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Synthesis,characterization,cytotoxic activity and DNA binding properties of the novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide
Rabia Eshkourfu a ,Bo židar Čobelji ća ,Miroslava Vuj či ćb ,Iztok Turel c ,Andrej Pevec c ,Kristina Sep či ćd ,Manja Zec e ,Sini ša Radulovi će ,Tatjana Srdi ć-Radi će ,Dragana Miti ća ,Katarina Andjelkovi ća ,Du šan Sladi ća ,⁎
a
Faculty of Chemistry,University of Belgrade,Studentski trg 12-16,11000Belgrade,Serbia
b
Institute of Chemistry,Technology and Metallurgy,University of Belgrade,Njego ševa 12,P.O.Box 815,11000Belgrade,Serbia c
Faculty of Chemistry and Chemical Technology,University of Ljubljana,A šker čeva 5,1000Ljubljana,Slovenia d
Biotechnical Faculty,University of Ljubljana,Department of Biology,Ve čna pot 111,1000Ljubljana,Slovenia e
Institute for Oncology and Radiology of Serbia,Department of Experimental Oncology,Laboratory for Experimental Pharmacology,Pasterova 14,11000Belgrade,Serbia
a b s t r a c t
a r t i c l e i n f o Article history:
Received 23February 2011
Received in revised form 25May 2011Accepted 26May 2011
Available online 13June 2011Keywords:
Dinuclear cobalt(III)complex Crystal structure Apoptosis DNA binding
A novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide,N ′,N ′2-bis[(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide was synthesized and characterized by elemental analysis,spectroscopy (NMR and infrared),and X-ray crystal analysis.The complex showed a moderate activity towards Artemia salina .The highest cytotoxic potential of the complex was observed on the epithelial breast cancer (MDA-361)cell line.The investigated complex induced apoptosis,the early apoptotic cells comprising 28.18%,compared to 5.64%of control cells in the same phase.The interaction of the complex with calf thymus DNA (CT-DNA)was monitored by blue shift and hyperchromism in the UV –vis spectra.The observed intrinsic binding constant (K b =4.2×105M −1)together with structural analysis of the complex indicate the groove binding.
©2011Elsevier Inc.All rights reserved.
1.Introduction
Although metal complex shows an immense versatility of structures,only few of them have entered the clinical praxis as antitumor agents.Most of these agents target DNA molecules.The most important ones are platinum agents (cisplatin,carboplatin,oxaliplatin)[1,2].Unfortu-nately,because of intrinsic and acquired resistance a limited spectrum of cancers can be treated by platinum drugs.In order to overcome this problem,polynuclear metal complexes in which the metal centers are linked by flexible alkyl chains [3,4]or positioned within a more rigid scaffold [5,6]have been synthesized.These agents can bind to DNA with larger af finity,impart unprecedented binding modes and recognize speci fic DNA sequences.
The complexes of polydentate dihydrazone ligands with adjacent coordination pockets,predominantly condensation derivatives of 2-acetylpyridine and malonic acid dihydrazide,are in the focus of interest of our research group [7–9].Dihydrazone type ligands have a large number of potential donor atoms and hence display versatile
behavior in metal coordination.The mode of coordination depends on the nature of the central metal atom,conformational flexibility of the ligand as well as on the presence of other species capable to compete for coordination pockets.Malonic dihydrazide based ligands,like N ′,N ′2-bis [(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide (H 2L,Scheme 1),are capable to bind to more than one metal center.In mononuclear octahedral Fe(III)complex,H 2L is coordinated through nitrogen atoms from both coordination pockets [7],while it binds two metal centers in bis-tridentate fashion in Cd(II)complex [8].Each Cd (II)atom has a pentagonal bipyramidal e of Zn(BF 4)2·6H 2O afforded formation of a dinuclear Zn(II)complex,with one molecule of the ligand bridging two Zn atoms,the remaining coordination sites being occupied by solvent molecules [9].In the reaction with Ni(ClO 4)2·6H 2O two binuclear complexes were obtained [3].Each complex cation is built of two ligands,each acting as a bis tridentate (NNO)system,bridging two metal centers.Such coordination results in octahedral environment around each Ni(II)center.Relative rigidity of the ligand might afford complxes with appropriate supramolecular architecture for ef ficient DNA binding.Malonic acid based dihydrazones and their complexes showed versatile biological activities [8,10].In comparison to other acyl hydrazones of 2-acetylpyridine [10],H 2L ligand shows better cytotoxic activity on HeLa (human cervix carcinoma cells)and B16(murine melanoma)tumor cell lines.The heptacoordinated binuclear Cd(II)
Journal of Inorganic Biochemistry 105(2011)1196–1203
⁎Corresponding author at:Faculty of Chemistry,University of Belgrade,Studentski trg 12-16,P.O.Box 158,11000Belgrade,Serbia.Tel.:+381113336679;fax:+381112636061.
E-mail address:dsladic@chem.bg.ac.rs (D.Sladi ć
).0162-0134/$–see front matter ©2011Elsevier Inc.All rights reserved.doi:
10.1016/j.jinorgbio.2011.05.024
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
j o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /j i n or g b i o
complex with H 2L showed very good cytotoxic activity already in the low micromolar range [8].The activity was,at least in part,a consequence of strong binding to DNA [11].These promising results prompted us to evaluate if other analogous metal complexes exhibit similar behavior.In this paper we report synthesis,characterization,cytotoxic activity and DNA binding properties of a novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide.Cobalt(III)was selected for its high positive charge and redox properties.2.Experimental
2.1.Materials and methods
2-Acetylpyridine (99%),and malonic acid dihydrazide (97%)were obtained from Aldrich.Starting salt Co(NO 3)2·6H 2O (purum p.a.≥99%)was obtained from Merck.All solvents (ethanol,DMSO)and chemicals used for DNA studies were reagent grade and used without further puri fication.All buffer solutions for investigations of in-teractions of compounds with DNA were prepared in deionized sterile water and filtered through 0.2μm filters (Nalgene,USA).Calf thymus DNA (CT-DNA)(lyophilized,highly polymerized),ethidium bromide and plasmid pUC18were purchased from Serva,Heidelberg.Stock solutions (10mM and 50mM)of salt Co(NO 3)2·6H 2O (S),ligand H 2L and complex [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OH were prepared in water and DMSO.Elemental analyses (C,H,and N)were performed by standard micro-methods using the ELEMENTARVario ELIII C.H.N.S O analyzer.IR spectra were recorded on the Perkin –Elmer FT-IR 1725X spectrometer using the KBr-pellet technique in the region 4000–400cm −1.The NMR spectra were recorded on Bruker Avance 500equipped with broad-band direct probe.All spectra were measured at 26°C.The complex was characterized on the basis of NMR spectroscopy results:1D (1H,13C,DEPT —distortionless enhancement by polarization transfer),2D (COSY —correlation spectroscopy)as well as 2D 1H –13C heteronuclear correlation spectra.Chemical shifts are given on δscale relative to tetramethylsilane (TMS)as internal standard for 1H and 13C.UV –visible (UV –vis)spectra were recorded on a UV Cintra 40UV –visible spectrometer operating from 200to 800nm in 1.0cm quartz cells.A Submarine Mini-gel Electrophoresis Unit (Hoeffer HE 33)with an EPS 300power supply was used for electrophoresis experiments.The stained gels were illuminated under a UV transilluminator Vilber-Lourmat (France)at 312nm and photographed with a Panasonic DMC-LZ5Lumix Digital Camera through filter DEEP YELLOW 15(TIFFEN,USA).2.2.Synthesis of [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OH
To a solution containing N ′,N ′2-bis[(1-E )-1-(2-pyridyl)ethylidene]propanedihydrazide (H 2L)(0.0338g,0.1mmol)in ethanol (15mL)were added solutions of 2,2′-dipyridylamine (0.0171g,0.1mmol)and Co(NO 3)2·6H 2O (0.0291g,0.1mmol)each dissolved in 5mL of ethanol.The mixture was re fluxed for 2h forming a brick-red solution.After five days dark brown –red crystals suitable for X-ray diffraction were formed.Yield 52%;(Found:C 42.80H 3.99N 19.79%.C 70H 78Co 4N 28O 25calcd.C 43.18H 4.04N 20.14%).IR spectrum (KBr;ν,cm −m =medium,
w =weak,s =strong,vs =very strong):3416(m),1602(w),1501(s),1457(s),1382(vs),1358(vs),1329(s),1257(m),1147(m),1074(m),1034(w),944(w),831(w),776(m),735(m),692(w),654(m),578(w).UV –vis (DMSO):λmax 370nm (ε,550M −1cm −1)1A 1g →1T 2g (0.275),λmax 320nm (ε,950M −1cm −1)1A 1g →1T 1g (0.475).2.3.Crystallographic structure determination
Crystal data:C 35H 39Co 2N 14O 12.5,M =973.66,triclinic,space group P 1,a =15.0282(4),b =16.3112(4),c =17.4548(5)Å,α=69.609(2),β=86.147(2),γ=88.014(2)°,V =4001.19(18)Å3,Z =4,D c =1.616g cm −3,μ=0.912mm −1.A red prism of compound [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OH with dimensions of 0.28×0.10×0.10mm was greased on a glass thread.Diffraction data were collected on a Nonius Kappa CCD diffractometer with an area detector at −123°C.A Cryo-stream Cooler (Oxford Cryosystems)was used for cooling the sample.A graphite monochromated MoK αradiation (λ=0.71073Å)was employed.The data were processed by using DENZO [12].A total of 25,530re flections were measured,14,384were independent,and 9066[I N 2σ(I )]were considered observed.The structure was solved by direct methods using SIR-92[13]and re fined by a full-matrix least-squares procedure based on F 2using SHELXL-97[14].All the non-hydrogen atoms were re fined anisotropically.All hydrogen atoms were included in the model at geometrically calculated positions and re fined using a riding model.The residual density peaks,observed in the difference Fourier map,were unre finable and probably can be attributed to disordered solvent molecules.In our final model,the scattering contributions from all of these diffuse moieties have been removed using the SQUEEZE routine in PLATON [15].A potential solvent-accessible volume of 236Å3was found.This volume corresponds to six additional molecules of water present in the unit cell,three per asymmetric unit.The empirical formula above is thus also in accordance with the elemental analysis and expresses two molecules of H 2O and 0.5molecules of EtOH per complex cation.All the calculations were performed by using the WinGX System,Ver.1.80.05[16].Final R indices [I N 2σ(I )]R1=0.0614,wR2=0.1317,and (all data)R1=0.1111,wR2=0.1504were found.2.4.Biological activity evaluation
A teaspoon of lyophilized eggs of the brine shrimp Artemia salina was added to 1L of the arti ficial sea water containing several drops of yeast suspension (3mg of dry yeast in 5mL distilled water),and air was passed through the suspension thermostated at 301K,under illumination for 24h.The tested substances were dissolved in DMSO.In a glass vial,into 1mL of arti ficial sea water 1–2drops of yeast extract solution (3mg in 5mL of distilled water)and 10–20hatched nauplii were added,and finally solutions of all derivatives to the appropriate concentrations.For each concentration,two determina-tions were performed.The vials were left at 28°C under illumination for 24h,and afterwards live and dead nauplii were counted.LC50was de fined as the concentration of a drug that causes death of 50%nauplii.DMSO was inactive under applied conditions.2.5.Cytotoxicity assay
Human epithelial breast cancer (MDA-361),human cervical carci-noma (HeLa),murine melanoma (B16),human leukemia cells (K562)and human lung fibroblasts (MRC 5)were maintained in RPMI 1640medium (Sigma-Aldrich,Cat.No.R7755),in a humidi fied atmosphere containing 5%(v/v)CO 2.Human endothelial cells EA.hy 926were maintained in high glucose DMEM —Dulbecco's modi fied eagles'medium (PAA,Cat.No.E15-883).Both media were supplemented with 10%heat-inactivated fetal bovine serum (Sigma-Aldrich,Cat.No.F4135),penicillin (100units/mL),and streptomycin (100μg/mL).
Except for the K562and MRC 5,all cell lines were seeded at a density of 2000cells/well in 96-well plates,whereas K562and MRC
5
Scheme 1.N ′,N ′2-bis[(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide.
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were seeded at a density of5000cells/well.The cells were left for24h to rest,and afterwards investigated agents were added.Preparation of tested solution was performed immediately before experiment,by dissolution in DMSO to the stock concentration of10mM,whereas further dilutions were performed with the RPMI—Roswell park memorial institute(or DMEM)medium.Final concentrations achieved in treated wells for all tested agents were3,9,30,90and 300μM,whereas1,3,10,30,and100μM for MDA-361,HeLa and K562cells.Each concentration was tested infivefold on particular cell line,and left for48h of incubation.Except for the K562cells,the cytotoxic activity of each agent was evaluated by means of sulphorhodamine B(SRB,Sigma-Aldrich,Cat.No.S1402-5G) colorimetric assay[17].Since the K562is the cell line that grows in suspension,the cytotoxicity potential was determined by means of MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide,Sigma)assay[18].
The%of control was further calculated as the following:%of control=[ODt−ODb]/[ODc−ODb],ODt—mean optical density of treated wells,ODb—mean optical density of blank wells,ODc—mean optical density of control wells.Results arefinally expressed as IC50 values(concentration of investigated agent that declines the number of viable cells by50%in treated cell population compared to non-treated control),determined using sigmoidal dose–response curve fitting on Graph Pad Prism software[19].Survival curves were determined using sigmoidal dose–response curvefitting on Graph Pad Prism software.
2.6.Flow-cytometric analysis of cell cycle phase distribution
Human epithelial breast cancer(MDA-361)cells were seeded in six well plates(200,000cells/well)in duplicate,and left overnight to rest,afterward the investigated substance was added.
For the cell cycle analysis the investigated compound was applied in two concentrations,thefirst one equal to IC50value,and the second one corresponding to2×IC50.For cell cycle analysis,cells were harvested after24h of drug treatment,washed once in phosphate buffered saline (PBS),andfixed in ice-cold ethanol(70%).Fixed specimens were kept at 4°C for24h,when ethanol was removed by two washing cycles in PBS. Following the last spinning cycle the cells were treated with0.2mL ribonuclease(1mg/mL)for30min at37°C.Then,cells were put on ice with propidium iodide(PI)(0.2mL,50μg/mL)added15min before analysis[20].
2.7.Apoptotic assay
Apoptosis of MDA-361cells treated with investigated complex [Co2L2](NO3)2·2H2O·0.5C2H5OH was evaluated by Annexin V–FITC—fluorescein isothiocyanate apoptosis detection kit(BD Biosciences Cat. No.65874x,Pharmingen San Diego,CA,USA).Briefly,1×106cells/mL treated with the concentration of1×IC50of investigated compound for 24h,were washed twice with cold PBS and then resuspended in200μL binding buffer(10mM HEPES/NaOH(HEPES—(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)pH7.4,140mM NaCl,2.5mM CaCl2). 100μL of the solution(1×105cells)were transferred to a5mL culture tube and5μL of Annexin V–FITC and5μL of PI was added.Cells were gently vortexed and incubated for15min at25°C in the dark.After that, 400μL of binding buffer was added to each tube and then analyzed using a FACS Calibur Becton Dickinsonflow cytometer and Cell Quest computer software[21].
2.8.DNA binding experiments
CT-DNA was dissolved in Tris buffer(10mM Tris–HCl pH7.9) overnight at4°C.DNA concentration was adjusted with the buffer to 3mg/mL.This stock solution was stored at4°C and was stable for several days.A solution of CT-DNA in water gave a ratio of UV absorbance at260and280nm,A260/A280of1.89–2.01,indicating that DNA was sufficiently free of protein.The concentration of DNA was determined from the UV absorbance at260nm using the extinction coefficientε260=6600M−1cm−1[22].The absorbance titrations were performed at afixed concentration of[Co2L2](NO3)2·2H2O·0.5C2H5OH complex and varying the concentration of double stranded CT-DNA.For an individual experiment,each DNA solution(1,2,3,4,5, 6,8,9,10,11,12,13,14and15μL of CT-DNA stock solution)was pipetted into a separate vial,to which10μL of a stock solution of compound were added and the volume adjusted to1mL with water. Reaction mixtures were incubated at37°C for2h with occasional vortexing.Primary spectra of all spectrometric measurements were imported into Microcal ORIGIN v8.0.
2.9.DNA cleavage experiments
Plasmid pUC18was prepared by transformation of a clone from Escherichia coli RR1(pUC18,2686bp,purchased from Sigma-Aldrich, USA)into electrocompetent E.coli DH5αstrain cells according to the protocol for growing E.coli culture overnight in LB medium at37°C by electroporation with the“Gene Pulser”(Bio-Rad)[23].The plasmid DNA from E.coli clones were isolated by modified method of alkali lysis[24]and purified with the“JetStar”kit(Genomed)using anion-exchange column.Afterfinal washing step with ice-cold70%ethanol, DNA pellet was air-dried andfinally resuspended in150μL sterile H2O and stored at−20°C.The concentration of plasmid DNA(0.10nM of pUC18)was determined by measuring the absorbance of the DNA-containing solution at260nm.One optical unit corresponds to50μg/ mL of double stranded DNA.
Plasmid DNA was incubated for4min at95°C,followed by incubation at4°C for the next20min.Then230ng of pUC18in a20μL reaction mixture in40mM sodium bicarbonate buffer,pH8.4,was incubated with different concentrations of Co(II),H2L and[Co2L2] (NO3)2·2H2O·0.5C2H5OH at37°C,for90min,or with10mM of [Co2L2](NO3)2·2H2O·0.5C2H5OH for2h.The reaction mixtures were vortexed from time to time.The reaction was terminated by short centrifugation at10,000rpm and adding7μL of loading buffer(0.25% bromophenol blue,0.25%xylene cyanol FF and30%glycerol in TAE buffer,pH8.24(40mM Tris-acetate,1mM EDTA)).The samples were subjected to electrophoresis on1%agarose gel(Amersham Pharma-cia-Biotech,Inc)prepared in TAE buffer pH8.24.The electrophoresis was performed at a constant voltage(80V)for about an hour(until bromophenol blue had passed through75%of the gel).After electrophoresis,the gel was stained for30min by soaking it in an aqueous ethidium bromide solution(0.5μg/mL),and after that was visualized under UV light.
2.10.Antibacterial activity
Antibacterial activity was evaluated using the standard agar-diffusion test,against four Gram positive(Bacillus subtilis,Bacillus cereus,Streptococcus pyogenes,Streptococcus agalactiae)and one Gram negative(Klebsiella pneumoniae)bacterial strain.All the strains were obtained from the Culture Collection of Extremophilic Microorgan-isms at the Department of Biology,Biotechnical Faculty,University of Ljubljana,Slovenia.The bacteria were allowed to grow overnight in LB medium,and their concentrations were determined by turbidimetric assay.After that,an appropriate volume of bacterial culture was added to LB nutrient agar previously cooled to42°C,to give thefinal concentrations of the bacteria approximately5×105CFU/ml.Twenty milliliters of inoculated medium were poured into Petri dishes and incubated at4°C for24h.The circles of agar(Ф=1cm)were cut out from the cooled medium.A stock solution of the complex in DMSO was gradually dissolved in deionized water.One hundred micro liters of the obtained solutions in various concentrations(1000,200,40,8, and0.16μg/mL)were pipetted into the holes cut in the inoculated
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medium,and the dishes were incubated at37°C for24h.The diameters of the inhibition zone were then measured.As controls,the inhibitory activities of the pure solvents(appropriate aqueous solutions of DMSO)were checked as well.Every measurement was repeated at least in three parallel samples.
3.Results and discussion
3.1.General
Starting from the ethanolic solution of Co(NO3)2·6H2O and H2L in the presence of2,2′-dipyridilamine,a dinuclear complex of cobalt(III) with two molecules of ligand L coordinated to each cobalt atom,and two nitrate ions in the outer sphere was obtained.Elemental analysis showed that the crystal substance contains0.5molecule of ethanol and2molecules of water per complex cation.The molar conductivity data showed that the complex is an electrolyte of1:2type.2,2′-Dipyridylamine was added into the reaction mixture during synthesis in order to change dielectric constant of the solution from which the complex crystallizes so that crystals suitable for X-ray analysis could be obtained.During the reaction,oxidation of Co(II)to Co(III)by air occurred,affording a diamagnetic complex,as shown by magnetic moment measurement.
3.2.Description of the crystal structure
Compound[Co2L2](NO3)2·2H2O·0.5C2H5OH crystallizes in mono-clinic P crystal system with two independent and slightly different ionic complexes[Co2L2]2+in the structure,one of which is shown in Fig.1.The complex cation[Co2L2]2+possesses a dinuclear double-stranded helicate structure in which each Co(III)center is coordinated by two tridentate N2O chelating units from different ligands.Each Co(III) center thus occupies a six-coordinated pseudooctahedral environment surrounded by two chelating units coordinated to Co(III)atom in mer configuration with pairs of carbonyl O atoms and pyridine N atoms in a cis relationship,whereas the hydrazide N atoms are trans to each other. The average Co\N bond distances(1.889Å)are slightly shorter than
average Co\O bond distances(1.902Å)but all are in the range usually reported.The C(O)\N and C(CH3)\N bond distances(av.1.310and av.
1.295Å,respectively),together with_N\N\bond distances(av.
1.393Å)suggest that the protons on hydrazide nitrogen atoms are lost and L acts as a dianionic bis-tridentate ligand during the coordination. The chelating ligands form dihedral angles between planes py\C(CH3) N_NC(O)at Co1of87.57and89.57°for the two independent molecules.The dihedral angles at Co2are89.23and86.15°for both ionic complexes in the structure.The separation distances between the metal ions are5.749and5.664Å,respectively.The bis(bidentate)ligand strands wrap around the cobalt centers,possessing pseudo two-fold rotation axis that passes through the metal ions.Interestingly,despite the presence of aromatic ligands in the crystal structure,the complex shows no obvious interactions between the aromatic rings.Each of the independent ionic complexes[Co2L2]2+in asymmetric unit is inherently chiral and possesses P for right-hand and M for left-hand helicity(Fig.2). Based on the fact that the complex[Co2L2](NO3)2·2H2O·0.5C2H5OH crystallized in achiral space group,the unit cell accommodates four molecules(both independent ionic complexes along with their enantiomers),thus forming a racemic solid product.
3.3.NMR studies
For further structural support the l H-NMR spectrum(Table1)of the cobalt(III)complex was compared with the l H-NMR spectra of free H2L.The NH proton of free H2L,observed at10.86ppm was absent in the spectrum of the complex,suggesting that deprotonation occurred.In the complex there are large relative shifts of all other protons,compared to ligand,indicating strong tridentate coordination to Co(III).
The above statement is further substantiated by comparing the 13C-NMR spectrum of free H
2
L with that of the complex,indicating N, N,O coordination(Table2).The NMR spectra are in full accordance with the crystal
structure.
Fig. 1.View of one of the two independent ionic complexes[Co2L2]2+found in asymmetric unit,with the atomic numbering scheme.Selected bond lengths(Å)and angles(°):Co1\N11.923(4),Co1\N21.851(4),Co1\N71.919(4),Co1\N81.851(4), Co1\O11.904(3),Co1\O31.907(3),Co2\N51.864(4),Co2\N61.923(4),Co2\N11 1.850(4),Co2\N12 1.931(4),Co2\O2 1.891(3),Co2\O4 1.905(3);N1\Co1\N2 83.11(19),N1\Co1\N792.92(16),N1\Co1\N898.39(18),N1\Co1\O1165.17(17), N1\Co1\O3165.17(17),N5\Co2\N682.35(17),N5\Co2\N11172.58(17), N5\Co2\N12103.02(17),N5\Co2\O282.50(16),N5\Co2\O4
92.04(16).
Fig.2.Space-filling representation of the two independent ionic complexes[Co2L2]2+ found in asymmetric unit with P-and M-helicity.In both representations the two strands in each complex are differently colored(green(light color)and blue(dark color))for clarity.Hydrogen atoms are omitted for clarity.
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3.4.Biological activity
Biological activity of the complex and the corresponding ligand was tested by the brine shrimp test(toxicity to A.salina).This test is in good correlation with cytotoxic activity[25].The complex showed a moderate activity,similar to that of the respective ligand(H2L)and Co (NO3)2·6H2O(Table3).
3.5.Cytotoxic activity
Cytotoxic potential of newly synthesized complex[Co2L2] (NO3)2·2H2O·0.5C2H5OH as well as corresponding ligand(H2L)and salt was investigated on six cell lines including human epithelial breast cancer(MDA-361),human cervical carcinoma(HeLa),murine melanoma(B16),human leukemia cells(K562),human endothelial cells(EA.hy926)and human lungfibroblasts(MRC5).The compounds were applied in range of concentrations1–100μM for MDA-361,HeLa,K562and3–300μM for B16,EA.hy926and MRC5, and cells left for48h.The activities of the compounds were determined by means of Sulphorhodamine B and MTT tests and the results were expressed in terms of IC50values.
In the investigated range of concentration,cytotoxic potential was observed for the complex on the epithelial breast cancer(MDA-361) cell line,where the IC50was50.9μM(Table4).The weak cytotoxic activity of the complex was observed with EA.hy926and B16cell line, reaching IC50values at258μM and221μM,respectively.The complex revealed low cytotoxicity against MRC5cells which are considered as normal,noncancer cell.
The corresponding H2L ligand also showed moderate cytotoxic activity against all cell lines except K562and MDA-361cells(Table4). The ligand reached IC50value on most of the cell lines including HeLa, EA.hy926,MRC5with the highest potency on the murine melanoma B16cell line.The corresponding salt showed weak cytotoxic activity only on human leukemia K562cells,EA.hy926,B16cells,and the weakest activity was observed with the MRC5cell line(Table4). K562cells were resistant to both complex and ligand,the complex even stimulating cells to grow.Fig.3represents the survival curves of the cells treated with complex[Co2L2](NO3)2·2H2O·0.5C2H5OH.
B16,EA.hy926and MRC5cells were treated with the complex in the range of concentration of3–300μM,whereas MDA-361,HeLa cells and K562cells were treated with the concentration of1–100μM.All cells showed dose-dependent inhibition of cell growth,except for the leukemic K562cells,where growth-stimulatory effect was observed.
Since the complex showed strong cytotoxic potential against epithelial breast cancer cell line MDA-361,we continued our research towards mechanism of the observed cytotoxicity.
3.6.Cell cycle and apoptosis
Results of cytotoxic activity provide only the information on the restriction of the population of viable cells,but not the mechanism by which this restriction is performed.Since low IC50value on MDA-361 cell line was obtained,it was necessary to examine whether the observed cytotoxic potential is due to modulation of cell cycle progression or induction of cell death.For that purpose,influence of the complex[Co2L2](NO3)2·2H2O·0.5C2H5OH on apoptosis of the most sensitive MDA-361cell line was tested.The cells were treated with the concentration corresponding to IC50and2×IC50,and left for 24h,afterwards harvested,collected and analyzed for cell cycle perturbations.There was no significant effect on cell cycle perturba-tions,as seen in the Fig.4.After24h of incubation of cells treated with the concentration of IC50and2×IC50,the investigated complex gave no significant changes in the percentage of cells in the G1,S and G2/M phases.
However,there is a clearly determined sub-G1peak in the histograms of treated cells,as shown in Fig.4.Those are hypodiploid cells,containing fragmentized DNA molecule,indicating that such cells are in late apoptotic phase.Therefore,the apoptotic potential of the complex[Co2L2]2+on MDA-361cell line was further examined, using double color staining with Annexin V–FITC/PI.The principle of this test lies in the exclusive characteristic of early apoptotic cells to expose phosphatidylserine(PS)molecule from the inner to the outer side of the cell membrane.The Annexin V is the Ca2+-protein dye with a strong preference towards negatively charged PS among other phospholipids.On the other hand,cells in late apoptosis and necrosis gain permeabilized cell membrane,which allows another dye used in this experimental procedure,propidium iodide,to enter the nucleus and bind DNA molecule.In this way,after analysis onflow cytometer, cells in early apoptotic stage can be clearly differentiated from necrotic and late apoptotic cells.The MDA-361cells were treated with the concentration of the complex corresponding to IC50and left for 24h,afterwards harvested and colored.The output histograms are shown in Fig.5.Obviously,the investigated complex induced a significant percent of early apoptotic cells,giving28.18%comparing to 5.64%of control cells in the same phase.There was no significant change in necrotic and late apoptotic phase cell population.
Table1
1H NMR spectral data.
Assignment(multiplicity,number of H-atoms,coupling constant,J in Hz)Chemical shift,δ(ppm)
C-1_C-17(d,2H,3J=5.0)7.89
C-2_C-16(t,2H,3J=6.5,6.0)7.49
C-3_C-15(t,2H,3J=8.0,7.5)8.20
C-4_C-14(d,2H,3J=7.5)8.13
C-7_C-11(s,6H) 3.10
C9(s,4H) 3.41 s—singlet;d—doublet;t—triplet.
Table2
13C NMR spectral data.
Assignment Chemical shift,δ(ppm)
C-1_C-17152.16
C-2_C-16128.80
C-3_C-15141.86
C-4_C-14126.55
C-5_C-13159.81
C-6_C-12162.54
C-7_C-1115.05
C-8_C-10183.74
C934.44
Table3
Toxic effect(expressed as LC50values in mM)of
ligand and complex on Artemia salina.
Substance LC50
[Co2L2]2+ 3.21
H2L 3.97
Co(NO3)2·6H2O 6.32
Table4
IC50values(μM)obtained with different cell lines.
Investigated
compounds
Cell line
EA.hy926MDA-361HeLa MRC5K562B16 [Co2L2]2+25850.9N100N300N100221 H2L124N10072.6107N10036.8 Co(NO3)2·6H2O108N100N10022262.1139
1200R.Eshkourfu et al./Journal of Inorganic Biochemistry105(2011)1196–1203。