DNA interactions and anticancer screening of copper(II) complexes of N-(methylpyridin-2-yl)-amidino-O-methylurea
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Cu(II) complexes of the tridentate ligand N-(methylpyridin-2-yl)-amidino-O-methylurea (L), namely [Cu(L)Cl2] and [Cu(L)ClO4]ClO4, have been investigated for interactions with DNA by spectroscopic methods and viscosity measurements. Both complexes bind to DNA through non-intercalative interactions. [Cu(L)Cl2] (Kb = 2.81 × 105 M−1) shows similar DNA-binding potential to [Cu(L)ClO4]ClO4 (Kb = 1.57 × 105 M−1). Investigation of the chemical nuclease properties toward plasmid pBR322 DNA by gel electrophoresis and atomic force microscopy (AFM) suggests that both complexes are able to cleave the supercoiled form (Form I) to the nicked (Form II) and linear forms (Form III) through an oxidative pathway. The possible reactive oxygen species have been investigated by the use of scavengers, indicating that hydroxyl radicals may be involved in the DNA cleavage mechanism. Both of these complexes show similar activities against selected human cancer cell lines.
The synthesis of molecules which can bind to DNA and/or show nuclease properties has received much attention in recent years [1, 2, 3]. Investigations of the interactions of DNA with metal complexes provide a basis to develop novel pharmaceuticals. Transition metal complexes have been widely exploited for these purposes, not only because of their distinctive spectral and electrochemical properties, but also due to the fact that by changing the ligand environment one can tune the interactions of such complexes with DNA. Platinum-based drugs such as cisplatin have dominated the treatment of various cancers. The design of new metal complexes that can interact with DNA requires a detailed understanding of how platinum and other metals interact and process DNA.
In the current work, the DNA-binding capabilities of [Cu(L)Cl2] and [Cu(L)ClO4]ClO4 have been studied with calf thymus (CT) DNA using absorption titration, viscosity measurements, thermal denaturation experiments, competitive DNA-binding studies, circular dichroism spectroscopy and stoichiometric determination. Their nuclease activities toward plasmid pBR322 DNA were also investigated by gel electrophoresis and atomic force microscopy (AFM). Furthermore, their in vitro anticancer activities were tested against three human cancer cell lines, specifically MCF-7 (breast cancer), NCI-H187 (small cell lung cancer), and KB (oral cavity cancer), by resazurin microplate assay (REMA).
Materials and instrumentation
Disodium salt of calf thymus DNA (CT-DNA, Type I fibrous) was purchased from Sigma-Aldrich. Plasmid pBR322 DNA was obtained from Vivantis. Ethidium bromide (EB) solution (10 mg mL−1) and tris(hydroxylmethyl)aminomethane (Tris base) were obtained from Promega. Agarose (D-1, Low EEO) was purchased from Pronadisa. DMSO and t-BuOH were purchased from Riedel-Haën and Univar, respectively. KI, glycerol, MeOH, and NaN3 were obtained from Sigma-Aldrich. All reagents were of molecular biology grade and used as received. [Cu(L)Cl2] and [Cu(L)ClO4]ClO4 were prepared by the previously reported procedure [10, 15].
Electronic absorption spectra were recorded using an Agilent 8453 UV–visible spectrophotometer. Fluorescence determinations were performed on a Shimadzu RF-5301PC spectrofluorophotometer. Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter. The amount of Cu for determination of the stoichiometry was determined with a Perkin Elmer AAnalyst 100 atomic absorption spectrometer. Electrophoretic band intensities were visualized with a Bio-Rad Gel Doc 2000 system using Lab Work software. AFM images were obtained using a Nanoscope V Multimode 8 AFM (Bruker AXS) operating in the PEAK FORCE tapping mode. Commercial Si-tip on nitride lever cantilevers (SNL, Bruker) with a force constant of 0.4 N m−1 were used.
The DNA stock solution, prepared in Tris buffer (containing 5 mM Tris–HCl and 50 mM NaCl at pH 7.1), gave a UV absorbance ratio A260/A280 of 1.8–1.9 (where A260 and A280 are the absorbances of a DNA sample at 260 and 280 nm, respectively), indicating that the DNA was sufficiently free of protein concentration . The DNA stock solution was kept at 4 °C and used within 4 days. A tenfold dilution of the DNA was determined spectrophotometrically at 260 nm by using the molar extinction coefficient value of 6600 M−1 cm−1 . All experiments were carried out in Tris buffer at pH 7.1 in sterile deionized water.
Electronic absorption titrations
Viscosity measurements were carried out on an Ubbelohde viscometer, immersed in a constant temperature bath at 37.0 ± 0.1 °C. The CT-DNA concentration was kept constant (100 μM), while the complex concentration was varied in the [Complex]/[DNA] (r) ratio of 0.0–1.0 with intervals of 0.1. Flow time was measured with a digital stopwatch. Each sample was measured three times and the average flow time was calculated. The viscosity at each ratio was determined by the flow time of the DNA-containing solutions (t) corrected by the flow time of buffer alone (t0), η = t − t0. Data are presented as relative viscosity (η/η0)1/3 versus r, where η is the viscosity of DNA in the presence of the complex, and η0 is the viscosity of DNA alone.
Circular dichroism spectroscopy
CD spectra were recorded over 220–320 nm at room temperature. All experiments were done using a quartz cell of 1 cm length and a scan rate of 100 nm min−1 with a response time of 4 s. The concentration of CT-DNA in Tris buffer was kept constant at 70 μM, while the complex concentration was varied over the [Complex]/[DNA] ratio of 0.5 and 1.0.
Thermal denaturation of CT-DNA was investigated on a UV–visible spectrophotometer with Peltier temperature-controlling cell holder and increment temperature rate of 0.5 °C min−1. The absorbances of CT-DNA (100 μM) in the absence and presence of each complex in [Complex]/[DNA] ratios of 0.0, 0.1, 0.2, 0.3, and 0.4 were recorded in Tris buffer. Melting curves were plotted between the relative absorption intensity (A/A0, A0 and A are the initial and the observed absorbances at 260 nm) and temperature range from 25 to 100 °C. The melting temperature (Tm) was determined from the maximum of the first derivative or tangentially from the graph at midpoint of the transition curves. ∆Tm is defined as the difference between the Tm values of bound and free DNA.
The stoichimetry of the interactions between the complexes and DNA was determined by a procedure similar to that described in the literature . The CT-DNA solution (5 mM, 1 mL) was added to the complex solution (5 mM, 1 mL), and the resulting mixture was incubated for 24 h at 37 °C. Precipitation of the copper(II)/DNA complex was obtained upon adding absolute ethanol (4 mL) and aqueous NaCl (2 M, 0.2 mL). The solution containing the precipitate was stored at −70 °C for 1 h, and the precipitate was subsequently isolated by centrifugation at 4 °C (10,000 rpm, 30 min). The supernatant was separated by slow decantation. Finally, deionized water (25 mL) was added to dissolve the copper(II)/DNA precipitate, and the DNA concentration was calculated (from triplicate experiments) by the absorption intensity at 260 nm using ε = 6600 M−1 cm−1 . The amount of copper(II) was measured by atomic absorption spectrometry (AAS), hence providing the Cu (mmol)/DNA (mol base) ratio.
Mechanistic studies of the DNA cleavage process were carried out in the presence of H2O2 (50 µM), and typical ROS scavengers DMSO (150 µM), KI (50 µM), glycerol (100 µM), MeOH (150 µM), and t-BuOH (150 µM) were used as hydroxyl radical (OH·) scavengers and NaN3 (100 µM) was used as a singlet oxygen (1O2) scavenger. These scavengers were added to plasmid pBR322 DNA (0.1 μg) prior to addition of the complexes (250 µM). All samples were prepared in HEPES buffer and incubated at 37 °C for 2 h and then analyzed according to the procedure described above.
Atomic force microscopy (AFM)
Plasmid pBR322 DNA (0.2 µg) was heated at 60 °C for 15 min to obtain the open circular form and then incubated with the complexes (50 and 100 µM for [Cu(L)Cl2] and 50 and 150 µM for [Cu(L)ClO4]ClO4) in the absence and presence of H2Asc (100 µM) in 20 µL HEPES buffer at 37 °C for 2 h. Milli-Q water and all solutions for the AFM studies were filtered through 0.2 µM FP030/3 filters (Scheicher and Schuell GMbH, Germany) to obtain clear AFM images. After incubation, a drop (8 µL) of each sample was placed onto peeled mica disks (PELCO Mica Discs, 9.9 nm diameter; Ted Pella, Inc. California, USA) and allowed to absorb for 2 min at room temperature. The samples were rinsed for 5 s with a stream of Milli-Q water directed onto the surface and subsequently blown dry with argon before imaging.
Anticancer activity assay
Results and discussion
Electronic absorption spectroscopy is widely employed to determine the binding affinities of metal complexes with DNA. Increasing absorbance or hyperchromicity results from base unstacking. Complexes which bind to DNA through intercalation usually manifest hypochromism (decrease in absorbance) and redshift (bathochromism) due to strong stacking interactions between the complex and the DNA base pairs . A classical intercalator like ethidium bromide shows hypochromism as .
Comparison of the DNA-binding ability between the two CuN3X2 complexes in this work and the previous works
Proposed binding modes
2.81 × 105
1.57 × 105
Ethidium bromide (EB)
1.00 × 107
1.24 × 104
2.00 × 104
8.00 × 104
5.20 × 103
4.00 × 103
Fluorescence spectroscopy is often used to investigate the competitive interactions between metal complexes and the classical DNA intercalator ethidium bromide. Free EB is only weakly fluorescent, but in the presence of DNA, its fluorescence is markedly increased, with an emission band at about 600 nm, resulting from intercalation of EB between adjacent DNA base pairs. This fluorescence can be competitively quenched by the addition of a second molecule .
Although the Kb and Ksv values of both complexes are different, they have the same trend. The Kb values apply to the binding capabilities of the complexes toward free double stranded DNA while the Ksv values are a measure of their competitive binding to the EB-bound DNA.
The effects of these complexes on the viscosity of CT-DNA are shown in Fig. 4, which reveals that the relative viscosity of CT-DNA in the presence of the complexes depends upon the complex concentration. There are three effects consisting of a reduction, enhancement and steadiness in viscosity. In the first effect, a dramatic reduction in viscosity was found for [Complex]/[DNA] ratios of 0.0–0.5 for both complexes. This outcome is opposite to the case of EB which raises the DNA viscosity. Consequently, the two complexes possibly bind to DNA through non-intercalative interactions. On the other hand, the second effect was a marked increase in viscosity, in the narrow r range of 0.5–0.8 for both complexes, thus indicating that intercalative binding of the complexes may occur in this concentration range. Finally, when raising the r ratio higher than 0.8, no further change in viscosity was observed. It is possible that the interactions may be electrostatic or groove binding  or that the binding sites may reach the saturation point at higher concentrations of the complexes. The similar behaviors of both complexes imply that they exhibit the same binding behaviors.
Circular dichroism spectroscopy is a useful technique to assess whether nucleic acids undergo conformational changes as a result of complex-DNA formation. The CD spectrum of CT-DNA consists of a positive band at 277 nm due to base stacking and a negative band at 245 nm because of helicity, which is a characteristic of the right-handed B form of DNA . Changes in the CD spectrum of DNA upon the complexes may often be assigned to corresponding changes in DNA structure . In general, non-intercalative interactions of small molecules with DNA show little or no perturbation on the base stacking and helicity bands of the CD spectrum because these binding modes do not significantly influence the secondary structure of DNA. On the other hand, a classical intercalator tends to enhance the intensities of both bands due to strong base stacking interactions and stable DNA conformations (right-handed B conformation of DNA).
DNA-melting analysis and stoichiometry
To better understand the function of these complexes when interacting with DNA in terms of stabilization or destabilization of DNA strands, thermal denaturation experiments were carried out. In such experiments, the stabilizing forces of the DNA double helix are overcome by melting or denaturation. A higher or lower Tm value of the complex-bound DNA compared to free DNA suggests that the complexes stabilize or destabilize the DNA strands, respectively. Hence, the melting temperature difference (ΔTm) can suggest the possible interaction type between complexes and DNA. Intercalation of natural or synthetic organics and metallointercalators generally results in a considerable increase in melting temperature and high ΔTm value. Neyhart et al. and Kumar et al. [38, 39] showed that the intercalation into DNA can increase the stability of the helix, resulting in ΔTm values of 5–14 °C. Low ΔTm values (close to zero) are indicative of non-intercalative binding modes [40, 41]. Moreover, negative ΔTm values suggest that groove and/or electrostatic interactions may be the primary binding modes .
Thermal melting temperature (Tm) and the melting temperature difference (ΔTm = Tm (of bound to DNA) − Tm (of the free DNA)) in the presence of the [Cu(L)Cl2] and [Cu(L)ClO4]ClO4 complexes
Determination of the DNA-binding stoichiometry of the complexes, expressed as Cu (mmol)/DNA (mol base), is an additional method to obtain information regarding these interactions. The stoichiometric ratio can be determined from atomic absorption and UV–Vis spectroscopic measurements. We found that [Cu(L)Cl2] and [Cu(L)ClO4]ClO4 gave Cu (mmol)/DNA (mol base) ratios of 26 and 12, respectively. These values can be compared with the Cu (mmol)/DNA (mol base) ratios of known copper(II) compounds. For example, [Cu(H2O)6]2+ with the ratio > 150 shows poorly selective DNA binding, most likely due to the interaction of this cationic species with the negatively charged phosphate groups on the DNA backbone, while a Cu(II)-dipeptide gave a ratio < 42, suggesting a more efficient DNA interaction . The two copper compounds in the present work shows similar values to the latter, suggesting similar DNA-binding affinity.
DNA cleavage studies
The nuclease activities of the complexes were studied by gel electrophoresis using plasmid pBR322 DNA in the absence and presence of ascorbic acid (H2Asc) as a reducing agent. In general, when plasmid DNA is subjected to electrophoresis, the fastest migration will be observed for the supercoiled form (Form I). If scission occurs on one strand, the supercoil will relax to generate a slower-moving nicked circular form (Form II). If both strands are cleaved, a linear form (Form III) that migrates between Forms I and II will be obtained.
It is known that the DNA-cleaving potential of transition metal complexes can be enhanced by adding exogenous redox reagents such as hydrogen peroxide and ascorbic acid. Herein, H2Asc was used to mimic the reducing environment, found inside the cells, and will induce the formation of copper(I) species from the present complexes. Under an aerobic atmosphere, this would allow the formation of reactive oxygen species (ROS) that are able to cleave DNA. In the presence of H2Asc, the nuclease activities of both complexes were dramatically improved (Fig. 7). Upon increasing the complex concentration (Lanes 4–8 in Fig. 7), Form I plasmid DNA was converted into Form II and then Form III, initially observed at 100 µM for [Cu(L)Cl2] and 150 µM for [Cu(L)ClO4]ClO4. At a complex concentration of 250 µM in the presence of H2Asc (Lane 8), [Cu(L)Cl2] gives exclusively DNA Form III (100%), while [Cu(L)ClO4]ClO4 gives a mixture of Form II (17.3%) and Form III (82.7%). These results follow the same trend as the DNA-binding abilities of the complexes.
Cancer cell growth inhibition of [Cu(L)Cl2] and [Cu(L)ClO4]ClO4 and cisplatin
IC50 (µg mL−1)
Two copper(II) complexes of N-(methylpyridin-2-yl)-amidino-O-methylurea containing different anionic co-ligands have been investigated for biological potential. Their DNA-binding abilities occur through non-intercalative modes. They also show chemical nuclease properties via oxidative DNA cleavage. Furthermore, the anticancer activities of both complexes appear to be superior to that of cisplatin. These results indicate that the DNA-interacting properties and cytotoxicities of such complexes are related. This may come from the fact that they contain the same cationic species in solution phase, [Cu(L)(H2O)x]2+ (x = 1–3), thus revealing similar properties to our previous work . We conclude that such complexes have potential as DNA-damaging agents for use in cancer therapy.
This work was financially supported by Center of Excellence for Innovation in Chemistry (PERCH-CIC) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Communication, through the Advanced Functional Materials Cluster of Khon Kaen University. P.G. acknowledges ICREA (Institució Catalana de Recerca i Estudis Avançats), the Ministerio de Economía y Competitividad of Spain (Project CTQ2011-27929-C02-01), and the support of COST Actions CM1003 and CM1105.
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