Investigation of DNA Binding, DNA Photocleavage, topoisomerase I inhibition and antioxidant activities of Water soluble titanium(IV) phthalocyanine compounds ¨ ¨ Arzu Ozel, Burak Barut, Umit Demirbas¸, Zekeriya Biyiklioglu PII: DOI: Reference:
S1011-1344(15)30174-3 doi: 10.1016/j.jphotobiol.2016.02.005 JPB 10242
To appear in: Received date: Accepted date:
25 November 2015 1 February 2016
¨ ¨ Please cite this article as: Arzu Ozel, Burak Barut, Umit Demirba¸s, Zekeriya Biyiklioglu, Investigation of DNA Binding, DNA Photocleavage, topoisomerase I inhibition and antioxidant activities of Water soluble titanium(IV) phthalocyanine compounds, (2016), doi: 10.1016/j.jphotobiol.2016.02.005
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ACCEPTED MANUSCRIPT Investigation of DNA Binding, DNA Photocleavage, Topoisomerase I Inhibition and Antioxidant Activities of Water Soluble Titanium(IV) Phthalocyanine
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Compounds
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Arzu Özela*, Burak Baruta, Ümit Demirbaşb, Zekeriya Biyiklioglub
Karadeniz Technical University, Faculty of Pharmacy, Department of Biochemistry,
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Karadeniz Technical University, Faculty of Science, Department of Chemistry, Trabzon,
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TURKEY
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b
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Trabzon, TURKEY
*Corresponding author: Tel: +90 462 377 88 18; Fax: +90 462 377 57 62 E-mail address: [email protected]
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ABSTRACT
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The bindig mode of water soluble peripherally tetra-substituted titanium(IV)
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phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal
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denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2
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and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-
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mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650 nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of
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supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation
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of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20 µM concentration. A series of antioxidant assays, including 2,2-diphenyl-1picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1,
ACCEPTED MANUSCRIPT Pc2 and Pc3 compounds are suitable agents for photo dynamic theraphy.
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Keywords: phthalocyanines, DNA-binding, DNA-cleavage, topoisomerase I
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inhibiton, antioxidant.
1. INTRODUCTION
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DNA is an important target for treating genetic diseases most notably cancer. The
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biological investigation of transition metal complexes as anticancer drugs have been increased in recent years because of their key impact in clinical therapy [1]. Mode of DNA interaction of a certain possible drug provides preliminary information about the
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possibility of its use as anticancer agent [2]. In that regard the versatile spectral and electrochemical properties of transition metal complexes enhance their DNA binding
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and cleavage activities [3, 4].
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Topoisomerases are essential enzymes in DNA replication, transcription, recombination, intensification and separation of chromosome both in eukaryotes and prokaryotes [5]. Topoisomerase I can break a single of DNA strand while topoisomerase II can break a double of DNA strands. It is well known that some of the anticancer drugs used clinically are involved in inhibition of topoisomerases such as daunomycin, bleomycin [6].
Over production of activated oxygen species generated by normal metabolic process, main contributes to oxidative damages of biomolecules such as DNA, lipids and proteins. Therefore, some diseases such as cancer, aging, inflammation,
ACCEPTED MANUSCRIPT cardiovascular and neurodegenerative are also accelerated. Depending on their structure
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and on the source of the oxidative stress, metal complexes can act as antioxidants [7].
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Because of their 18-π electron structure and high thermal and chemical stability
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phthalocyanines can be used in the various field such as chemical sensor [8], solar cells [9], semiconductors [10], liquid crystals [11], optical storage devices [12], dyes [13], catalyst [14]. In addition, phthalocyanine compounds have been investigated as
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photodynamic therapy agents in the cancer treatment due to their photosensitizer and
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antioxidant properties [15-17]. Water soluble metallophthalocyanines that are capable of binding and cleaving DNA have attracted considerable interest over the last years for their potential application in biotechnology, therapeutic approaches and the study of
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nucleic acid conformations [18-24]. And then, a variety of increasingly effective titanium compounds have been developed for use as chemotherapeutic agents [25].
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However, no attention has yet been given to titanium phthalocyanine compounds in this area. Considering the promising anticancer properties of phthalocyanines and titanium
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compounds, it seemed reasonable to investigate the titanium compounds with phthalocyanines.
In this study, we investigated DNA-binding, DNA-cleavage, topoisomerase I inhibition and antioxidant properties of three different water soluble peripherally tetrasubstituted titanium(IV) phthalocyanines using absorption spectroscopy, thermal denaturation, gel electrophesis.
2. MATERIALS AND METHODS
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Materials and Equipments
ninato
oxotitanium(IV)iodide
(Pc1),
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(dimethylamino)phenoxy]ethoxy}phthalocya
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2(3),9(10),16(17),23(24)-tetrakis-{2-[3-
oxotitanium(IV)iodide(Pc2)
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2(3),9(10),16(17),23(24)-tetrakis-{2[3(diethylamino)phenoxy]ethoxy}phthalocyaninato and
2(3),9(10),16(17),23(24)-tetrakis-(2-{2-[3-
(dimethylamino)phenoxy]ethoxy}ethoxy)-phthalocyaninato
oxotitanium(IV)iodide
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(Pc3) were synthesized by Z. Biyiklioglu [26, 27]. Disodium salt of CT DNA, ethidium
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bromide (EB), agarose, bromophenol blue, sodium dodecyl sulfate, xylene cyanol, glycerol, gallic acid, butylhydroxyanisole (BHA), nitro blue tetrazolium (NBT), Lmethionine, riboflavin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), bovine serum albumin
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(BSA), disodium ethylenediaminetetraacetate dihidrate (EDTA) were purchased from Sigma-Aldrich. Topoisomerase I Human Assay Kit was purchased from Topogen.
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Supercoiled plasmid pBR322 DNA was obtained from Fermantas.
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Studies of DNA binding and antioxidant activities were performed by Perkin Elmer Lambda 25 UV-Vis spectrophotometer. Electrophoresis images were photographed with BioRad Gel Doc XR system for DNA cleavage studies. Photocleavage studies were performed using a General Electric quartz line lamp (300 W). A 650 nm glass cut off filter (Schott) and a water filter were used to filter out ultraviolet and infrared radiations, respectively. Thermal denaturation studies were carried out using Cary 100 Bio UV-Visible Spectrophotometer (containing Varian Cary Temperature Controller).
2.2.
DNA Binding Studies
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The stock solutions of Pc compounds were prepared in water and stored at
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room temperature. The stock solution of CT-DNA was prepared in 5 mM Tris-HCl
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buffer (containing 50 mM NaCl, pH 7.2) followed by exhaustive stirring for three days
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and kept at 4 °C up to a week. UV absorbance ratio of the DNA solution at 260 and 280 nm (A260/A280) was 1.8-1.9 indicating that the DNA solution was protein free. The DNA concentration was measured by UV absorbance at 260 nm using the molar extinction
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coefficient (ɛ) of 6600 M-1 cm-1. To determine the binding of Pc compounds to DNA,
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absorption spectra of Pc compounds (37.5 μM) without DNA were recorded first, and then changes in the absorption spectrum were monitored following the addition and 10 min equilibration with various concentrations of DNA (2.5-20 μM). For the Pc
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compounds, the binding constants (Kb) were determined from the spectroscopic titration
(F. 1)
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data using the following equation [28]:
Where the apparent absorption coefficient Ɛa, Ɛf and Ɛb correspond to
Aobsd/[Compound], the extinction coefficient of the free compound and the extinction coefficient of the compound when fully bound to DNA, respectively. In plots of [DNA]/( Ɛa−Ɛf) versus [DNA], Kb is given by the ratio of slope to the intercept.
2.3.
Thermal Denaturation Studies
Thermal melting denaturation were examined for CT-DNA (60 µM) and Pc compounds (60 µM) in buffer (5 mM Tris-HCl / 50 mM NaCl pH 7.2) heating from 60-
ACCEPTED MANUSCRIPT 90 °C at rate of 1 °C/min, recording the UV absorbance at 260 nm every 0.5 °C using
DNA-Photocleavage Studies
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thermal melting programme [29].
The DNA cleavage activity of Pc compounds were evaluated in the absence and presence of irradiation at 650 nm by their ability to catalyze the conversion of the
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supercoiled pBR322 DNA (Form I) to nicked circular DNA (Form II) and linear DNA
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(Form III) using agarose gel electrophoresis. Total volume of reaction mixtures were 10 µL and contained Tris-HCl buffer (pH 7.0), supercoiled plasmid pBR322 DNA (250 ng) and Pc compounds (20 μM). Then irradiated 10 min at 650 nm and incubated at 37 ºC
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for 1 hour. The reaction was stopped by adding 5 µL of loading buffer (0.2% bromophenol blue, 4.5% sodium dodecyl sulfate, 0.2% xylene cyanol, 30% glycerol),
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samples were then loaded on 0.8% agarose gel containing EB (1 mg/ml in TAE (Trisacetate-EDTA), electrophoresis was carried out at 100 V for 90 min and resulting image
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was visualized with BioRad Gel Doc XR system [30].
DNA cleavage activities were also tested in the presence of oxidizing agents such
as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method as described above at irradiation 650 nm.
2.5.
Topoisomerase I Inhibition Assay
The topoisomerase I inhibition activities of Pc compounds were measured as relaxation of supercoiled plasmid DNA using agarose gel electrophoresis. Total volume
ACCEPTED MANUSCRIPT of reaction mixtures were 10 µL and contained 35 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM DTT, 2 mM spermidine, 0.1 mg/mL BSA, supercoiled pBR322
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DNA (250 ng), 1 Unit topoisomerase I and Pc compounds (20 µM). These reaction
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mixtures were incubated at 37 ºC for 30 min. After the reaction was stopped by adding
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5 µL of loading buffer (0.2% bromophenol blue, 4.5% sodium dodecyl sulfate, 0.2% xylene cyanol, 30% glycerol) samples were loaded on 0.8% agarose gel containing EB (1 mg/mL in TAE (Tris-acetate-EDTA). Electrophoresis was carried out at 45 V for 180
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min and resulting image was visualized with BioRad Gel Doc XR system [31].
Superoxide Radical Scavenging Assay
enzymatic
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Superoxide scavenging activities of the Pc compounds were tested in a nonsuperoxide
radicals
(O2−•)
generation
assay
using
a
modified
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spectrophotometrical NBT photoreduction method [32]. Total volume of assay mixtures were 1 mL and contained riboflavin (2 µM), methionine (13 mM), NBT (75 mM),
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EDTA (0.1 mM), and Pc compounds (50 mM in phosphate buffer, pH 7.8). After illuminating with a fluorescent lamp at 30 ºC for 10 min, the absorbance of the samples (Asample) were measured at 560 nm. Assay mixture without the compounds (Pc) were used as a control (control absorbance, Acontrol). All experiments were in triplicates and results were expressed as the mean ± standard deviation (S.D.). BHA was used as a positive control. Free O2−•radical scavenging effect was calculated using the following Formula 2:
(F. 2)
ACCEPTED MANUSCRIPT 2.7.
DPPH Radical Scavenging Assay
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Pc compounds were tested for in vitro antioxidant activities by DPPH free radical
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scavenging assay method [33]. Total volume of assay mixtures were 1 mL and
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contained methanolic DPPH solution (0.1 mM) and different concentrations of Pc compounds. After incubation in the dark at room temperature for 30 min, absorbance of the samples (Asample) were measured at 517 nm. Assay mixture without Pc compounds
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were used as a control (control absorbance, Acontrol). All experiments were in triplicates
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and results were expressed as the mean ± standard deviation (S.D.). Gallic acid was used as a positive control. Free radical scavenging effect was calculated using the
Metal Chelating Effects Assay
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2.8.
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Formula 2.
Metal chelating activities of the Pc compounds were examined using the method
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described [7] compared to EDTA as the reference compound. 500 µL of different concentrations (25-100 μM) of compounds were prepared and added to 2 mM 50 µL FeCl2, 5 mM 100 μL ferrozine. Then the reaction was incubated at room temperature for 10 min and the absorbance of the samples (Asample) were measured at 562 nm. Assay mixture without the compounds (Pcs) were used as a control (control absorbance, Acontrol). All experiments were in triplicates and results were expressed as the mean ± standard deviation (S.D.). Metal chelating effect was calculated using the Formula 2.
3. RESULTS AND DISCUSSION
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Determination of Binding of Pc compounds with CT-DNA
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The absorption spectra of the Pc compounds in the absence and presence of
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CT-DNA were performed to determine the binding mode of DNA with Pc compounds.
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A compound can bind to DNA via covalent or noncovalent (intercalation, electrostatic or groove binding) interactions. In general, DNA intercalative binder agents cause large shifts at wavelength because of π-stacking interactions between their aromatic
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chromophore groups while DNA groove binder or stacking agents trigger small shifts in
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absorbances and wavelengths in spectra [18].
The absorpstion spectra of the Pc compounds and Pc compounds-DNA were
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recorded between the range of 300-750 nm. The absorption spectra of Pc1, Pc2 and Pc3 (Fig2., Fig3., Fig4.) showed both hypochromic and small red shifts in the presence of
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DNA. Upon increasing the concentration of CT-DNA to the Pc1, Pc2 and Pc3 was observed with small red-shift of 2 nm (λmax 647 → 649) of Pc1 and small red-shift of 1
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nm (λmax 645 → 646) of Pc2 and no shift for Pc3. The hypochromic and small red shifts support non-intercalative binding mode between Pc compounds and DNA. The apparent binding constant (Kb) values of Pc1, Pc2 and Pc3 were determined as 3.75x104, 2.3x104, 4.57x104, respectively. These values are lower than the apparent binding constant (Kb) normally associated with intercalation (Kb < 106) [34]. These results indicates that Pc compounds represent non-intercalative binding mode.
3.2.
Thermal Denaturation Studies
ACCEPTED MANUSCRIPT The thermal melting temperature (Tm) is the temperature that half of the DNA strands are separated into single strand [35]. Tm value is very informative about stability
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of the DNA double helix with temperature. Increasing in the Tm values indicate the
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strength of interaction between DNA and metal complexes [36]. The Tm values can be
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used to determine binding mode of DNA-complexes as intercalative or external. General, in the presence of classical intercalation Tm value increases about 8-12 ˚C [37]. On the other hand, groove binders do not trigger a remarkable change in Tm value [38].
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In this study, Tm values of CT-DNA in the absence and presence of Pc compounds were
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determined by monitoring the absorbance of DNA at 260 nm as a function of temperature. As shown Table 1, the Tm value of CT-DNA in the absence of any Pc compounds were found 80.2 ˚C while Tm values of CT-DNA in the presence of Pc1, Pc2
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and Pc3 were found 81.7 ˚C, 82.2 ˚C, 82.9 ˚C respectively. This small changes in Tm
DNA-Photocleavage Studies
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values indicated the groove binding between CT-DNA and Pc1, Pc2 and Pc3.
Pc compounds contain metal ions are well suited for application as
metallonucleases, because of their possibility to tune the redox potential through the choice of proper metal ion [39]. Interactions of Pc compounds with supercoiled pBR322 DNA were investigated using agarose gel electrophoresis. It has been well known when circular plasmid DNA is subjected to electrophoresis, the fastest migrating is the supercoiled Form I, the slowest moving is the open circular Form II and the linear Form III runs in between the other two forms [40]. When one strand is cleaved, the supercoils relax to generate a slower-moving open circular Form II and both strands are cleaved, Form III is generated that migrates between Form I and Form II [30] .
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In our preliminary studies, the cleavage experiments were carried out in the
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absence and presence of irridiation at 20 µM concentration of Pc compounds. The
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results were shown Fig. 5. It was observed that all of Pc compounds exhibited
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remarkable cleavage activities in the presence of irradiation at 650 nm. Therefore, all of these studies were performed in the presence of irradiation at 650 nm.
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The nuclease effiency of a compound mainly depends on the activators used to
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initiate the DNA cleavage [41]. As shown Fig. 6. at irradiation of wavelength 650 nm in the presence of oxidizing agents (hydrogen peroxide (H2O2), ascorbic acid (AA), 2mercaptoethanol(ME)) Pc compounds exhibited more effective cleavage activity to
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convert the supercoiled DNA into the open circular and linear forms than in the absence of oxidizing agents. All of the Pc compounds showed more DNA cleavage activity in
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the presence of the cleavage activators. The nuclease effiency of Pc compounds in the presence of the cleavage activators were followed as H2O2>AA>ME, respectively.
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These results indicated that in presence of irradiation at 650 nm and oxidizing agents Pc1, Pc2 and Pc3 can be suitable agents for cleavage of DNA.
3.4.
Topoisomerase I Inhibition Assay
It is well known that topoisomerase I can affect topological changes of DNA by transiently cleaving one DNA strand and leads to relaxation of the supercoiled DNA Form III to open circular DNA Form I and lineer DNA Form II [42]. When topoisomerase I enzyme retains its catalytic activity, the disappearance of Form I and the appeareance of bands for Form II and Form I will be observed [31].
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In this study, the inhibiton activities of all Pc compounds were investigated
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against topoisomerase I by pBR3222 DNA relaxation assay. At the concentration of 20
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μM, Pc compounds exhibited remarkable topoisomerase I inhibitory activity (Fig. 7.).
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Pc3 showed the highest and Pc1 showed the lowest inhibition activity. These results showed that Pc compounds have potential as anticancer agents because of their
Superoxide Radical Scavenging Assay
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3.5.
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topoisomerase I inhibition activities.
Oxidation is important living organisms for generation of energy in biological
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proceses. However reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anions are precursors to active free radicals have generated reacting with
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biological macromolecules including fatty acid, DNA, proteins and thereby causing tissue damage [44]. Therefore, superoxide radical scavenging compounds are essential
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substances with ability to protect cells against damage various disseases. In this study, The SOD radical scavenging activities of Pc compounds were determined antioxidative behaviour in the riboflavin-NBT system at 560 nm and compared to BHA.
The stock solution of the Pc compounds were prepared in water at 1 mM and their superoxide scavenging activity tested at 25, 50, 75, 100 μM concentrations. The SOD activities of the Pc compounds were investigated by NBT assay and the results were shown in Table 2. Pc1 showed the highest activity (85.01 ± 0.15%) at 100 μM. The order of superoxide radical scavenging activity of compounds and refercence compound were found to Pc1 > Pc3 > Pc2 > BHA at 100 μM. According to the results
ACCEPTED MANUSCRIPT Pc compounds exhibited excellent activities for superoxide radical scavenging assay.
DPPH Radical Scavenging Assay
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The radicals are inactivated by the antioxidants by their free-radical scavenging or hydrogen donation abilities [45]. DPPH is a stable nitrogen-centered free radical and is
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well known scavenger used in antioxidant assay because of fast and simple method [46].
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The radical scavenging activities of Pc compounds in comparison with gallic acid were determined by DPPH assay and the results were shown in Table 3. The stock solution of the Pc compounds were prepared in water at 1 mM and their DPPH radical
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scavenginig activity tested at 25, 50, 75, 100 μM concentrations. As shown Table 3, of DPPH free radical scavenging values of Pc1 ranged from 13.49 ± 0.40% (25 μM) to
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32.73 ± 0.90% (100 μM). Pc2 compound showed poor DPPH free radical scavenging activity at all of the concentration (5.12 ± 0.10%, 11.64 ± 0.21%, 18.65 ± 0.09%, 30.05 Pc3 exhibited the highest DPPH free radical scavenging
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± 0.13%, respectively).
activitiy from 16.71 ± 0.32% to 43.14 ± 0.30% inhibiton from 25 μM to 100 μM of compound. Gallic acid showed more DPPH free radical scavenging activity than all of Pc compounds.
3.7.
Metal Chelating Effects Assay
Transtions metals are essential elements to provide enzyme activity in the human body. But at the same time unpaired electrons can react quickly with peroxides and form alkoxyl radicals. Therefore, the chelation of transition metals by antioxidants can
ACCEPTED MANUSCRIPT be considered an important mechanism in the oxidation process [47]. In this work, metal
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chelating activities were determined compared to EDTA as the reference compound.
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The stock solution of the Pc compounds were prepared in water at 1 mM and
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their metal chelating activity were tested at 25, 50, 75, 100 μM concentrations. As shown Table 4 the order of metal chelating activity of compounds were found Pc3 > Pc1 > Pc2 at all of the concentrations. When the concentrations of Pc3 was increased
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from 25 µM to 100 µM, metal chelating activities were increased from 10.7 ± 0.19% to
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36.83 ± 0.13%. It was found that Pc1 had 33.07 ± 0.11% activity at 100 µM while Pc2 had 30.65 ± 0.04% activity at 100 µM. These results showed that all of Pc compounds
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have lower chelating activities than EDTA as standart compound.
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4. CONCLUSION
In this work the DNA binding of Pc compounds were determined with UV-Vis
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spectroscopy and thermal melting denaturation studies. The apparent binding constant (Kb) of Pc compounds and thermal melting denaturation studies exhibited that Pc compounds bind to calf thymus DNA via minor groove binding. The DNAphotocleavage studies showed that Pc compounds have strong cleavage activity to DNA in presence of irradiation at 650 nm. The topoisomerase I inhibition studies of Pc compounds showed that they have strong inhibitory activity, especially Pc3 compound. The antioxidant activity investigation of Pc compounds showed that they have moderate DPPH radical scavenging activity, metal chelating effects and significant superoxide radical scavenging activity. All of these results exhibited that Pc compounds can be suitable agent for the cancer treatment especially photodynamic therapy because of their
ACCEPTED MANUSCRIPT appreciable cleavage and inhibition of topoisomerase I activities to plasmid pBR322
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DNA.
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5. ACKNOWLEDGMENTS
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We thank Sena F. Sezen, PhD (Department of Pharmacology, Facult of Pharmacy,
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Karadeniz Technical University, Trabzon, Turkey) for critical reading for manuscript.
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heterocyclic bases, Polyhedron 27 (2008) 1343-1352.
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CT-DNA
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Table 1. Tm values of CT-DNA in the absence and presence of Pc compounds.
81.7 ˚C
CT DNA + Pc2
82.2 ˚C
CT DNA + Pc3
82.9 ˚C
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CT DNA + Pc1
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Tm values 80.2 ˚C
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Table 2. Superoxide radical scavenging activity (%) of the Pc compounds. μM 25
Pc1 55.71 ± 0.35
Pc2 40.41 ± 0.30
50
64.79 ± 0.15
55.55 ± 0.19
75
75.35 ± 0.26
100
85.01 ± 0.15
BHA 17.18 ± 0.30
70.73 ± 0.36
32.11 ± 0.35
67.64 ± 0.24
72.67 ± 0.32
46.65 ± 0.32
70.43 ± 0.14
77.49 ± 0.27
59.16 ± 0.24
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Pc3 55.77 ± 0.29
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Table 3. Radical-scavenging activity on DPPH radicals (%) of the compounds. μM 25
Pc1 13.49 ± 0.40
Pc2 5.12 ± 0.10
50
20.13 ± 0.41
11.64 ± 0.21
75
23.70 ± 0.44
18.65 ± 0.09
37.14 ± 0.11
81.02 ± 0.03
100
32.73 ± 0.90
30.05 ± 0.13
43.14 ± 0.30
83.16 ± 0.06
GA 71.70 ± 0.07
23.58 ± 0.17
77.44 ± 0.13
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Pc3 16.71 ± 0.32
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Table 4. Metal chelating effect (%) of compounds on ferrous ions. Pc1
Pc2
Pc3
EDTA
25
6.17 ± 0.17
3.31 ± 0.81
10.70 ± 0.19
16.92 ± 0.10
50
11.56 ±0.43
9.83 ± 0.15
16.97 ± 0.04
48.19 ± 0.09
75
26.24 ±0.66
21.04 ± 0.46
21.19 ± 0.12
84.10 ± 0.20
100
33.07 ±0.11
30.65 ± 0.04
36.83 ± 0.13
95.70 ± 0.13
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µM
ACCEPTED MANUSCRIPT FIGURE CAPTION Fig. 1. Chemical structures of Pc1, Pc2 and Pc3.
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Fig. 2. Absorption spectrum of Pc1 upon increasing amounts of CT-DNA.
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Fig. 3. Absorption spectrum of Pc2 upon increasing amounts of CT-DNA.
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Fig. 4. Absorption spectrum of Pc3 upon increasing amounts of CT-DNA. Fig. 5. Agarose gel electrophoresis of pBR322 DNA (250 ng) in the absence and presence of light Pc compounds 50 mM Tris-HCl buffer (pH 7.0).
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Fig. 6. Oxidative pBR322 DNA (250 ng) cleavage in the absence and presence of
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different agents.
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Fig. 7. The inhibition activity of topoisomerase I of Pc compounds.
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Fig. 1. Chemical structure of Pc1, Pc2 and Pc3.
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Fig. 2. Absorption spectrum of Pc1 upon increasing amounts of CT-DNA.
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Fig. 3. Absorption spectrum of Pc2 upon increasing amounts of CT-DNA.
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Fig. 4. Absorption spectrum of Pc3 upon increasing amounts of CT-DNA.
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Fig. 5. Agarose gel electrophoresis of pBR322 DNA (250 ng) in the absence and presence of light Pc compounds 50 mM Tris-HCl buffer (pH 7.0). Lane 1, DNA control; lane 2, DNA + Pc1 (20 μM); lane 3, DNA + Pc2 (20 μM);
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lane 4, DNA + Pc3 (20 μM); lane 5, DNA control + 10 min irradiation; lane 6,
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DNA + Pc1 (20 μM) + 10 min irradiation; lane 7, DNA + Pc2 (20 μM) + 10 min
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irradiation ; lane 8, DNA + Pc3 (20 μM) + 10 min irradiation.
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Fig. 6. Oxidative pBR322 DNA (250 ng) cleavage in the absence and presence of
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different agents.
Lane 1: DNA control + 10 min. irradiation; lane 2: DNA + Pc1 (20 μM) + 10 min
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irradiation ; lane 3: DNA + Pc2 (20 μM) + 10 min irradiation; lane 4: DNA + Pc3 (20 μM) + 10 min irradiation; lane 5: DNA + H2O2 (0.4 M) + 10 min irradiation;
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lane 6: DNA + AA (0.25 mM) + 10 min irradiation; lane 7: DNA + ME (0.4 M) + 10 min irradiation; lane 8: DNA + Pc1 (20 μM) + H2O2 (0.4 M) + 10 min
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irradiation ; lane 9: DNA + Pc2 (20 μM) + H2O2 (0.4 M) + 10 min irradiation;
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lane 10: DNA + Pc3 (20 μM) + H2O2 (0.4 M) + 10 min irradiation; lane 11: DNA + Pc1 (20 μM) + AA (0.25 mM) + 10 min irradiation; lane 12: DNA + Pc2 (20
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μM) + AA (0.25 mM) + 10 min irradiation; lane 13: DNA + Pc3 (20 μM) + AA (0.25 mM) + 10 min irradiation; lane 14: DNA + Pc1 (20 μM) + ME (0.4 M) + 10
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min irradiation; lane 15: DNA + Pc2 (20 μM) + ME (0.4 M) + 10 min irradiation; lane 16: DNA + Pc3 (20 μM) + ME (0.4 M) + 10 min irradiation
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Fig. 7. The inhibition activity of topoisomerase I of Pc compounds.
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Lane 1: DNA control; lane 2: DNA + 1 Unit topoisomerase; lane 3: DNA + 1 Unit topoisomerase + Pc1 (20 μM); lane 4 : DNA + 1 Unit topoisomerase + Pc2 (20
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μM); lane 5: DNA + 1 Unit topoisomerase + Pc3 (20 μM)
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Graphical abstract
ACCEPTED MANUSCRIPT Highlights 1. The Kb of Pc compounds were 3.75x104, 2.3x104 and 4.57x104. 2. Pc compounds exhibited significant inhibitory effects against topoisomerase I.
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3. These phthalocyanine compounds bind to CT-DNA via minor groove binding.
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4. Pc3 compound had the highest DNA binding affinity and DNA cleavage activity.
S1011-1344(15)30174-3 doi: 10.1016/j.jphotobiol.2016.02.005 JPB 10242
To appear in: Received date: Accepted date:
25 November 2015 1 February 2016
¨ ¨ Please cite this article as: Arzu Ozel, Burak Barut, Umit Demirba¸s, Zekeriya Biyiklioglu, Investigation of DNA Binding, DNA Photocleavage, topoisomerase I inhibition and antioxidant activities of Water soluble titanium(IV) phthalocyanine compounds, (2016), doi: 10.1016/j.jphotobiol.2016.02.005
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ACCEPTED MANUSCRIPT Investigation of DNA Binding, DNA Photocleavage, Topoisomerase I Inhibition and Antioxidant Activities of Water Soluble Titanium(IV) Phthalocyanine
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Compounds
a
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Arzu Özela*, Burak Baruta, Ümit Demirbaşb, Zekeriya Biyiklioglub
Karadeniz Technical University, Faculty of Pharmacy, Department of Biochemistry,
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Karadeniz Technical University, Faculty of Science, Department of Chemistry, Trabzon,
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TURKEY
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b
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Trabzon, TURKEY
*Corresponding author: Tel: +90 462 377 88 18; Fax: +90 462 377 57 62 E-mail address: [email protected]
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ABSTRACT
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The bindig mode of water soluble peripherally tetra-substituted titanium(IV)
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phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal
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denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2
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and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-
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mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650 nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of
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supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation
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of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20 µM concentration. A series of antioxidant assays, including 2,2-diphenyl-1picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1,
ACCEPTED MANUSCRIPT Pc2 and Pc3 compounds are suitable agents for photo dynamic theraphy.
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Keywords: phthalocyanines, DNA-binding, DNA-cleavage, topoisomerase I
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inhibiton, antioxidant.
1. INTRODUCTION
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DNA is an important target for treating genetic diseases most notably cancer. The
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biological investigation of transition metal complexes as anticancer drugs have been increased in recent years because of their key impact in clinical therapy [1]. Mode of DNA interaction of a certain possible drug provides preliminary information about the
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possibility of its use as anticancer agent [2]. In that regard the versatile spectral and electrochemical properties of transition metal complexes enhance their DNA binding
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and cleavage activities [3, 4].
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Topoisomerases are essential enzymes in DNA replication, transcription, recombination, intensification and separation of chromosome both in eukaryotes and prokaryotes [5]. Topoisomerase I can break a single of DNA strand while topoisomerase II can break a double of DNA strands. It is well known that some of the anticancer drugs used clinically are involved in inhibition of topoisomerases such as daunomycin, bleomycin [6].
Over production of activated oxygen species generated by normal metabolic process, main contributes to oxidative damages of biomolecules such as DNA, lipids and proteins. Therefore, some diseases such as cancer, aging, inflammation,
ACCEPTED MANUSCRIPT cardiovascular and neurodegenerative are also accelerated. Depending on their structure
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and on the source of the oxidative stress, metal complexes can act as antioxidants [7].
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Because of their 18-π electron structure and high thermal and chemical stability
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phthalocyanines can be used in the various field such as chemical sensor [8], solar cells [9], semiconductors [10], liquid crystals [11], optical storage devices [12], dyes [13], catalyst [14]. In addition, phthalocyanine compounds have been investigated as
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photodynamic therapy agents in the cancer treatment due to their photosensitizer and
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antioxidant properties [15-17]. Water soluble metallophthalocyanines that are capable of binding and cleaving DNA have attracted considerable interest over the last years for their potential application in biotechnology, therapeutic approaches and the study of
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nucleic acid conformations [18-24]. And then, a variety of increasingly effective titanium compounds have been developed for use as chemotherapeutic agents [25].
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However, no attention has yet been given to titanium phthalocyanine compounds in this area. Considering the promising anticancer properties of phthalocyanines and titanium
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compounds, it seemed reasonable to investigate the titanium compounds with phthalocyanines.
In this study, we investigated DNA-binding, DNA-cleavage, topoisomerase I inhibition and antioxidant properties of three different water soluble peripherally tetrasubstituted titanium(IV) phthalocyanines using absorption spectroscopy, thermal denaturation, gel electrophesis.
2. MATERIALS AND METHODS
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Materials and Equipments
ninato
oxotitanium(IV)iodide
(Pc1),
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(dimethylamino)phenoxy]ethoxy}phthalocya
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2(3),9(10),16(17),23(24)-tetrakis-{2-[3-
oxotitanium(IV)iodide(Pc2)
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2(3),9(10),16(17),23(24)-tetrakis-{2[3(diethylamino)phenoxy]ethoxy}phthalocyaninato and
2(3),9(10),16(17),23(24)-tetrakis-(2-{2-[3-
(dimethylamino)phenoxy]ethoxy}ethoxy)-phthalocyaninato
oxotitanium(IV)iodide
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(Pc3) were synthesized by Z. Biyiklioglu [26, 27]. Disodium salt of CT DNA, ethidium
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bromide (EB), agarose, bromophenol blue, sodium dodecyl sulfate, xylene cyanol, glycerol, gallic acid, butylhydroxyanisole (BHA), nitro blue tetrazolium (NBT), Lmethionine, riboflavin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), bovine serum albumin
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(BSA), disodium ethylenediaminetetraacetate dihidrate (EDTA) were purchased from Sigma-Aldrich. Topoisomerase I Human Assay Kit was purchased from Topogen.
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Supercoiled plasmid pBR322 DNA was obtained from Fermantas.
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Studies of DNA binding and antioxidant activities were performed by Perkin Elmer Lambda 25 UV-Vis spectrophotometer. Electrophoresis images were photographed with BioRad Gel Doc XR system for DNA cleavage studies. Photocleavage studies were performed using a General Electric quartz line lamp (300 W). A 650 nm glass cut off filter (Schott) and a water filter were used to filter out ultraviolet and infrared radiations, respectively. Thermal denaturation studies were carried out using Cary 100 Bio UV-Visible Spectrophotometer (containing Varian Cary Temperature Controller).
2.2.
DNA Binding Studies
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The stock solutions of Pc compounds were prepared in water and stored at
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room temperature. The stock solution of CT-DNA was prepared in 5 mM Tris-HCl
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buffer (containing 50 mM NaCl, pH 7.2) followed by exhaustive stirring for three days
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and kept at 4 °C up to a week. UV absorbance ratio of the DNA solution at 260 and 280 nm (A260/A280) was 1.8-1.9 indicating that the DNA solution was protein free. The DNA concentration was measured by UV absorbance at 260 nm using the molar extinction
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coefficient (ɛ) of 6600 M-1 cm-1. To determine the binding of Pc compounds to DNA,
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absorption spectra of Pc compounds (37.5 μM) without DNA were recorded first, and then changes in the absorption spectrum were monitored following the addition and 10 min equilibration with various concentrations of DNA (2.5-20 μM). For the Pc
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compounds, the binding constants (Kb) were determined from the spectroscopic titration
(F. 1)
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data using the following equation [28]:
Where the apparent absorption coefficient Ɛa, Ɛf and Ɛb correspond to
Aobsd/[Compound], the extinction coefficient of the free compound and the extinction coefficient of the compound when fully bound to DNA, respectively. In plots of [DNA]/( Ɛa−Ɛf) versus [DNA], Kb is given by the ratio of slope to the intercept.
2.3.
Thermal Denaturation Studies
Thermal melting denaturation were examined for CT-DNA (60 µM) and Pc compounds (60 µM) in buffer (5 mM Tris-HCl / 50 mM NaCl pH 7.2) heating from 60-
ACCEPTED MANUSCRIPT 90 °C at rate of 1 °C/min, recording the UV absorbance at 260 nm every 0.5 °C using
DNA-Photocleavage Studies
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2.4.
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thermal melting programme [29].
The DNA cleavage activity of Pc compounds were evaluated in the absence and presence of irradiation at 650 nm by their ability to catalyze the conversion of the
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supercoiled pBR322 DNA (Form I) to nicked circular DNA (Form II) and linear DNA
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(Form III) using agarose gel electrophoresis. Total volume of reaction mixtures were 10 µL and contained Tris-HCl buffer (pH 7.0), supercoiled plasmid pBR322 DNA (250 ng) and Pc compounds (20 μM). Then irradiated 10 min at 650 nm and incubated at 37 ºC
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for 1 hour. The reaction was stopped by adding 5 µL of loading buffer (0.2% bromophenol blue, 4.5% sodium dodecyl sulfate, 0.2% xylene cyanol, 30% glycerol),
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samples were then loaded on 0.8% agarose gel containing EB (1 mg/ml in TAE (Trisacetate-EDTA), electrophoresis was carried out at 100 V for 90 min and resulting image
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was visualized with BioRad Gel Doc XR system [30].
DNA cleavage activities were also tested in the presence of oxidizing agents such
as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method as described above at irradiation 650 nm.
2.5.
Topoisomerase I Inhibition Assay
The topoisomerase I inhibition activities of Pc compounds were measured as relaxation of supercoiled plasmid DNA using agarose gel electrophoresis. Total volume
ACCEPTED MANUSCRIPT of reaction mixtures were 10 µL and contained 35 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM DTT, 2 mM spermidine, 0.1 mg/mL BSA, supercoiled pBR322
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DNA (250 ng), 1 Unit topoisomerase I and Pc compounds (20 µM). These reaction
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mixtures were incubated at 37 ºC for 30 min. After the reaction was stopped by adding
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5 µL of loading buffer (0.2% bromophenol blue, 4.5% sodium dodecyl sulfate, 0.2% xylene cyanol, 30% glycerol) samples were loaded on 0.8% agarose gel containing EB (1 mg/mL in TAE (Tris-acetate-EDTA). Electrophoresis was carried out at 45 V for 180
2.6.
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min and resulting image was visualized with BioRad Gel Doc XR system [31].
Superoxide Radical Scavenging Assay
enzymatic
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Superoxide scavenging activities of the Pc compounds were tested in a nonsuperoxide
radicals
(O2−•)
generation
assay
using
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modified
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spectrophotometrical NBT photoreduction method [32]. Total volume of assay mixtures were 1 mL and contained riboflavin (2 µM), methionine (13 mM), NBT (75 mM),
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EDTA (0.1 mM), and Pc compounds (50 mM in phosphate buffer, pH 7.8). After illuminating with a fluorescent lamp at 30 ºC for 10 min, the absorbance of the samples (Asample) were measured at 560 nm. Assay mixture without the compounds (Pc) were used as a control (control absorbance, Acontrol). All experiments were in triplicates and results were expressed as the mean ± standard deviation (S.D.). BHA was used as a positive control. Free O2−•radical scavenging effect was calculated using the following Formula 2:
(F. 2)
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DPPH Radical Scavenging Assay
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Pc compounds were tested for in vitro antioxidant activities by DPPH free radical
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scavenging assay method [33]. Total volume of assay mixtures were 1 mL and
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contained methanolic DPPH solution (0.1 mM) and different concentrations of Pc compounds. After incubation in the dark at room temperature for 30 min, absorbance of the samples (Asample) were measured at 517 nm. Assay mixture without Pc compounds
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were used as a control (control absorbance, Acontrol). All experiments were in triplicates
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and results were expressed as the mean ± standard deviation (S.D.). Gallic acid was used as a positive control. Free radical scavenging effect was calculated using the
Metal Chelating Effects Assay
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2.8.
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Formula 2.
Metal chelating activities of the Pc compounds were examined using the method
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described [7] compared to EDTA as the reference compound. 500 µL of different concentrations (25-100 μM) of compounds were prepared and added to 2 mM 50 µL FeCl2, 5 mM 100 μL ferrozine. Then the reaction was incubated at room temperature for 10 min and the absorbance of the samples (Asample) were measured at 562 nm. Assay mixture without the compounds (Pcs) were used as a control (control absorbance, Acontrol). All experiments were in triplicates and results were expressed as the mean ± standard deviation (S.D.). Metal chelating effect was calculated using the Formula 2.
3. RESULTS AND DISCUSSION
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Determination of Binding of Pc compounds with CT-DNA
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The absorption spectra of the Pc compounds in the absence and presence of
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CT-DNA were performed to determine the binding mode of DNA with Pc compounds.
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A compound can bind to DNA via covalent or noncovalent (intercalation, electrostatic or groove binding) interactions. In general, DNA intercalative binder agents cause large shifts at wavelength because of π-stacking interactions between their aromatic
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chromophore groups while DNA groove binder or stacking agents trigger small shifts in
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absorbances and wavelengths in spectra [18].
The absorpstion spectra of the Pc compounds and Pc compounds-DNA were
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recorded between the range of 300-750 nm. The absorption spectra of Pc1, Pc2 and Pc3 (Fig2., Fig3., Fig4.) showed both hypochromic and small red shifts in the presence of
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DNA. Upon increasing the concentration of CT-DNA to the Pc1, Pc2 and Pc3 was observed with small red-shift of 2 nm (λmax 647 → 649) of Pc1 and small red-shift of 1
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nm (λmax 645 → 646) of Pc2 and no shift for Pc3. The hypochromic and small red shifts support non-intercalative binding mode between Pc compounds and DNA. The apparent binding constant (Kb) values of Pc1, Pc2 and Pc3 were determined as 3.75x104, 2.3x104, 4.57x104, respectively. These values are lower than the apparent binding constant (Kb) normally associated with intercalation (Kb < 106) [34]. These results indicates that Pc compounds represent non-intercalative binding mode.
3.2.
Thermal Denaturation Studies
ACCEPTED MANUSCRIPT The thermal melting temperature (Tm) is the temperature that half of the DNA strands are separated into single strand [35]. Tm value is very informative about stability
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of the DNA double helix with temperature. Increasing in the Tm values indicate the
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strength of interaction between DNA and metal complexes [36]. The Tm values can be
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used to determine binding mode of DNA-complexes as intercalative or external. General, in the presence of classical intercalation Tm value increases about 8-12 ˚C [37]. On the other hand, groove binders do not trigger a remarkable change in Tm value [38].
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In this study, Tm values of CT-DNA in the absence and presence of Pc compounds were
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determined by monitoring the absorbance of DNA at 260 nm as a function of temperature. As shown Table 1, the Tm value of CT-DNA in the absence of any Pc compounds were found 80.2 ˚C while Tm values of CT-DNA in the presence of Pc1, Pc2
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and Pc3 were found 81.7 ˚C, 82.2 ˚C, 82.9 ˚C respectively. This small changes in Tm
DNA-Photocleavage Studies
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values indicated the groove binding between CT-DNA and Pc1, Pc2 and Pc3.
Pc compounds contain metal ions are well suited for application as
metallonucleases, because of their possibility to tune the redox potential through the choice of proper metal ion [39]. Interactions of Pc compounds with supercoiled pBR322 DNA were investigated using agarose gel electrophoresis. It has been well known when circular plasmid DNA is subjected to electrophoresis, the fastest migrating is the supercoiled Form I, the slowest moving is the open circular Form II and the linear Form III runs in between the other two forms [40]. When one strand is cleaved, the supercoils relax to generate a slower-moving open circular Form II and both strands are cleaved, Form III is generated that migrates between Form I and Form II [30] .
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In our preliminary studies, the cleavage experiments were carried out in the
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absence and presence of irridiation at 20 µM concentration of Pc compounds. The
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results were shown Fig. 5. It was observed that all of Pc compounds exhibited
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remarkable cleavage activities in the presence of irradiation at 650 nm. Therefore, all of these studies were performed in the presence of irradiation at 650 nm.
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The nuclease effiency of a compound mainly depends on the activators used to
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initiate the DNA cleavage [41]. As shown Fig. 6. at irradiation of wavelength 650 nm in the presence of oxidizing agents (hydrogen peroxide (H2O2), ascorbic acid (AA), 2mercaptoethanol(ME)) Pc compounds exhibited more effective cleavage activity to
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convert the supercoiled DNA into the open circular and linear forms than in the absence of oxidizing agents. All of the Pc compounds showed more DNA cleavage activity in
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the presence of the cleavage activators. The nuclease effiency of Pc compounds in the presence of the cleavage activators were followed as H2O2>AA>ME, respectively.
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These results indicated that in presence of irradiation at 650 nm and oxidizing agents Pc1, Pc2 and Pc3 can be suitable agents for cleavage of DNA.
3.4.
Topoisomerase I Inhibition Assay
It is well known that topoisomerase I can affect topological changes of DNA by transiently cleaving one DNA strand and leads to relaxation of the supercoiled DNA Form III to open circular DNA Form I and lineer DNA Form II [42]. When topoisomerase I enzyme retains its catalytic activity, the disappearance of Form I and the appeareance of bands for Form II and Form I will be observed [31].
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In this study, the inhibiton activities of all Pc compounds were investigated
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against topoisomerase I by pBR3222 DNA relaxation assay. At the concentration of 20
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μM, Pc compounds exhibited remarkable topoisomerase I inhibitory activity (Fig. 7.).
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Pc3 showed the highest and Pc1 showed the lowest inhibition activity. These results showed that Pc compounds have potential as anticancer agents because of their
Superoxide Radical Scavenging Assay
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3.5.
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topoisomerase I inhibition activities.
Oxidation is important living organisms for generation of energy in biological
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proceses. However reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anions are precursors to active free radicals have generated reacting with
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biological macromolecules including fatty acid, DNA, proteins and thereby causing tissue damage [44]. Therefore, superoxide radical scavenging compounds are essential
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substances with ability to protect cells against damage various disseases. In this study, The SOD radical scavenging activities of Pc compounds were determined antioxidative behaviour in the riboflavin-NBT system at 560 nm and compared to BHA.
The stock solution of the Pc compounds were prepared in water at 1 mM and their superoxide scavenging activity tested at 25, 50, 75, 100 μM concentrations. The SOD activities of the Pc compounds were investigated by NBT assay and the results were shown in Table 2. Pc1 showed the highest activity (85.01 ± 0.15%) at 100 μM. The order of superoxide radical scavenging activity of compounds and refercence compound were found to Pc1 > Pc3 > Pc2 > BHA at 100 μM. According to the results
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DPPH Radical Scavenging Assay
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3.6.
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The radicals are inactivated by the antioxidants by their free-radical scavenging or hydrogen donation abilities [45]. DPPH is a stable nitrogen-centered free radical and is
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well known scavenger used in antioxidant assay because of fast and simple method [46].
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The radical scavenging activities of Pc compounds in comparison with gallic acid were determined by DPPH assay and the results were shown in Table 3. The stock solution of the Pc compounds were prepared in water at 1 mM and their DPPH radical
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scavenginig activity tested at 25, 50, 75, 100 μM concentrations. As shown Table 3, of DPPH free radical scavenging values of Pc1 ranged from 13.49 ± 0.40% (25 μM) to
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32.73 ± 0.90% (100 μM). Pc2 compound showed poor DPPH free radical scavenging activity at all of the concentration (5.12 ± 0.10%, 11.64 ± 0.21%, 18.65 ± 0.09%, 30.05 Pc3 exhibited the highest DPPH free radical scavenging
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± 0.13%, respectively).
activitiy from 16.71 ± 0.32% to 43.14 ± 0.30% inhibiton from 25 μM to 100 μM of compound. Gallic acid showed more DPPH free radical scavenging activity than all of Pc compounds.
3.7.
Metal Chelating Effects Assay
Transtions metals are essential elements to provide enzyme activity in the human body. But at the same time unpaired electrons can react quickly with peroxides and form alkoxyl radicals. Therefore, the chelation of transition metals by antioxidants can
ACCEPTED MANUSCRIPT be considered an important mechanism in the oxidation process [47]. In this work, metal
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chelating activities were determined compared to EDTA as the reference compound.
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The stock solution of the Pc compounds were prepared in water at 1 mM and
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their metal chelating activity were tested at 25, 50, 75, 100 μM concentrations. As shown Table 4 the order of metal chelating activity of compounds were found Pc3 > Pc1 > Pc2 at all of the concentrations. When the concentrations of Pc3 was increased
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from 25 µM to 100 µM, metal chelating activities were increased from 10.7 ± 0.19% to
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36.83 ± 0.13%. It was found that Pc1 had 33.07 ± 0.11% activity at 100 µM while Pc2 had 30.65 ± 0.04% activity at 100 µM. These results showed that all of Pc compounds
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have lower chelating activities than EDTA as standart compound.
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4. CONCLUSION
In this work the DNA binding of Pc compounds were determined with UV-Vis
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spectroscopy and thermal melting denaturation studies. The apparent binding constant (Kb) of Pc compounds and thermal melting denaturation studies exhibited that Pc compounds bind to calf thymus DNA via minor groove binding. The DNAphotocleavage studies showed that Pc compounds have strong cleavage activity to DNA in presence of irradiation at 650 nm. The topoisomerase I inhibition studies of Pc compounds showed that they have strong inhibitory activity, especially Pc3 compound. The antioxidant activity investigation of Pc compounds showed that they have moderate DPPH radical scavenging activity, metal chelating effects and significant superoxide radical scavenging activity. All of these results exhibited that Pc compounds can be suitable agent for the cancer treatment especially photodynamic therapy because of their
ACCEPTED MANUSCRIPT appreciable cleavage and inhibition of topoisomerase I activities to plasmid pBR322
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DNA.
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5. ACKNOWLEDGMENTS
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We thank Sena F. Sezen, PhD (Department of Pharmacology, Facult of Pharmacy,
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Karadeniz Technical University, Trabzon, Turkey) for critical reading for manuscript.
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CT-DNA
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Table 1. Tm values of CT-DNA in the absence and presence of Pc compounds.
81.7 ˚C
CT DNA + Pc2
82.2 ˚C
CT DNA + Pc3
82.9 ˚C
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CT DNA + Pc1
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Tm values 80.2 ˚C
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Table 2. Superoxide radical scavenging activity (%) of the Pc compounds. μM 25
Pc1 55.71 ± 0.35
Pc2 40.41 ± 0.30
50
64.79 ± 0.15
55.55 ± 0.19
75
75.35 ± 0.26
100
85.01 ± 0.15
BHA 17.18 ± 0.30
70.73 ± 0.36
32.11 ± 0.35
67.64 ± 0.24
72.67 ± 0.32
46.65 ± 0.32
70.43 ± 0.14
77.49 ± 0.27
59.16 ± 0.24
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Pc3 55.77 ± 0.29
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Table 3. Radical-scavenging activity on DPPH radicals (%) of the compounds. μM 25
Pc1 13.49 ± 0.40
Pc2 5.12 ± 0.10
50
20.13 ± 0.41
11.64 ± 0.21
75
23.70 ± 0.44
18.65 ± 0.09
37.14 ± 0.11
81.02 ± 0.03
100
32.73 ± 0.90
30.05 ± 0.13
43.14 ± 0.30
83.16 ± 0.06
GA 71.70 ± 0.07
23.58 ± 0.17
77.44 ± 0.13
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Pc3 16.71 ± 0.32
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Table 4. Metal chelating effect (%) of compounds on ferrous ions. Pc1
Pc2
Pc3
EDTA
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6.17 ± 0.17
3.31 ± 0.81
10.70 ± 0.19
16.92 ± 0.10
50
11.56 ±0.43
9.83 ± 0.15
16.97 ± 0.04
48.19 ± 0.09
75
26.24 ±0.66
21.04 ± 0.46
21.19 ± 0.12
84.10 ± 0.20
100
33.07 ±0.11
30.65 ± 0.04
36.83 ± 0.13
95.70 ± 0.13
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Fig. 2. Absorption spectrum of Pc1 upon increasing amounts of CT-DNA.
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Fig. 3. Absorption spectrum of Pc2 upon increasing amounts of CT-DNA.
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Fig. 4. Absorption spectrum of Pc3 upon increasing amounts of CT-DNA. Fig. 5. Agarose gel electrophoresis of pBR322 DNA (250 ng) in the absence and presence of light Pc compounds 50 mM Tris-HCl buffer (pH 7.0).
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Fig. 6. Oxidative pBR322 DNA (250 ng) cleavage in the absence and presence of
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Fig. 7. The inhibition activity of topoisomerase I of Pc compounds.
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Fig. 1. Chemical structure of Pc1, Pc2 and Pc3.
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Fig. 2. Absorption spectrum of Pc1 upon increasing amounts of CT-DNA.
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Fig. 3. Absorption spectrum of Pc2 upon increasing amounts of CT-DNA.
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Fig. 4. Absorption spectrum of Pc3 upon increasing amounts of CT-DNA.
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Fig. 5. Agarose gel electrophoresis of pBR322 DNA (250 ng) in the absence and presence of light Pc compounds 50 mM Tris-HCl buffer (pH 7.0). Lane 1, DNA control; lane 2, DNA + Pc1 (20 μM); lane 3, DNA + Pc2 (20 μM);
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lane 4, DNA + Pc3 (20 μM); lane 5, DNA control + 10 min irradiation; lane 6,
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DNA + Pc1 (20 μM) + 10 min irradiation; lane 7, DNA + Pc2 (20 μM) + 10 min
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irradiation ; lane 8, DNA + Pc3 (20 μM) + 10 min irradiation.
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Fig. 6. Oxidative pBR322 DNA (250 ng) cleavage in the absence and presence of
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different agents.
Lane 1: DNA control + 10 min. irradiation; lane 2: DNA + Pc1 (20 μM) + 10 min
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irradiation ; lane 3: DNA + Pc2 (20 μM) + 10 min irradiation; lane 4: DNA + Pc3 (20 μM) + 10 min irradiation; lane 5: DNA + H2O2 (0.4 M) + 10 min irradiation;
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lane 6: DNA + AA (0.25 mM) + 10 min irradiation; lane 7: DNA + ME (0.4 M) + 10 min irradiation; lane 8: DNA + Pc1 (20 μM) + H2O2 (0.4 M) + 10 min
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irradiation ; lane 9: DNA + Pc2 (20 μM) + H2O2 (0.4 M) + 10 min irradiation;
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lane 10: DNA + Pc3 (20 μM) + H2O2 (0.4 M) + 10 min irradiation; lane 11: DNA + Pc1 (20 μM) + AA (0.25 mM) + 10 min irradiation; lane 12: DNA + Pc2 (20
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Fig. 7. The inhibition activity of topoisomerase I of Pc compounds.
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Lane 1: DNA control; lane 2: DNA + 1 Unit topoisomerase; lane 3: DNA + 1 Unit topoisomerase + Pc1 (20 μM); lane 4 : DNA + 1 Unit topoisomerase + Pc2 (20
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Graphical abstract
ACCEPTED MANUSCRIPT Highlights 1. The Kb of Pc compounds were 3.75x104, 2.3x104 and 4.57x104. 2. Pc compounds exhibited significant inhibitory effects against topoisomerase I.
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3. These phthalocyanine compounds bind to CT-DNA via minor groove binding.
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SC R
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4. Pc3 compound had the highest DNA binding affinity and DNA cleavage activity.
