absorption spectra of the ketoimine Pt(II) complex at a fixed concen-tration (23.2 lM) in the presence or absence (dark line) of increased concentrationsof CT-DNA (maximum concentration indicated by the red line), in 5 mM Tris–HCl,50 mM NaCl (pH 7). The arrow indicates the variation in absorbance with increasedconcentrations of CT-DNA (0–22.7 lM). Inset: Plot of a linear fitting of½DNA�ðea�ef Þ

¼ ½DNA�ðeb�ef Þ

þ 1Kb ðeb�ef Þ

to determine the value of the DNA affinity binding constant(Kb) of the complex. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

162 J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167

(Fig. 5), electrophoretic mobility assays were performed by incu-bating pBSKII plasmid DNA with increasing concentrations of theketoimine Pt(II) complex (Fig. 6). This assay allows the observationof conformational changes in the supercoiled form (Form I) of plas-mid DNA, and then evaluate if there is a relationship between theplasmid-DNA interaction and the cytotoxicity of the complex

[65,66]. It can be observed a reduction in the electrophoreticmobility of the band corresponding to the Form I of plasmid DNAat increasing concentrations of the complex, which may be associ-ated with an unwinding capacity of the complex (Fig. 6). Also wecan observe an increase of Form III (linear) in a dose dependentmanner (Fig. 6).

Intercalating agents can cause unwinding of the double helix asa result of its placement between the bases of DNA, causing a delayin its migration on agarose gel [67,68]. In contrast, complexes thatestablish an interaction to DNA by groove binding, usually in theminor groove, cannot trigger pronounced conformational changesin DNA as do the intercalating agents, once groove binding com-plexes adjust its structure to the minor groove and follow thetwisting of the DNA molecule along the central axis [69,70]. Thusit appears that the ketoimine Pt(II) complex may interact withDNA through an intercalative biding mode (Fig. 6). In the otherhand, cisplatin induces a decrease in the electrophoretic mobilityof the band corresponding to the Form I of the pBR322 plasmidDNA and an increase in the migration of the band correspondingto the relaxed circular form (Form II) at increasing concentrationsof cisplatin [66]. Pt-DNA adducts are associated to a DNA destabi-lization and then a DNA distortion, such as the one produced bycisplatin, the 1,2-d(GpG) DNA intra-strand cross-link, that in theend leads to a local unwinding of the DNA double helix [71,72].Thus, compared to cisplatin, the complex possibly induces DNAadducts, and those can be monofunctional adducts or interstrandcross-links [8,9]. Accounting for this possible interaction, an enzy-matic restriction assay was performed in order to characterize the

λ/HindIII pBSKII 60 80 100 150 200 250 300 350 L DMSO λ/HindIII

Ketoimine Pt(II) complex (µM)

Form IIForm III

Form I

Form I – Supercoiled form; Form II – relaxed circular form; Form III – linear form

λ/HindIII pBSKII 60 80 100 150 200 250 300 350 L DMSO λ/HindIII

Ketoimine Pt(II) complex (µM)

Form IIForm III

Form I

Form I – Supercoiled form; Form II – relaxed circular form; Form III – linear form

Fig. 6. Electrophoretic mobility pattern of the interaction between pBSKII plasmid DNA and the ketoimine Pt(II) complex. pBSKII plasmid DNA (200 ng) was incubated in thepresence of increasing concentrations of the Pt(II) complex (ranging from 60 to 350 lM) or in the absence (0 or 1.5% DMSO; vehicle control), in 50 mM Tris–HCl, 10 mM NaCl,pH 7.2, for 24 h at 37 �C. Electrophoresis was performed in a 0.8% (w/v) agarose gel. k/HindIII – Molecular ladder; pBSKII – untreated pBSKII plasmid DNA; L – linearizedpBSKII plasmid DNA; DMSO – 1.5% DMSO.

J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167 163

specific binding to purines (adenine and guanine) or pyrimidines(thymine and cytosine) by the complex. This assay is based onthe inhibition of SmaI and DraI restriction if the complex selectivitybinds to the bases in the recognition sites of these enzymes, -CCCGGG- and -TTTAAA- for SmaI and DraI, respectively. As shownin the Fig. 7, there was no inhibition of the two restriction enzymessince pUC18 was totally linearized in the presence of the complexat the concentrations tested, with one fragment at the size of2686 bp and two fragments of 1975 and 692 bp for SmaI and DraIrestriction, respectively. The third fragment of 19 bp resulting fromdigestion with DraI was not seen in the agarose gel.

Cisplatin shows the ability of inhibit BamHI restriction activity(recognition site -GGATCC-) which is associated with high affinityfor GG regions by cisplatin [66]. Thus, the possible formation ofDNA adducts by the Pt(II) complex differ from those induced bycisplatin, showing no greater affinity to any base of the DNAmolecule.

3.4.3. Chromosomal aberrationsThe induction of genotoxicity by anti-tumor compounds with the

production of chromosomal aberrations is presents as a mechanismof anti-tumor activity of these agents [73]. Thus, it was evaluated theability of the complex to induce chromosomal aberrations in V79cells, assessing the interaction between the complex and the DNAmolecule in the complexity of a cell system. It was observed thatthe complex induces chromosomal aberrations in the three concen-trations tested, however de level of chromosomal aberrations at the

Fig. 7. Electrophoretic pattern of SmaI and DraI restriction of the pUC18 plasmid DNA in9 lM (r = 0.3) of the ketoimine Pt(II) complex and DMSO (1.5%), for 1 h at 37 �C. ElectropUC18 – untreated pUC18 plasmid DNA.

concentrations studied were not significant (Fig. 8). Indeed, incuba-tion of V79 cells with 5 lM of the Pt(II) complex induce 4% of CTG,while at 10 lM of the complex there are 1% of CSB, 1% of MA, 1% ofCTG, 1% of CSG and 2% of ENDO, and at 20 lM we were able to visu-alized 1% of ENDO and 2% of CTB (Fig. 8). Mitomycin C (MMC), anantibiotic with anti-tumor potential that promotes chromosomalaberrations by the formation of crosslinks in both strands of the dou-ble helix of DNA [74], induced approximately 50% of chromosomalaberrations in V79 cells (Fig. 8): CTG (7%), CTB (22%), CSB (7%), TRI(9%), TETRA (2%) and MA (3%).

Comparing to MMC, the ketoimine Pt(II) complex is not highlygenotoxic (Fig. 8). DMSO induced 2% of CTG and it is also consid-ered not genotoxic (Fig. 8). Additionally, it was evaluated the pos-sible interference of the ketoimine Pt(II) complex in cellproliferation by the determination of the Mitotic Index (MI) (thenumber of mitotic cells comparatively to the total number of cells)(Fig. 9). The MI value of the complex decreased in a concentrationdependent manner (Fig. 9) and these results indicate that the keto-imine Pt(II) complex is able to delay cell cycle progression, inagreement with those results obtained above for cell cycle assay(Fig. 4C).

In the presence of MMC the MI value was much more reducedthen that of the complex, indicating once again the greater extentof genotoxicity induced by this agent, and that the ketoimine Pt(II)complex presents a low genotoxic potential, that might beexplained by the preferential binding of the Pt(II) atom to sulfurcontaining ligands, like glutathione in the cytoplasm, preventing

the presence of the complex. pUC18 at 30 lM was incubated with 3 lM (r = 0.1) andphoresis was performed in a 0.8% (w/v) agarose gel. k/HindIII – Molecular ladder;

Fig. 8. Types of chromosomal aberrations in metaphase cells of a chinese hamsterpulmonary fibroblasts cell line (V79) in the presence of the ketoimine Pt(II) complex.Cells were treated with 0.1% (v/v) DMSO (vehicle control), 1.5 lM of MMC (positivecontrol) and the ketoimine Pt(II) complex at 5, 10 and 20 lM for 16 h. CTG –chromatid gap; CSG – chromosome gap; CTB – chromatid break; CSB – chromosomebreak; TRI – triradial chromosome; TETRA – tetraradial chromosome; ENDO –endoreduplication; MA – cells with multiple chromosomal aberrations. The resultsshowed are expressed as mean ± SEM of two independent assays.

0123456789

0.1 % DMSO

1.5 µM MMC

5 10 20

Mito

tic In

dex

(MI)

Ketoimine Pt(II) complex (µM)

Fig. 9. Mitotic index (MI) variation in V79 cells after incubated in the presence ofthe ketoimine Pt(II) complex. Cell were incubated with 5, 10 and 20 lM of thecomplex, 0.1% (v/v) DMSO (vehicle control) and 1.5 lM of MMC (positive control)for 16 h.

164 J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167

the ketoimine Pt(II) complex of reaching the DNA in the nucleus[31,75,76]. In fact, cisplatin has a major capacity to interact withproteins (75–85%) after enter the cell, compared to DNA (5–10%)[77]. Then, it can be inferred that proteins could be a preferentialcellular target to the complex, more than the DNA molecule.

3.5. Inhibition of topoisomerase II decatenation activity

The topoisomerase II is involved in processes such as DNA rep-lication, transcription and recombination, condensation/deconden-sation and segregation of chromosomes [78]. This enzyme allowsthe relaxation of DNA by inducing a transient break in the DNAdouble-stranded molecule, enabling the passage of another DNAdouble-stranded molecule (not broken), and in the end the reseal-ing of the break, a process called decatenation [78–80]. Cisplatinand DOX are capable of inhibit the activity of decatenation of topo-isomerase II [78,80]. The ability of the complex to inhibit the decat-enation activity of topoisomerase II was evaluated in kDNA(Supplementary Fig. S2). At 75 lM the ketoimine Pt(II) complexappears to have no ability to inhibit the activity of topoisomeraseII since the incubation of kDNA with the complex allowed to gen-erate two bands corresponding to two molecules of double-stranded circular DNA that normally results from the decatenationactivity of topoisomerase II, the circular and relaxed forms of kDNA(Supplementary Fig. S2). In contrary, DOX exhibit just one bandcorresponding to the kDNA (Supplementary Fig. S2). Additionally,DMSO was not able to inhibit this activity. Furthermore, once topo-isomerase II is important to chromosome segregation, inhibition of

its activity may lead to endoreduplication [80]. Cisplatin inhibitstopoisomerase II activity promoting cases of endoreduplication[80,81]. As observed in Supplementary Fig. S2 the complex in studydoes not have the ability to inhibit the decatenation activity oftopoisomerase II, and in this regard, the 2% of endoreduplicationobserved before during chromosome aberrations analysis (Fig. 8)may not be correlated with the inhibition of this activity.

Since DNA may not be in vivo the main target of the ketoiminePt(II) complex the analysis of the effect of the complex in proteinexpression was considered.

3.6. Proteomic studies

HCT116 cells exposed to the ketoimine Pt(II) complex showedan overexpression of proliferation-associated protein 2G4(PA2G4) and stratifin (14-3-3r) (Fig. 10B; Table 1).

PA2G4 induces cell cycle arrest possibly by repressing E2A acti-vation of cell cycle related genes [82]. 14-3-3r is also involved in anegative regulation of the cell cycle progression by the inhibitionof cyclin-dependent kinases (CDKs) and can also be involved in apositive regulation of the tumor suppressor protein p53 ([83–85]). Thus, the overexpression of both proteins is in agreementwith the flow cytometry assays with PI staining and that the keto-imine Pt(II) complex may be involved in inducing a cell cycle delay(Fig. 4C). Contrary, in the presence of cisplatin both proteins wererepressed (Fig. 10D; Table 1), which can be related to the fact thatcisplatin induces its anti-tumor activity mainly by inducing celldeath by apoptosis instead of cell cycle arrest [1]. The ketoiminePt(II) complex induces a repression of the voltage-dependentanion-selective channel protein 1 (VDAC1) (Fig. 10B; Table 1), apositive regulator of apoptosis by the formation of a mitochondrialchannel that releases pro-apoptotic proteins, such as cytochrome c,from the mitochondrial membrane space [86]. However, as it wasobserved early in flow cytometry assays with a double stainingwith annexin V-FITC and PI (Fig. 4A), the complex shows the capac-ity to induce apoptosis in a moderate level. Thus it can possiblyindicates that apoptosis is trigger by an extrinsic mechanism ratherthan an intracellular pathway. In the presence of cisplatin there isan overexpression (Fig. 10D; Table 1) in agreement of the involve-ment of VDAC1 in apoptosis induction by cisplatin [87]. Prohibitin(PHB) is overexpressed in the presence of both complexes, theketomine Pt(II) complex and cisplatin (Fig. 10; Table 1). This pro-tein appears with different expression levels and also differentmechanisms of action depending on cell type, acting sometimesas a tumor suppressor, as a molecular controller of cell prolifera-tion and apoptosis [88,89]. For example, PHB is known to suppressthe colonic tumor formation in mice with colitis-associated cancer,interacting with p53 to promote apoptosis [90]. This possibly indi-cates that the ketoimine Pt(II) complex has anti-tumor potentialand confirm the well establish chemotherapeutic activity ofcisplatin.

The heat shock protein HSP 90-beta (HSP90B) is a chaperoneand promotes the correct folding of proteins [91]. In the presenceof the ketoimine Pt(II) complex (Fig. 10B and A; Table 1) there isa decrease in the level of expression of HSP90B protein comparedto control cells. This possibly indicates that the Pt(II) complexmay interfere with this mechanism to prevent the folding of uncor-rected synthesized proteins. In the other hand, this protein is over-expressed in tumor cancer cells [91], and then this decrease ofexpression also demonstrates the antiproliferative potential ofthe complex. Cells exposed to cisplatin also demonstrate adecreased expression of HSP90B (Fig. 10D; Table 1).

Cytoskeleton-associated proteins such as t-complex protein 1subunit zeta (TCPZ), tubulin alpha-1B chain (TBA1B), tropomyosinalpha-3 chain (TPM3), actin cytoplasmic 2 (ACTG), ezrin (EZRI) andfructose-biphosphate aldolase A (ALDOA) are overexpressed in the

Fig. 10. Two-dimensional gel electrophoresis for total protein extracts from HCT116 cells exposed for 24 h to: (A) 0.2% (v/v) DMSO (vehicle control); (B) 35 lM ketoiminePt(II) complex; (C) 0.75% (v/v) of NaCl at 0.9% (w/v) (vehicle control for cisplatin); (D) 25 lM cisplatin.

Table 1Proteins whose expression was significantly changed relatively to the control (more than 1.5-fold or less than 0.7-fold) in HCT116 cells exposed to 35 lM of ketoimine Pt(II)complex and 25 lM of cisplatin for 24 h.

Spot ID Protein identificationa Protein Reference (HUMAN) Ketoimine Pt(II) complex Cisplatin

16 stratifin/14-3-3 protein r 14-3-3r 2.41 �0.3730 voltage-dependent anion-selective channel protein 1 VDAC1 -0.58 1.4820 prohibitin PHB 2.35 1.545 proliferation-associated protein 2G4 PA2G4 3.24 �0.357 heat shock protein HSP 90-beta HSP90B -0.57 �0.393 T-complex protein 1 subunit zeta TCPZ 1.64 �0.334 tubulin alpha-1B chain TBA1B 4.39 �0.415 tropomyosin alpha-3 chain TPM3 1.69 1.0411 actin, cytoplasmic 2 ACTG 2.62 0.772 ezrin EZRI 6.37 �0.2129 fructose-biphosphate aldolase A ALDOA 2.75 2.0825 peroxiredoxin-6 PRDX6 2.99 �0.4422 peroxiredoxin-2 PRDX2 1.58 0.9426 thioredoxin-dependent peroxide reductase mitochondrial PRDX3 1.65 1.1127 heat shock cognate 71 kDa protein PARK7 1.51 1.0323 superoxide dismutase [Cu-Zn] SODC 1.70 0.49

a http://www.uniprot.org/.

J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167 165

presence of the ketoimine Pt(II) complex (Fig. 10B; Table 1). Theseresults may indicate that the complex has a positive effect on themaintenance of the cellular structure. On the contrary for cisplatinthe expression of these proteins are mostly decreased comparedwith control cells (Fig. 10C and D; Table 1). However, major struc-tural changes in cell structure occur in the apoptotic process, andin a lesser extension during cell cycle progression, being the firstthe preferential process adopted by cisplatin [1,92].

In the presence of the ketoimine Pt(II) complex an overexpres-sion of oxidative stress enzymes, such as peroxiredoxin-6 (PRDX6),peroxiredoxin-2 (PRDX2), thioredoxin-dependent peroxide reduc-tase mitochondrial (PRDX3), heat shock cognate 71 kDa protein(PARK7) and superoxide dismutase [Cu–Zn] (SODC), could beobserved (Fig. 10B; Table 1). These results possibly indicate thatthe complex can induce reactive oxygen species (ROS) formationin HCT116 cells. Indeed in Fig. 6 we were able to see an increase

in the linear form of pDNA with the increase of the complex. Thisincrease in the linear form may be related with this high level ofROS as observed by our group with other coordination compounds[38]. Although cisplatin promotes the induction of ROS, for exam-ple as a mechanism to promote the apoptotic process [93], theseproteins were not overexpressed in the presence of cisplatin(Fig. 10D; Table 1).

4. Conclusions

The discovery of new compounds with antiproliferative proper-ties is of most importance to promote a more efficient therapywithout promoting toxicity and resistance problems that are thetwo major drawbacks for the currently chemotherapeutics agentsclinically applied. The knowledge of the mechanisms and biologicaltargets of those chemotherapeutics agents can lead to the design of

166 J. Silva et al. / Inorganica Chimica Acta 423 (2014) 156–167

new compounds with more promising anti-tumor activities thatcan overcome these issues. The ketoimine Pt(II) complex of thisstudy shows a cytotoxic potential against two tumor cell lines ofcolorectal and hepatocellular carcinomas, respectively, HCT116and HepG2 cells, and a higher antiproliferative effect regardingtumor cells compared to normal fibroblasts and mammary epithe-lial cells (MCF-10A). The mechanisms underlying its antiprolifera-tive activity are the induction of a cell cycle delay and apoptosis asobserved by flow cytometry. Although the complex shows the abil-ity to interact in vitro with DNA and might have the ability to formcomplex-DNA adducts as seen by a decrease of the electrophoreticmobility of the supercoiled plasmid DNA in a concentration-depen-dent manner, the complex does not demonstrate a significantcapacity to promote genotoxicity in V79 cells.

To investigate if the complex can interact with proteins, proteo-mic assays were performed and was observed that the complexpromotes an overexpression of PA2G4 and 14-3-3r, two cell cycleinhibitors, and induces a decrease of VDAC1 expression, a proteinthat can trigger apoptosis. The ketoimine Pt(II) complex inducesalso an overexpression of PHB, that in conjugation with a decreaseof HSP90B expression may correlate with its observed antiprolifer-ative properties. The complex showed also the ability to induce anoverexpression of cytoskeleton-associated proteins and oxidativestress enzymes that demonstrate respectively the ability to main-tain the cell structure and to induce the formation of ROS (alsoobserved through EMSA assays). These results demonstrate thepotential of this trans-platinum complex as an interesting antipro-liferative compound for further investigation. The Pt(II) complexin vitro affinity for DNA could be further used to study the possibil-ity of its encapsulation into nanovectorization systems (e.g. goldnanoparticles functionalized with oligonucleotides and targetingmoieties [94,95]) for in vivo targeting cancer cells even morespecifically.

Acknowledgements

We thank the project Silence is golden (siAu) - silencing the silenc-ers via multifunctional gold nanoconjugates towards cancer therapy(PTDC/BBB-NAN/1812/2012) for financial support. The work wasalso partially supported by project PEst-OE/QUI/UI0100/2013(Fundação para a Ciência e a Tecnologia). We also thank Dr. Joa-quim Henriques (Faculty of Veterinary Medicine, UniversidadeLusófona de Humanidades e Tecnologias (ULHT), Portugal) for thekind gift of cisplatin, Dr. Isabel Carreira (Laboratory of Cytogeneticsand Genomics, Faculty of Medicine, University of Coimbra, Portu-gal) for the kind gift of normal fibroblasts cells; Dr. Guadalupe Cab-ral (CEDOC, FCM/UNL, Portugal) for helping with flow cytometryassays and Dr. Susana Santos (ULHT, Portugal) for discussions.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ica.2014.07.067.

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