A synthetic dinuclear copper(II) hydrolase and its potential as antitumoral: cytotoxicity, cellular uptake, and DNA cleavage

Journal of Inorganic Biochemistry 103 (2009) 1323–1330

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A synthetic dinuclear copper(II) hydrolase and its potential as antitumoral: Cytotoxicity, cellular uptake, and DNA cleavage Nicolás A. Rey a,1, Ademir Neves a, Priscila P. Silva b, Flávia C.S. Paula b, Josianne N. Silveira c, Françoise V. Botelho b, Leda Q. Vieira d, Claus T. Pich e, Hernán Terenzi e, Elene C. Pereira-Maia b,* a

Department of Chemistry, Universidade Federal de Santa Catarina, Florianópolis SC, Brazil Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte MG, Brazil c Department of Clinical and Toxicological Analyses, Universidade Federal de Minas Gerais, Belo Horizonte MG, Brazil d Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Belo Horizonte MG, Brazil e Department of Biochemistry, Universidade Federal de Santa Catarina, Florianópolis SC, Brazil b

a r t i c l e

i n f o

Article history: Received 14 December 2008 Received in revised form 21 April 2009 Accepted 8 May 2009 Available online 20 May 2009 Keywords: Dicopper(II) complex Cytotoxicity Nuclease activity Cellular uptake DNA binding

a b s t r a c t We have studied the protonation equilibria of a dicopper(II) complex [Cu2(l-OH)(C21H33ON6)] (ClO4)2H2O, (1), in aqueous solution, its interactions with DNA, its cytotoxic activity, and its uptake in tumoral cells. C21H33ON6 corresponds to the ligand 4-methyl-2,6-bis[(6-methyl-1,4-diazepan-6-yl)iminomethyl]phenol. From spectrophotometric data the following pKa values were calculated 3.27, 4.80 and 6.10. Complex 1 effectively promotes the hydrolytic cleavage of double-strand plasmid DNA under anaerobic and aerobic conditions. The following kinetic parameters were calculated kcat of 2.73  104 s1, KM of 1.36  104 M and catalytic efficiency of 2.01 s1 M1, a 2.73  107 fold increase in the rate of the reaction compared to the uncatalyzed hydrolysis rate of DNA. Competition assays with distamycin reveal minor groove binding. Complex 1 inhibited the growth of two tumoral cell lines, GLC4 and K562, with the IC50 values of 14.83 lM and 34.21 lM, respectively. There is a good correlation between cell growth inhibition and intracellular copper content. When treated with 1, cells accumulate approximately twice as much copper as with CuCl2. Copper–DNA adducts are formed inside cells when they are exposed to the complex. In addition, at concentrations that compound 1 inhibits tumoral cell growth it does not affect macrophage viability. These results show that complex 1 has a good therapeutic prospect. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Considerable progress has been attained in cancer chemotherapy with the combined use of drugs that work by different mechanisms of action. However, chemotherapy still presents some inconveniences such as the development of resistance and undesirable side effects. As a consequence, much effort has been made to find new agents more selective and less toxic. DNA is an important target of antitumoral drugs because it plays a central role in replication, transcription, and regulation of genes. The presence of metal-binding sites in its structure makes it a good target for metal-containing drugs such as cisplatin [1,2]. Different types of interactions are possible such as intercalation between base-pairs, minor groove binding, and major groove binding [3]. Antitumoral metal complexes can also, as in the case

* Corresponding author. Fax: +55 31 34095700. E-mail addresses: [email protected], [email protected] (E.C. PereiraMaia). 1 Present address: Department of Chemistry, Pontifícia Universidade Católica do Rio de Janeiro, Rio de Janeiro RJ, Brazil. 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.05.008

of bleomycin, generate radical species that abstract a hydrogen from the sugar moiety and cleave DNA molecule [4]. Several copper complexes were also described to cleave DNA by an oxidative mechanism that requires the presence of oxygen [5,6]. The best studied example is the [Cu(Phen)2]2+ (Phen = 1,10-phenanthroline), which is reduced in situ leading to the [Cu(Phen)2]+ species that subsequently binds to the minor groove of DNA, combines with molecular oxygen, generates a non-diffusible oxidant and finally induces strand scission by oxidation of the ribose backbone [7]. Certain copper complexes that break DNA strands by an oxidative pathway were reported to be cytotoxic against tumoral cells [8–11] and to trigger cellular apoptosis [12,13]. However it is important to emphasize that oxidative DNA cleavage agents do not produce fragments consistent with those produced by natural hydrolases, (50 -phosphates and 30 -hydroxyls) and thus nucleic acids cleaved oxidatively cannot be enzymatically religated [14] limiting their utilization in molecular biology and therefore synthetic hydrolytic DNA cleavage agents which does not have these drawbacks are preferable to oxidative cleavage agents. Dinuclear metallohydrolases are a structurally diverse group of enzymes that use dinuclear metal ion centers to catalyze the

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hydrolysis of amides and esters of carboxylic and phosphoric acids. DNA cleavage can occur by a nucleophilic attack to a phosphate in nucleic acid backbone [15,16]. Many efforts have been made by several groups aiming to design small-molecules capable to catalyze DNA hydrolysis at physiological conditions. Some copper complexes have demonstrated the ability to promote a hydrolytic cleavage of the DNA backbone [17–21]. We have been exploring the antitumoral potential of the DNA cleaving activity mediated by metal compounds by a hydrolytic pathway. In a previous work, we studied the cytotoxic activity of a dinuclear copper compound and found that GLC4/CDDP cells that are 6-fold resistant to cisplatin are only slightly resistant to it [22]. We proposed a hydrolytic mechanism for DNA cleavage and that the cytotoxic action was related to this activity. The mechanism of action of almost all the DNA-targeting agents that are used in cancer chemotherapy had been established only after their antitumour activity. Besides the elucidation of the mechanism of action, another important concern is the study of cellular uptake because a sufficient amount of the drug should accumulate inside target cells and, more specifically, in the cell nucleus [3]. In this work, we studied the chemical equilibrium of a dicopper(II) l-hydroxo complex, [Cu2(l-OH)(C21H33ON6)](ClO4)2H2O, in aqueous solution, its interactions with DNA, its cytotoxic activity, and its uptake in tumoral cells. In a previous work, we have described the synthesis, characterization, catecholase-like activity, and preliminary results on DNA cleavage [23]. The complex was initially designed as a promiscuous synthetic model since it is able to catalyze the oxidation of catechols as well as the hydrolysis of diesters such as 2,4-bis(dinitrophenyl)phosphate and DNA.

2. Experimental 2.1. Reagents The synthesis and characterization of the [Cu2(l-OH) (C21H33ON6)](ClO4)2H2O (complex 1) is described in details elsewhere [23]. All reagents and solvents used were of reagent grade or HPLC grade and used without further purification. 2.2. Spectrophotometric measurements A Diode Array Hewlett Packard 8451 A spectrometer equipped with a Masterline 2095 thermostat at 25 °C was used for UV and visible absorption measurements. Experiments were carried out under nitrogen atmosphere and the temperature was kept constant at 25 °C. The ionic strength was maintained at 0.1 M with sodium chloride. The concentration of complex 1 used in the spectroscopic measurements was 2.5  104 M. Acidified solutions of 1 in water were spectrophotometrically titrated with NaOH. These data were treated by means of the SQUAD program [24], which searches for the best combination of stability constants of the species to fit the data and simultaneously calculates the molar absorptivities based on the current value of the stability constant, b. Results are expressed in terms of the molar absorption coefficient e related to the total concentration of complex 1. The species distribution curves were calculated from spectrophotometric measurements by the SCECS software [25]. 2.3. DNA cleavage assays Plasmid DNA was used to assay cleavage activity of complex 1 as previously described [26–28]. Different concentrations of complex 1 were incubated with 0.6 lg of pBSK II plasmid DNA, at pH 6.0, 6.5, 7.0 and 7.5 in

25 mM PIPES buffer (piperazine-N,N0 -bis[ethanesulfonic acid) and 8.0 in 25 mM HEPES buffer [N-(2-hydroxyethyl)piperazine-N0 -(2ethanesulfonic-acid)], at 50 °C for 16 h. Complex 1 concentrations were 0, 5, 10, 20 and 40 lV. All incubations were performed in triplicates. Samples were submitted to agarose gel electrophoresis and stained with ethidium bromide. The resulting gels were digitalized with a photodocumentation system (UVP, CA, USA) and DNA bands were quantified using Lab-Works Software version 4.0 (UVP, Inc.). For distamycin competition assays, DNA was pre-incubated in the presence of 30 lM distamycin for 30 min and then incubated for 4 h with 1 at 0, 150 and 300 lM, at 50 °C, pH 7.5. Samples were submitted to agarose gel electrophoresis and analyzed as described above. For anaerobic DNA cleavage assays, a two-step procedure was used to obtain deoxygenated water [27,28]. All solutions and reaction mixtures containing 0, 100 and 200 lM of complex 1 were prepared in an argon-filled glove bag. Samples were then incubated at 50 °C in a sealed argon-filled vacuum desiccator, at pH 7.5 for 2 h. Fe(EDTA) was used as a positive control for DNA damage via radical processes. All other conditions and procedures were essentially the same as those described for aerobic reactions. Samples were submitted to agarose gel electrophoresis and analyzed. Kinetic analysis of DNA cleavage was also performed. The velocity of DNA hydrolysis as a function of complex 1 concentration was monitored at pH 7.5. Supercoiled plasmid DNA (0.6 lg) was incubated at 50 °C with 1 at concentrations of 0, 50, 100, 150, 200 and 250 lM, in PIPES buffer at pH 7.5. At fixed time intervals up to 90 min, samples were collected and analyzed by gel electrophoresis. TM

2.4. Cell lines and cultures The GLC4 cell line was derived from pleural effusion of a patient with small cell lung carcinoma. Culture of GLC4 cells was performed in RPMI 1640 (Sigma Chemical Co.) medium supplemented with 10% of fetal calf serum (Biomedia Co.) at 37 °C in an humidified 5% CO2 atmosphere. The K562 cell line was purchased from the Rio de Janeiro Cell Bank (number CR083 of the RJCB collection). The K562 cell line was established from the pleural effusion of 53 yearold female with chronic myelogenous leukemia in terminal blast crisis. K562 cell line was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (CULTLAB, São Paulo, Brazil) at 37 °C in a humidified 5% CO2 atmosphere. Cultures grow exponentially from 105 cells/mL to about 106 cells/mL in 3 days. Cell viability was checked by Trypan Blue exclusion. The cell number was determined by Coulter counter analysis. For cytotoxicity assessment, 1  105 cells/mL were cultured for 72 h in the absence and the presence of various concentrations of the copper compound. The sensitivity to the compound was evaluated by the concentration that inhibits cell growth by 50%, IC50. A stock solution of the compound was prepared in DMSO (dimethyl sulphoxide). The final concentration of DMSO in the experiments with cells was below 0.5% and we have checked that the solvent has no effect on cell growth at this concentration. 2.5. Long-term accumulation K562 cells at the concentration of 1  105 cells/mL were incubated for 72 h in the absence and presence of various concentrations of the copper compound. After incubation, the cell number was determined. An aliquot was taken, washed twice with ice-cold isotonic buffer and the pellet was re-suspended in 33% HNO3. Copper concentration was determined by GFAAS (graphite furnace atomic absorption spectrometry) in a Varian model Zeeman 220 spectrophotometer equipped with a graphite tube atomizer and an autosampler. The total copper concentration in cells determined in the absence of compound 1 was subtracted from the samples incubated with the compound.

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2.6. Short-term accumulation The uptake of the dinuclear copper compound and that of CuCl2 was followed in K562 cells as a function of the incubation time. Cell culture was initiated at 5  105 cells/mL, and cells were used 24 h later, when they were at about 8  105 cell/mL. In a typical experiment, 1  106 cells/mL were incubated with 1  105 M of tested compounds up to 1 h in an isotonic buffer (132 mM NaCl, 3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM HEPES, 5 mM glucose) at pH 7.20 and 37 °C, under continuous stirring. At fixed time intervals, an aliquot was taken, washed twice with ice-cold isotonic buffer without glucose and the pellet was re-suspended in 33% HNO3. Copper concentration was determined by atomic absorption spectroscopy in a Varian model Zeeman 220 spectrophotometer equipped with a graphite tube atomizer and an autosampler.

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The absorbance at 550 nm of each sample well was measured using an automated plate reader [29]. The experiments were repeated at least twice.

3. Results and discussion Complex (1) contains a symmetric Schiff base derived from the facial tridentate ligand, 6-amino-6-methylperhydro-1,4-diazepine (AAZ). The molecular structure of the dinuclear cation in complex 1, determined by X-ray diffractometry [23] revealed two pentacoordinated cupric ions, which are bridged by the phenolate oxygen of the ligand and by an exogenous hydroxo ion. The geometry around each Cu(II) is distorted square-pyramidal, with bridging atoms in equatorial positions. 3.1. Protonation studies

2.7. DNA binding 6

K562 cells (5  10 ) were incubated with different concentrations of 1 ranging from 1 to 10 lM for 2 h. Afterwards, DNA was extracted from cells, by using a kit from Sigma (Sigma’s GenElute Mammalian Genomic DNA Miniprep Kit). Briefly, cells were incubated with RNase A in order to obtain a RNA-free genomic DNA. Subsequently, cells were treated with proteinase K and lysed in a chaotropic salt-containing solution to insure the thorough denaturation of macromolecules. Addition of ethanol caused DNA to bind to a silica membrane in a microcentrifuge tube. After washing to remove contaminants, DNA was eluted in a Tris–EDTA solution. DNA concentration per nucleotide was determined by spectrophotometric analysis (e = 6600 M1 cm1 at 260 nm). The ratio of absorbance at 260–280 nm was between 1.6 and 1.9. Copper concentration was determined by GFAAS. 2.8. Mice and macrophages Males and females of 4–8 weeks of the mouse strain C57BL/6 (CEBIO – UFMG, Belo Horizonte, Brazil) were used. Experimental animals were kept in a conventional animal house with barriers, temperature and light control. Food and water were offered ad libitum. Inflammatory macrophages were obtained from the peritoneal cavity, 3 days after injection of 2 mL of 3% thioglicolate medium, containing 1% sterile agar (Biobrás SA, Montes Claros, MG, Brazil). The animals were then sacrificed and 10 mL of sterile RPMI 1640 medium, without phenol red (Sigma Chemical Co., St. Louis, MO, USA), were injected into the peritoneal cavity. The largest possible content was aspirated, and the cells were centrifuged at 4 °C. Supernatant medium was discarded and cells were re-suspended in RPMI 1640 medium without phenol red, supplemented with 10% fetal bovine serum (CultLab São Paulo, Brazil), 0.05 mM bmercaptoethanol (Sigma Chemical Co.), 0.2% gentamicin and 200 mM L-glutamine. The cells were counted in a Newbauer chamber, and the final concentration was adjusted for each experiment. 2.9. Macrophages viability assays To evaluate the effect of compound 1 on macrophages viability, 2  105 cells/mL were incubated in the absence and presence of different concentrations of compound 1 for 24, 48 or 72 h. Cell viability was assessed using the MTT (3-[4,5-dimethylthiazol-2yl]diphenyltetrazolium bromide) reduction assay. 100 lL of 5 mg/ mL MTT were added to 2  105 cells in duplicate wells of a 96-well flat-bottom tissue culture plate and cells were incubated for 4 h at 37 °C. After 4 h, 100 lL of SDS detergent was added to each well.

Spectra of solutions containing 2.5  104 M of 1 were recorded as a function of the pH in the range 3–7 (Fig. 1). Around pH 3, the spectrum of 1 exhibits a broad absorption band centered at about 370 nm with a shoulder at approximately 390 nm, which can be assigned to a ligand-to-metal charge transfer from the bridging phenolate to copper ions. By increasing the pH up to 4, the absorption centered at 370 nm undergoes a batochromic shift to 394 nm and an isosbestic point can be observed at 369 nm, which indicates the presence of only two absorbing species in solution (Fig. 1A). From pH 4.3 to 5.4, a hypsochromic shift to 380 nm and a new isosbestic point at 384 nm occur (Fig. 1B). Finally, by raising the pH up to 7, one observes an increase in the intensity of the absorption (Fig. 1C). These results correspond to the titration of three ionizable protons. From these spectrophotometric data, the following protonation constants were determined: [Cu2(H2O)2(H1L)]5+, log b = 14.17 ± 0.02; [Cu2(H2O)2(H2L)]4+, log b = 10.90 ± 0.02; and [Cu2(H2O)2(H3L)]3+, log b = 6.10 ± 0.02, which correspond to the pKa values of 3.27, 4.80 and 6.10. The calculated spectra are in a good accordance with the experimental data (Fig. 1D). In all detected protonation states of 1, the phenol group is deprotonated and coordinated in a bridging mode to the copper(II) ions. As two of the nitrogen atoms (those occupying apical positions in the structure of 1) are more distant from the metal centers as a result of the Jahn–Teller distortion, they can be easily protonated by lowering the pH. So, we attributed the first two pKa values to the deprotonation and re-coordination of these donor-atoms. The last pKa corresponds to the deprotonation of a water molecule to give the l-hydroxo species 1 (Fig. 2). The species distribution curves calculated for the same experimental conditions are shown in Fig. 3. At pH 3, the main species is [Cu2(H2O)2(H1L)]5+, in which a nitrogen atom from each one of the ligand pendant arms is protonated. As a result, both copper ions are four-coordinated and should adopt a square planar geometry. By raising the pH, one nitrogen deprotonates yielding [Cu2(H2O)2(H2L)]4+, which dominates the system from pH 3.3 to 4.8. Another diaqua species, [Cu2(H2O)2(H3L)]3+, is formed by deprotonation of [Cu2(H2O)2 (H2L)]4+ and predominates from pH 4.8 to 6.1. A similar species was observed by Torelli et al. for a dicopper(II) complex of the symmetric ligand 2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4methylphenol (H-BPMP) [30]. Above pH 6.2, the main species is the l-hydroxo species 1, Fig. 2. It should be noted that the spectrum obtained at pH 7 is virtually identical to that published for complex 1 in the solid state (diffuse reflectance) [23], whose structure was determined by X-ray diffractometry. This suggests that the coordination sphere around Cu(II) centers is maintained after dissolution. This hydroxo-bridged species begins to be formed at pH 4.5 and, at pH 7.5, constitutes 95% of the total complex concentration.

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A

B

6000

Molar Absorptivity (M-1 cm-1)

Molar Absorptivity (M-1 cm-1)

6000 5000 4000 3000 2000 1000 0

5000 4000 3000 2000 1000 0

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Wavelenght (nm)

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D

6000 5000 4000 3000 2000 1000 0

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Wavelenght (nm)

Fig. 1. Spectrophotometric titration data for a solution containing 2.5  104 M of 1. A – the pH values ranged from 3.0 to 4.0; B – the pH values ranged from 4.3 to 5.4; C – the pH values ranged from 5.5 to 7.0. D – molar absorptivities calculated from the experimental spectra N – [Cu2(H2O)2(H1L)]5+; s – [Cu2(H2O)2(H2L)]4+; – [Cu2(H2O)2(H3L)]3+; 5 – [Cu2(l-OH)(H3L)]2+.

5+

N

N

O

H NH

Cu

Cu

NH H2O

H HN

4+

N

-H+ +H+

H N

Cu

Cu NH H2O

OH2 HN

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H HN

OH2 HN

+H+ -H+ 2+

N

N

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H N

Cu

Cu NH

H N

O H

HN

+H+ -H+

3+

N H N

N

O Cu

NH H2O

Cu

H N

OH2 HN

1 Fig. 2. Chemical equilibrium between different protonation states of complex 1.

N.A. Rey et al. / Journal of Inorganic Biochemistry 103 (2009) 1323–1330

2.5

Species concentration x10 4 (M)

D 2.0

B

C

A

1.5

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3

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pH Fig. 3. Species distribution curves showing percentages of species with respect to the total concentration of complex 1 in the solution. Total concentration of 1 is 2.5  104 M. A = [Cu2(H2O)2(H1L)]5+; B = [Cu2(H2O)2(H2L)]4+; C = [Cu2(H2O)2 (H3L)]3+; D = [Cu2(l-OH)(H3L)]2+.

3.2. DNA cleavage The nuclease activity of compound 1 towards diester bonds was investigated using plasmid DNA as the substrate. Plasmids are circular DNA molecules present as the supercoiled form, or FI. If an agent cleaves the DNA molecule in a single strand it can adopt the circular form, or FII. If cleavage involves two DNA strands in regions one close to the other, it can be converted to linear form, or FIII. Complex 1 cleaved the DNA molecule in all pH and concentrations tested, but the pH 7.5 is by far the one in which the activity is most pronounced (Fig. 4). At pH 7.5, FI is completely hydrolyzed to FII (90%) and FIII (10%) by incubating DNA with 40 lM of 1. The species distribution curves as a function of the pH (Fig. 3) showed that at pH 7.5 the main species is [Cu2(l-OH)(H3L)]2+, which indicates that this species is the active form. At pH 8.0 a significant decrease in the activity is observed. Increasing complex 1 concentration enhances cleavage activity. We have verified that CuCl2 at the same experimental conditions does not cause DNA cleavage.

Fig. 4. Cleavage of pBSK II plasmid DNA by 1 at different pH values. For each pH value, incubations were performed with 0, 5 10, 20, and 40 lM complex 1 at 50 °C for 16 h.

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Distamycin protected DNA from cleavage, which was approximately 50% of the control values in its presence (Fig. 5). Distamycin is a typical minor groove binder that interacts with DNA sequences riches in adenine and thymine base pairs. This inhibition is indicative that complex 1 also interacts with the DNA molecule in the minor groove. Complex 1 effectively promoted the hydrolytic cleavage of double-strand plasmid DNA under anaerobic and aerobic conditions, confirming that cleavage occurs by a hydrolytic process (Fig. 6). Kinetic parameters of the DNA cleavage reaction were determined (Fig. 7). Complex 1 effectively promoted the hydrolytic cleavage of supercoiled (form I) to nicked circular (form II) DNA, with the following kinetic parameters: kcat of 2.73  104 s1, KM of 1.36  104 M and catalytic efficiency of 2.01 M1 s1. When compared to the estimated uncatalyzed hydrolysis rate of DNA (1011 s1), there is a 2.73  107 fold increase in the rate of the reaction. Based on the spectrophotometric titration and kinetic data obtained at pH optimum (7.5), we propose a mechanism in which the phosphate of DNA binds to 1 through an oxygen atom in a monodentate fashion. The rate-limiting step must involve an intramolecular nucleophilic attack by the bridging hydroxo ion on the phosphorous atom of the metal-bound phosphate ester, resulting in substrate hydrolysis (P–O bond cleavage). It is worth noting that, for the hydrolysis of the activated phosphate diester BDNPP, bis(2,4-dinitrophenyl)phosphate, a kinetic pKa of 6.0 ± 0.2 was found [23], which is in complete agreement with the spectrophotometric pKa reported here for the formation of the l-OH species 1, equal to 6.10 ± 0.02. This reinforces the idea that the exogenous bridging hydroxo group is, in fact, the attacking nucleophile. Another complex very active is the mononuclear copper(II) complex of a tridentate Schiff base [(2-(imidazol-4-yl)ethyl)(1(imidazol-2-yl)methyl)imine] described by Scarpellini et al. which cleaved DNA with a kcat of 7.8  105 s1, about a 107 rate increase compared with the estimated uncatalyzed rate of hydrolysis [28]. Rossi et al. showed that the dicopper complex [Cu2(Hbtppnol)(lCH3COO)](ClO4)2 mediates a hydrolytic cleavage of DNA with a rate constant of 6.1  106 s1, which represents a 6.1  105-fold rate enhancement compared with the uncatalyzed hydrolysis rate of DNA [22]. A trinuclear copper(II) complex [Cu3L]6+, in which L is N,N,N0 ,N0 -tetra[(2-pyridyl)methyl]-5,50 -bis(aminomethyl)-2,20 -

Fig. 5. Cleavage profile of pBSK II plasmid DNA in the presence of increasing concentrations of 1 (left panel) and inhibition of the cleavage by distamycin, a typical minor groove binder (right panel). DNA was pre-incubated in the presence of 30 lM distamycin for 30 min and then incubated for 4 h with 1 at 0, 150 and 300 lM, at 50 °C, pH 7.5.

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Fig. 6. Plasmid DNA cleavage in anaerobic and aerobic conditions. Incubations were performed for 2 h with 0.6 lg plasmid pBSK II DNA and 1 at 0, 100 and 200 lM at pH 7.5, 50 °C.

dipyridyl, exhibited a very high cleaving activity with a kcat of 1.68  103 s1, 1.7  108-fold rate enhancement compared with the uncatalyzed hydrolysis [21]. Complex 1 is one of the most active dinuclear Cu(II) complexes in hydrolytically DNA cleaving described to date, comparable to the Cu neamine complex described by Sreedhara and coworkers [31]. 3.3. Cancer cells sensitivity The fact that compound 1 is able to cleave DNA molecule encouraged us to study its effect on the growth of two tumoral cell lines. We have obtained the IC50 values of 14.83 lM and 34.21 lM in GLC4 and K562 cells, respectively (Table 1). We have also made the cytotoxicity studies using acetonitrile as solvent and found the same values. Values obtained with cisplatin and carboplatin are also shown for the sake of comparison. In a previous work, we studied the cytotoxic activity of a synthetic dicopper nuclease, [Cu2(Hbtppnol)(l-CH3COO)](ClO4)2, in the GLC4 cell line and found

the IC50 value of 18.3 lM, e.g., compound 1 is slightly more active than it [22]. 3.4. Cellular accumulation To investigate if the cytotoxicity activity of compound 1 was related to its accumulation within the cells, we determined the intracellular compound concentration. K562 cells were incubated with increasing concentrations of 1 for 3 days (long-term accumulation). The total copper concentration determined in the absence of 1 was subtracted from the concentrations of samples. The average value was 1  1016 mol/cell. Considering that the volume of one cell equals to 1012 L, it gives an intracellular concentration of copper of 1  104 M. This value is within the range of total cellular copper content (10–100 lM) reported by González-Guerrero and Argüello [32]. There is a good correlation between cell growth inhibition and intracellular copper content, shown in Fig. 8. Incubation with the IC50 concentration yields an intracellular copper concentration of 3  1014 mol/cell or, taking into account that the complex is dinuclear, 1.5  1014 mol/cell of the complex. It indicates that the complex is being concentrated within cells by some mechanism of transport, possibly the main copper influx transporter in human cells, the 190 amino acid Cu transporter 1 (hCtr1) [33]. For the sake of comparison, similar experiments were reported with antitumoral platinum compounds: for cisplatin and carboplatin, at IC50 the intracellular platinum concentration was about 1.0 

Table 1 Growth inhibition of GLC4 and K562 cells by compound 1. Compound

Complex 1 Cisplatin Carboplatin [Cu2(Hbtppnol)(l-CH3COO)] (ClO4)2

Fig. 7. Kinetics analysis of DNA cleavage promoted by 1. Incubations were performed at pH 7.5, 50 °C with 0.6 lg plasmid pBSK II DNA and 1 at 0, 50, 100, 150, 200 and 250 lM. Samples were collected each 15 min and analyzed.

IC50a (lM ± s.d.) GLC4 cells

K562 cells

14.83 ± 0.15 0.40 ± 0.05b 9.9 ± 1.1b 18.3 ± 1.8c

34.21 ± 0.30 – – –

a IC50 is the concentration required to inhibit 50% of cell growth, after 3 days of incubation. The values are the mean of triplicate determinations. b Value from Ref. [34]. c Value from Ref. [22].

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lar uptake is a crucial feature to drug effectiveness. Filomeni et al. described the pro-apoptotic activity of two isatin-Schiff base copper(II) complexes, [Cu(isapn)] and [Cu(isaepy)2], and found a relation between cellular accumulation and activity, namely, the latter compound is more effective in inducing cell death and more permeating than the former [12].

Growth inhibition (%)

125

100

75

3.5. DNA binding

50

25

0 0

10

20

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40

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Fig. 8. Correlation between cell growth inhibition and intracellular copper content. K562 cells at the concentration of 1  105 cells/mL were incubated for 72 h in the absence and presence of various concentrations of the copper compound, at pH 7.20 and 37 °C, under continuous stirring. Copper concentration was determined by GFAAS. The total copper concentration in cells determined in the absence of compound 1 was subtracted from the samples incubated with the compound. Four separate measurements are represented.

3.6. Macrophages viability As macrophages play an important role in antitumor immunity, due to the production of important antitumor molecules such as TNF-a and nitric oxide, we investigated the effect of our compound on mouse peritoneal macrophage viability. Up to 64 lM the complex does not affect the viability of mouse peritoneal macrophages after a 24 h or 48 h-incubation. After 72 h of incubation, complex 1 does not affect macrophages viability up to 32 lM (Fig. 11). Thus, compound 1, at concentrations in which it inhibits tumoral cell growth, does not affect macrophage viability. 4. Final considerations Compound 1 is one of the most efficient dinuclear copper complexes in promoting cleavage of DNA under hydrolytic conditions described to date. Compound 1 enters tumoral cells, binds to DNA inside cells and inhibits tumoral cell growth. At the concen-

12

700

DNA-Cu adducts per million nt

Cellular concetration of copper x 1016 (mol/cell)

1016 mol/cell [34] and for four complexes derived from N-alkylpropanediamines at IC50 doses the intracellular platinum concentration was about 4.0  1016 mol/cell [35]. Interestingly, the uptake of cisplatin is reported to be mediated by the copper transporter Ctr1 in yeast and mammals [36,37]. In addition, Kabolizadeh et al. showed that the uptake of a cationic trinuclear platinum drug, that is being evaluated in phase II clinical trials for cancer treatment, is also mediated by hCtr1 [38]. Compound 1 uptake was followed in K562 cells as a function of the time up to 60 min (short-term measurements) and compared to the uptake of CuCl2, after incubation with a fixed concentration of 1  105 M for both compounds. The intracellular copper concentration versus the time of incubation is plotted in Fig. 9. When treated with compound 1, cells accumulate approximately twice as much copper as with CuCl2. The fact that compound 1 can easily enter and accumulate inside cells is very important because cellu-

We have shown that complex 1 enters and is accumulated in cells. Once inside cells, different metal binding domains are present and DNA is a potential target. We have extracted the DNA of cells, after incubation with 1 and determined copper concentration by AAS. Cells were incubated with increasing concentrations of complex 1 for 2 h prior to DNA extraction and analysis of copper content by AAS. In Fig. 10, the amount of copper bound to DNA is plotted as a function of the concentration of 1 in the incubation medium. The number of Cu–DNA adducts increases with complex concentration. Therefore, copper– DNA adducts are formed inside cells when they are exposed to the complex.

10

8

6

4 Compound 1 CuCl2

2 0

15

30

45

60

Incubation time (min) Fig. 9. The intracellular copper concentration versus the time of incubation. 1  106 cells/mL were incubated with 1  105 M of tested compounds up to 1 h, at pH 7.20 and 37 °C, under continuous stirring. Copper concentrations were determined by GFAAS. The values are mean of four separate measurements.

600 500 400 300 200 1

2

3

4

5

6

7

[complex 1] µM Fig. 10. Amount of copper bound to DNA as a function of the concentration of compound 1 added to the incubation medium. 5  106 K562 cells were incubated with different concentrations of 1 ranging from 1 to 10 lM for 2 h. After the incubation period, DNA was extracted, the DNA concentration per nucleotide (per nt) was determined by spectrophotometric analysis and the copper concentration by GFAAS. The values are mean of three separate measurements.

N.A. Rey et al. / Journal of Inorganic Biochemistry 103 (2009) 1323–1330

Cellular viability (%)

1330

105 85 65 45 25 5 control

64

32

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[1] X 10 M Fig. 11. Effect of compound 1 on macrophages viability. 2  105 cells/mL were incubated in the absence and presence of different concentrations of compound 1 for 72 h. Cell viability was assessed using the MTT tetrazolium dye reduction assay. The values are means of two independent experiments.

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