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Re(I) tricarbonyl complex of 1,10-phenanthroline-5,6-dione: DNA binding, cytotoxicity, anti-inflammatory and anti-coagulant effects towards platelet activating factor.
Re(I) tricarbonyl complex of 1,10-phenanthroline-5,6-dione: DNA binding,
cytotoxicity, anti-inflammatory and anti-coagulant effects towards Platelet
Activating Factor
Michael Kaplanis, George Stamatakis, Vasiliki D. Papakonstantinou,
Maria Paravatou-Petsotas, Constantinos A. Demopoulos, Christiana A.
Mitsopoulou
PII:
DOI:
Reference:
S0162-0134(14)00042-7
doi: 10.1016/j.jinorgbio.2014.02.003
JIB 9469
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
22 October 2013
5 February 2014
6 February 2014
Please cite this article as: Michael Kaplanis, George Stamatakis, Vasiliki D. Papakonstantinou, Maria Paravatou-Petsotas, Constantinos A. Demopoulos, Christiana A. Mitsopoulou, Re(I) tricarbonyl complex of 1,10-phenanthroline-5,6-dione: DNA binding,
cytotoxicity, anti-inflammatory and anti-coagulant effects towards Platelet Activating
Factor, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.02.003
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Re(I) tricarbonyl complex of 1,10-phenanthroline-5,6-dione: DNA binding, cytotoxicity, antiinflammatory and anti-coagulant effects towards Platelet Activating Factor.
Michael Kaplanis a, George Stamatakis b, Vasiliki D Papakonstantinou b, Maria Paravatou-
. Laboratory of Inorganic Chemistry, Faculty of Chemistry, National and Kapodistrian University
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Petsotas c, Constantinos A. Demopoulos b, Christiana A. Mitsopoulou a, *
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of Athens, Panepistimiopolis Zografou, 157 71 Athens, Greece
b
. Laboratory of Biochemistry, Faculty of Chemistry, National and Kapodistrian University of
Athens, Athens 15771, Greece
c
. Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National
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Centre for Scientific Research ‘‘Demokritos’’, 15310 Athens, Greece
Abstract
The complex fac-[Re(CO)3(phendione)Cl] (1) (where phendione=1,10-phenanthroline-5,6dione) has been synthesized and fully characterized by UV-visible, FTIR, and NMR techniques.
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The DNA binding properties of 1 are investigated by UV-spectrophotometric (melting curves),
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covalent binding assay, CV (cyclic voltammetry), circular dichroism (CD) and viscosity
measurements. Experimental data indicate that 1 fits into the major groove without disrupting the
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helical structure of the B-DNA in contrast to the free phendione which intercalates within the base
pairs of DNA. Upon irradiation, complex 1 promotes the cleavage of plasmid pBR322 DNA from
supercoiled form I to nicked form II via a proton coupled electron transfer mechanism. This comes
as a result of experimental data in anaerobic /aerobic conditions and in the presence of DMSO. The
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biological activities of 1 and its precursors [Re(CO)5Cl] and phendione are tested towards a series
of cancerous cell lines as glioblastoma (T98G), prostate cancer (PC3) and breast cancer (MCF-7) as
well as platelet activating factor (PAF)-aggregation. Moreover, all the aforementioned compounds
are tested for their ability to modulate PAF-basic metabolic enzymes activities in preparations of
rabbit leukolytes. The in vitro experiments indicate that phendione has a better antitumor effect than
cisplatin whereas [Re(CO)5Cl] is a better PAF inhibitor than both the phendione ligand and 1.
Moreover, for the first time it is indicated that [Re(CO)5Cl], with a IC50 of 17 nM is comparable to
the widely used PAF receptor antagonists, BN52021 and WEB2170 with IC50 of 30 and 20 nM,
respectively, whereas 1 affects PAF-catabolism.
Key words: Re(I) carbonyl, phendione, DNA binding, DNA photocleavage, anticancer activity,
PAF, PAF-metabolism.
*Corresponding author: Email: cmitsop@chem.uoa.gr; Fax: +30210 7274435
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1. Introduction
The antitumor activity of cisplatin and its associated side effects and resistance continue to
fuel research in organometallic complexes in an effort to formulate new, specific metallodrugs with
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fewer or no side effects [1]. In this context, transition metal complexes are studied for their efficient
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DNA binding ability [2-6] and it is well documented that they can be used as probes capable to
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utilize nucleic acid structures [2-6], DNA-molecular light switches [7,8], oxidative cleaving agents
for DNA [4-6,8], and potential anti-cancer drugs [9-11]. Rhenium belonging in the same period as
platinum have attracted researchers attention not only due to the 188-Re beneficial
radiopharmaceutical activities but also because several Re(I) complexes were found to be very
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active against suspended cancer cell lines (as MOLT-4 and HL-60) and solid tumors like MCF-7
and SK2 [12]. Moreover, significant cancer cell cytotoxicity has been reported for rhenium (I)
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tricarbonyl complexes [13,14] whereas a plenty of Re(I) polypyridine complexes have been
designed not only for targeting biomolecules, such as DNA [8,15-20] and protein [9,21,22] but also
as cellular probes with cellular uptake like cytotoxicity and bioimaging application [9,22].
On the other hand, the coordinated ligand of a complex plays an important role since it
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controls the way the complex interacts to DNA; this can occur by intercalation, groove binding or
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external electrostatic binding [2-4]. An interesting class of ligands is phenanthrolines and
substituted derivatives which interact with DNA by aromatic π stacking between the base pairs,
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both when they are free or coordinated to a metal centre. Among the phenanthroline derivatives,
1,10-phenanthroline-5,6-dione (phendione), displays significant anticancer role with and without a
coordinated metal [23,24], whereas it also displays antibacterial and antifungal activity [23-27].
Actually, its biological activity is enhanced or reduced when it is coordinated to different metal
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centers [27, 28] even if it keeps its planar geometry, indicating that the role of the metal moiety is
crucial for the electronic properties both of the metal center and the phendione resulting in
significant differences in biological activity. Recently, the anticancer activity of phendione and its
Cu2+ and Ag+ complexes has been studied in a wide spectrum of cell lines, containing not only
mouse’s and monkeys’ cells, but also human-derived cancer cell lines [23,24,26]. In the majority of
cases, these complexes exhibit excellent biological activity and represent a class of DNA-targeting
compounds capable of inhibiting nucleotide synthesis. In order to ascertain the likely cyto-selective
nature of these agents, C. Deegan et al. [23] included two cell lines, both derived from normal
human cells. The results suggest that these compounds are incapable of selectively killing cancer
cells. Additionally, the metal complexes are more cytotoxic against all tested cell lines. Lately, it
has been proved that the complex Pt(phendione)Cl2 is as active as cisplatin against a variety of
human cancer [27].
Little is known about the anti-inflammatory and anti-coagulant effects of Re(I) tricarbonyl
complexes. Actually, in cancer, malignant situations such as angiogenesis and metastatic
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progression are strongly promoted by the inflammatory tumor microenvironment due to the high
levels of cytokine and chemokine secretion by the recruited inflammatory and stromal cells while
coagulation of blood, inflammatory activation of platelets, endothelial cells and monocytes are
typical steps in such cases. These occur as a common mechanistic effect of an inflammatory
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network of mediators like thrombin, platelet-activating factor etc [29,30].
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Platelet activating factor (PAF), characterized mainly as 1-O-alkyl-2-acetyl-sn-glycero-3-
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phosphochloline, is an ether phospholipid mediator, synthesised and released by different types of
cells, such as mast cells, neutrophils, monocytes, eosinophils, macrophages, platelets and
endothelial cells. Its biological activity is exhibited through specific membrane PAF-receptors,
coupled with G-proteins. The biosynthetic enzymes of PAF are PAF-cholinephosphotransferase
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(PAF-CPT, EC 2.7.8.16) and lyso PAF-acetyletransferase (lyso PAF-AT, EC 2.3.1.67), regulating
PAF production in blood and cells. Concerning PAF catabolism, the basic enzyme of PAF
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degradation is PAF-acetylhydrolase (PAF-AH, EC 3.1.1.47), with its isoform in plasma also known
as Lp-PLA2 [31,32].
Studies have shown that transformed cancer cell lines able to express PAF-AH directly in
the tumor microenvironment, reduced in growth and other malignant activities such as proliferation,
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motility and metastatic angiogenesis. This reduction was attributed to the blockage of PAF-induced
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signaling pathways. In addition, in patients with renal cell carcinoma treated with INF-a, a cytokine
untreated [29].
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with anti-cancer activities, PAF-CPT activity was significantly reduced in comparison to those
Up to now, only organic compounds are used as PAF antagonists such as ginkolide BN
52012 and hetrazepines WEB 2170 whereas only recently the use of metal complexes has been
referred as potential pharmaceutics against RAF activities [29].
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In this study taking into consideration all the above, we examined for the first time the in
vitro effects of phendione and its corresponding Re(I) complex against antitumor and PAF-induced
biological activities. For this purpose, the antitumor activities of the aforementioned compounds
were tested on three cell lines, namely human breast cancer MCF-7, human prostate cancer PC3 and
human glioblastoma T98G and compared to cisplatin complex. The potent inhibitory effect of these
compounds was studied on PAF-induced platelet aggregation towards washed rabbit platelets
(WRPs). In addition, they were tested for their ability to modulate PAF-basic metabolic enzymes
activities in preparations of rabbit leukolytes. Moreover, in an attempt to investigate the role of the
metal centre on DNA binding mode of phendione, the interaction of the synthesized
[Re(CO)3(phendione)Cl] complex with calf thymus DNA was studied with a range of techniques
whereas its photocleavage reaction on DNA was monitored by agarose gel electrophoresis.
2. Experimental
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2.1. Materials and Methods
The chemicals and the solvents were purchased by Sigma Aldrich and used as received.
Calf thymus DNA (CT-DNA) was purchased from Sigma. All the experiments involving interaction
of the complexes with DNA were conducted in phosphate buffer (5 mM), pH = 7.00 and NaCl (4
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mM). 1H NMR spectra were recorded on a Varian Unity Plus 300 MHz spectrometer. Samples were
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run in a 5 mm probe with deuterated solvents as internal lock and reference. The assignment of the
H NMR spectra of the free ligand and complex is based on 2D NMR experiments ( 1H–1H COSY).
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Electronic absorption spectra were recorded on a Varian Cary 300E spectrometer at 25.0 ± 0.2 oC.
2.2. Synthesis
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purified by crystallization from ethanol [34].
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The ligand phendione was synthesized according to literature procedure [33] and was
Synthesis of fac-[Re(CO)3(phendione)Cl] (1)
The complex 1 was synthesized based on the following report by Stoeffler [8] with some
modifications [18]. A mixture of Re(CO)5Cl (0.1211 g, 0.33 mmol) and phendione (0.1040 g, 0.50
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mmol) were suspended in 5 mL toluene and refluxed under N2 atmosphere for 4 h. Then, the
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mixture was allowed to cool to room temperature and the solid was collected by filtration under
vacuum. The crude product was purified by column chromatography on silica gel using THF
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(tetrahydrofuran) as an eluent. THF evaporated under reduced pressure and the brown solid
recrystallized from CH2Cl2 by the addition of diethyl ether. The NMR spectrum agrees well with the
literature spectra. Yield: 74%. 1H NMR (300 mHz, DMSO-d6, δ ppm) 9.23 (d, 2H), 8.74 (d, 2H),
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7.95 (t, 2H). FT-IR (cm-1, KBr disc): 2033, 1910, 1891 (ν C O ), 1704 (νC=O). Anal. Calc. for
C15H6N2O5ClRe: C, 34.72; H, 1.75; N, 5.40; found: 34.80; H, 1.72; N, 5.33
2.3. DNA binding studies
All experiments involving DNA interactions with complex 1 and its components were
carried out in phosphate buffer pH=7.0 (the buffer was prepared by diluting 0.449g (3,3 mmol) of
KH2PO4 and 0.296g (1.7 mmol) of K2HPO4 in 1 L of double distilled water containing
NaCl 4mM ). The ionic strength was 4 mM ensuring that the concentration of NaCl was at
least 100-fold more than that of DNA. A solution of calf thymus DNA (CT-DNA) in buffer gave
a ratio of UV absorbance at 260 and 280 nm of ca. 1.9:1, indicating that the DNA was sufficiently
free of protein [35]. The DNA concentration per nucleotide was determined by UV absorption
spectroscopy using the molar absorption coefficient (ε260 = 6600 M-1cm-1) at 260 nm [35]. Stock
solution was stored at 4 οC and used within 4 days since at low concentrations DNA tends to
absorb onto the surfaces of plastic tubes (from www.sigmaaldich.com). In all of our
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experiments, the compounds and DNA are incubated for 24h at 25 oC since the equilibrium is very
slow as it was found by UV-visible (UV-Vis) spectroscopy.
2.3.1. Thermal denaturation studies (Tm measurements)
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Samples for thermal denaturation experiments were obtained by adding a freshly prepared
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solution of rhenium compound, dissolved in methanol, to a constant DNA concentration (15 μM) in
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phosphate buffer. The resulting solutions, (with ratios r= [complex]/ [DNA] = 0, 0.05 0.1, 0.2 and
0.5) were incubated for 24h at 25 oC, before measurements were recorded. DNA melting
experiments were carried out by monitoring the absorbance of CT-DNA at 260 nm in the
temperature range from 25.0 oC to 95.0 oC, on increasing temperature in 0.5 oC , with a Varian
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Cary 300 spectrophotometer. Samples were allowed to equilibrate for 1 min at each temperature.
The melting temperature (Tm) of DNA was determined as the mid point of the optically detected
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transition.
2.3.2. Circular Dichroism (CD) measurements
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CD measurements were carried out on a Jasco J700 spectropolarimeter interfaced with a
PC. Data were analyzed through the standard Jasco software package. Each sample, which prepared
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similarity to Tm experiment ([DNA]=20 μM), was scanned in triplicate in a range of wavelengths
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between 200 nm to 400 nm. The drawn spectra are the means of three independent scans.
2.3.3. Cyclic Voltammetry (CV) experiment
CV measurements were carried out using a Pine AFCBP1 (pine Instrument Company,
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Groove City, PA, USA). The CV experiments were performed in a one compartment cell equipped
with platinum working electrode, platinum wire as the auxiliary electrode and Ag/AgCl as reference
electrode and the scan rate was 100 mV∙s-1. The potential was expressed as V vs. ferrocenium–
ferrocene, and NaCl (10 mM) of the buffer was the supporting electrolyte.
2.3.4. Absorption titration
Absorption titration experiments were performed as follows: a constant metal complex
concentration (20 μM) (dissolved in methanol) was incubated with varying nucleotide concentration
(0-400 μM) in phosphate buffer solution (final percentage of MeOH = 5%). The data of the
absorption titration experiments were fit in eq 1 to obtain intrinsic binding constant Kb,
[DNA] (ε a ε f ) [DNA] (ε b ε f ) 1 K b (ε b ε f )
(1)
where [DNA] is the concentration of DNA in base pairs, εa is the extinction coefficient of the
observed absorption band at the given DNA concentration (correspond to Aobs/[complex]), εf is the
extinction coefficient of the complex free in solution, and εb is the extinction coefficient of the
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complex when fully bound to DNA. A plot of [DNA]/[εa - εf] versus [DNA] gave a slope 1/[εa - εf]
and Y intercept equal to 1/Kb[εb - εf]. The intrinsic binding constant Kb is the ratio of the slope to the
intercept. The linear least-squares analysis was done using Origin Lab 8.0.
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2.3.5. Viscosity measurements
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Viscometric experiments were performed using Schott Gerate AVS 310 Automated
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Viscometer, maintained at 25.0 (±0.1) oC in a thermostatic water bath. The concentration of DNA
was 100 μM and that of metal complex was varied from 5 to 50 μM. Flow time of solutions in
phosphate buffer (pH 7.0) was recorded in triplicate for each sample, and an average flow time was
calculated. Data were presented as (η/η0)1/3 versus binding ratio r, where η is the viscosity of DNA
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in the presence of complex and η0 is the viscosity of DNA alone. The relative viscosity of DNA in
the presence and absence of the metal complex was calculated using the following
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expression: 0 (t t 0 ) (t DNA t 0 ) , where t0 and tDNA are the observed flow time of the buffer
solution alone and DNA, respectively, while t the flow rate of the samples in the presence of the
complex. The relationship between the relative solution viscosity (η/η0) and contour length (L/L0) is
given by the equation L L0 ( 0 ) , where L0 denotes the apparent molecular length in the
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2.3.6. DNA electrophoresis
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absence of the metal complex [36].
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For the gel electrophoresis experiments, supercoiled pBR322 plasmid DNA (0.15 μg) was
treated with different concentrations of complex 1 in 50 mM Tris∙HCl and 18 mM NaCl buffer
(pH= 7.2). The samples, which were incubated for 24 h in the dark at 25 oC, were irradiated for 2 h
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using 1000 W Xenon lamp (Oriel, mod 68820, Universal Arc. Lamp) and λir> 335 nm. The samples
were analyzed by electrophoresis for 1 h at 100 V on 1 % agarose gel in TBE (tris-borate-EDTA)
buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH=8.0), followed by staining with
ethidium bromide (0.5 μg/ mL final concentration). The gels were imaged with a BioSure UVTransilluminator and photographed using a Picture Works, Photo Enhancer v3.2 digital camera.
2.4. In vitro cytotoxicity studies
2.4.1. Cell lines and culture conditions
Human cell lines of breast cancer (MCF-7), prostate cancer (PC3) and glioblastoma (T98G)
were cultured in high glucose D-MEM medium with L-glutamine (Biochrom) supplemented with
10% foetal bovine serum (Biochrom) and penicillin/streptomycin (Biochrom) 10 U/mL / 10μg/mL.
Cells were maintained at 37°C in a humidified 5% CO2 incubator and were subcultured every 2-4
days.
2.4.2. Cytotoxicity of the ligand and the complexes (MTT assay)
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In-vitro cytotoxicity studies were performed using the MTT (3-[4,5-dimethylthiazol-2-yl]2,5 diphenyl tetrazolium bromide) colorimetric method, according to the literature [37,38]. Briefly,
cells were seeded in 96-well flat-bottomed plates at a density of ~6000 cells per well and incubated
for 24 h at 37 oC. The day after seeding, exponentially growing cells were incubated for 72 h with
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various concentrations of each compound. Cisplatin was added as positive control and wells with no
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metal compound as negative control. Tetraplicate wells were set up for each condition. After
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incubation the medium was removed and MTT solution (1 mg/mL in medium) was added for 4
hours. The resulting purple formazan crystals were dissolved by the addition of isopropanol (100
μL) and absorbance was measured for each well by a spectrophotometric [ELISA (enzyme- linked
immunosorbent assay)] plate reader at 540 nm. Background absorption was measured at 620 nm
mean OD of treated cells
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mean OD of untreaded cells (control)
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% cell viability
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and subtracted. The % cell viability was expressed as:
The data were plotted against the corresponding compound concentration in a semi-log chart and
the IC50 (the concentration causing 50% inhibition of cell proliferation) was determined from the
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dose–response curve, using GraphPad Prism 5.0. MTT assay was performed three independent
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times and in all experiments DMSO was lower than 1%.
2.5. PAF analysis
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2.5.1 Materials and instruments
Centrifugations were performed on a Heraeus Labofuge 400R. Homogenizations were
conducted in 30% of the power of a supersonic Bandelin Sonoplus HD 2070 sonicator
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(Heinrichstraze 3-4, D-12207 Berlin, Germany). The liquid scintillation counter used was a 1209
Rackbeta (Pharmacia, Wallac, Finland). Platelet aggregation studies were performed in a ChronoLog model 400 VS aggregometer (Havertown, PA, USA) coupled to a Chrono-Log recorder at 37
°C with constant stirring at 1200 rpm.
BSA (bovine serum albumin), PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine),
trichloroacetic acid (TCA), CDP-choline, lyso-PAF, acetyl-CoA, dithiothreitol (DTT), EDTA,
MgCl2, Tris HCl and analytical reagents were purchased from Sigma (St. Louis, MO, USA). 1-Ohexadecyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine ([3H]-PAF) with a specific activity of 10
Ci/mmol was obtained from New England Nuclear (Dupont, Boston, MA, USA). 1-O-alkyl- 2-snacetyl-glycerol (AAG) was purchased from BIOMOL International LP (Palatine House, Matford
Court, Exeter, UK). 2,5-Diphenyloxazole (PPO) and 1,4-bis(5-phenyl-2-oxazolyl) benzene
(POPOP) were purchased from BDH Chemicals (Dorset, England). Scintillation liquid cocktail
(dioxane base) was prepared by diluting 7 g PPO, 0.3 g POPOP and 100g Napthalene in 200 mL
H2O and then transferred to 1 L of dioxane. Liquid chromatography grade solvents and silica G for
TLC were purchased from Merck KGaA (Darmstadt, Germany).
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2.5.2 Isolation of plasma and leukocytes from blood samples
9 mL of blood were collected from the marginal ear vein of a white New Zealand rabbit in
1 mL of an anticoagulant solution of sodium citrate / citric acid. The sample was centrifuged at
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630×g for 13 min at 25 oC. The supernatant of the centrifugation (plasma rich in platelets) was
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centrifuged at 1400×g for 20 min at 25 oC in order to obtain plasma. Saline was added in the pellet
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until the volume of 10 mL and mixed by inversion. The leukocytes were isolated after the
sedimentation of the erythrocytes using 3.4 mL of dextran solution (3 % dextran in NaCl 0.15 M)
for 1 h at room temperature. The leukocyte-rich supernatant was centrifuged at 500×g for 10 min at
room temperature. The supernatant was removed and contaminating erythrocytes of the sediment
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pellet were lysed by the addition of 5 mL of a lysis solution consisted of 155 mM NH4Cl, 10 mM
KHCO3, and 0.1 mM EDTA and then removed by centrifugation at 300×g for 10 min at room
temperature. The isolated leukocytes were re-suspended in 1 mL of a buffer containing 50 mM Tris-
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HCl (pH 7.4), sonicated into ice (4 times of 15 s each) and centrifuged at 500×g for 10 min at 4 oC.
Leukocytes homogenates after protein determination by Bradford method were aliquoted and stored
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at -80 oC.
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2.5.3 Washed rabbit platelet aggregation assay
Biological assay on washed rabbit platelets was performed as previously described [31].
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Briefly, PAF was dissolved in 12.5 mg of bovine serum albumin (BSA) per 1 mL of saline at a final
concentration of 1.846 nM. The complexes and the ligand were tested in several concentrations,
diluted when necessary in the BSA saline, for their ability to cause or inhibit platelet aggregation.
After 1 minute of sample addition, stimulation with PAF (2.5 × 10-11 mol/L, final concentration in
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the cuvette) followed. The platelet aggregation induced by PAF was measured as PAF-induced
aggregation, before (considered as 0% inhibition) and after the addition of various concentrations of
the examined sample, creating a linear curve of percentage inhibition (ranging from 0 to 100%)
versus different concentrations of the sample. From this curve, the concentration of the sample that
inhibited 50% PAF induced aggregation was calculated, and this value was defined as IC50.
2.5.4. PAF-CPT activity assay
The assay was performed on the homogenate of leukocytes isolated as previously described
[39]. Briefly, the reaction was carried out at 37 oC for 20 min in a final volume of 200 μL
containing 100 mM Tris-HCl (pH 8.0), 15 mM dithiothreitol (DTT), 0.5 mM EDTA, 20 mM
MgCl2, 1mg/ mL BSA, 100 μM CDP-Choline, 100 μΜ 1-O-alkyl-2-sn-acetyl-glycerol (AAG,
added in the assay mixture in ethanol), and the sample (0.05 mg/ mL final concentration of protein).
The mixture of Tris, DTT, EDTA, MgCl2 and BSA was incubated in 37 oC for 5 min. Initially, the
homogenate was added in the mixture. After 30 s, AAG was added and 30 s later the reaction was
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started by addition of CDP-Choline. The reaction was stopped by adding 0.5 mL of cold methanol
after 20 min.
2.5.5. Lyso-PAF-AT activity assay
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The assay was performed on the homogenate of leukocytes isolated from New Zealand
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White rabbits as previously described. Briefly, the reaction was carried out at 37 oC for 30 min in a
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final volume of 200 μL containing 50 mM Tris-HCl (pH 7.4), 0.25 mg/ mL BSA, 20 μMΜ lysoPAF and 200 μΜ acetyl-CoA and the sample (0.125 mg/ mL final concentration of protein). The
reaction started by the addition of the homogenated sample and was stopped after 30 min by adding
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0.5 mL of cold methanol.
2.5.6. Determination of produced PAF and biosynthetic enzymes (PAF-CPT and lyso-PAF-AT)
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activities
The extraction, purification and determination of the produced in each assay PAF was
carried out as previously described [39]. Briefly, PAF was extracted according to the Bligh–Dyer
method and was separated by thin-layer chromatography (TLC) on Silica Gel G coated plates with a
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development system consisting of chloroform:methanol:acetic:acid:water (100:57:16:8, v/v/v/v).
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PAF bands were scrapped off, extracted and the amount of PAF was determined by the washed
rabbit platelet aggregation assay. All assays were performed in triplicate and specific activities of
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PAF-CPT and lyso-PAF-AT were expressed as pmol of produced PAF/min/mg of sample protein
present in each assay.
2.5.7. Plasma PAF-AH (LpPLA2) activity assay
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Plasma PAF-AH was determined in rabbit plasma by the trichloroacetic acid precipitation
method using [3H] PAF as a substrate, as previously described [39]. Briefly, 2 μL of plasma were
incubated with 4 nmol of [3H]-PAF (20 Bq per nmol) for 30 min at 37 oC in a final volume of 200
μL of 50 mM Tris/HCl buffer (pH 7.4). The reaction was terminated by the addition of BSA
solution (final concentration 0.75 mg/mL) and followed by precipitation with trichloroacetic acid
(TCA final concentration 9.6% v/v). The samples were placed in an ice bath for 30 min and
subsequently centrifuged at 16000×g for 5 min at 4 oC. The [3H]-acetate released into the aqueous
phase was measured on a liquid scintillation counter. All assays were performed in triplicate and the
enzyme specific activity was expressed as pmol of degraded PAF/min/µL of plasma.
3. Results and discussion
3.1. Synthesis and characterization
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The Re(I) complex was synthesized according to the general method reported by Ruiz et al
[18] with some modifications. The preparation included the reaction of the precursor [Re(CO)5Cl]
with slight excess of the ligand phendione (1:1.2) under reflux in toluene for 4 h. The crude product
was purified using column chromatography on silica gel and the red band recrystallized from
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CH2Cl2 by the addition of ether. In Table S1 (supporting material) are reported the proton chemical
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shifts of phendione and the corresponding Re(I) complex, and are compatible with those of the
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literature [8,18,33,34]. The deshielding of the diimine protons can attributed to the electron
density’s removal from phendione towards [Re(CO)3Cl] core after complexation [40].
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Please insert here Fig 1.
The IR spectra, recorded in KBr pellets, are rather complicated but despite this, there are
some features quite common to aromatic rings corresponded to the stretches of C–H and C–C and
1
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the out of plane bending of C–H. The most characteristic band of the phendione appears at 1687 cmand assigned as v(C=O). The corresponding band for complex 1 appears at 1704 cm-1. The
characteristic peaks of complexes of the type fac-[Re(α-diimine)(CO)3Cl] observed in the region
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1890 – 2040 cm-1, due to the C O stretching vibrations. For 1, the highest band appears at 2033
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cm-1 and corresponds to a totally symmetric in-phase v(CO) vibration. The two lower bands belong
to the out of-phase totally symmetric vibration and the asymmetric vibration of the equatorial CO
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ligands, and for 1 appear at 1891 and 1910 cm-1, respectively [41].
Electronic absorption spectra of complex 1 in various solvents with different polarity are
indicated in Fig. 2. The spectrum consists of two main bands; a broad low-energy band and a
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narrower, more intense, high-energy band at the UV region. The UV bands resemble in shape and
position the lowest-energy ones in the corresponding spectrum of the free diimine ligand, despite
they are found at higher wavelengths and are more structured. The shape along with the position
and the solvent dependence of the low-energy band denote its (Re)dπ→π*(phendione) chargetransfer (MLCT) character, which agrees with previous interpretations of many mononuclear
tricarbonyl Re complexes [41,42]. From Fig. 2 it is obvious that the polarity of the solvent affects
the shape and the place of the bands not only because of the solvatochromic effect but also due to
the hydrogen bonds that the oxygen atoms of the phendione can form. To the existence of these
bonds can be attributed the observed changes over time to the UV-Vis of complex 1 in methanolic
buffer solution. (Fig. S1). This is further supported by the 1H-NMR experiments in a mixture of
10% CD3OD and 90% D2O that shows substantial line broadening. This behaviour also suggests
that complex 1 is in equilibrium with a hydrated species. The hydration reaction occurs in carbonyl
groups, when the ligand coordinate to a metal centre or a proton ion binds to the nitrogen atoms of
the phendione [43]. Indeed, this hydration observed for complexes [Os(bpy)2(phendione)]2+ [43]
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and for [Ru(phen)2(phendione)]2+ [44], with the latter to be responsible for the photocleavage of
supercoiled pUC18 plasmid DNA [44].
T
Please insert here Fig 2.
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3.2. DNA binding studies
3.2.1 Tm measurements
DNA thermal denaturation experiments show only a minor shift in the DNA melting
temperature (Tm), giving ΔTm values of 1-3 oC upon addition of the complex 1 to CT- DNA (Table
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1). Particularly, a relative low destabilization of the double helix is observed at the ratio 0.5 (r =
[complex]/ [DNA]) with ΔTm= 2.9 oC. The data suggest a primarily external or groove binding
nature of the complex. Non classical intercalating complexes show similar behaviour [45-47], while
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intercalating ligands like ethidium bromide show significantly high ΔTm values [48].
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Please insert here Table 1.
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3.2.2. Circular dichroism experiments
The CD spectral technique is useful in monitoring the conformational variations of DNA in
CE
P
solution. CD of native CT-DNA is conservative, giving a negative band at ~246 nm, arising due to
the B-form right handed helicity, and a positive band at ~278 nm originating from uniform
nucleobase stacking in the B-form conformation [49]. Both intensities and wavelengths are quite
sensitive to the mode of the interaction with small molecules. It is generally accepted that the
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groove binding or electrostatic interaction does not change significantly the intensities of both the
bands, while the classical intercalation enhances the intensities due to the strengthening of the basestacking and stabilization of the right-handed B conformation.
Upon addition of the rhenium(I) complex no significant modification is observed either to
the ellipticity of the bands or to their position (Fig. S2). Particularly, only a little change in
ellipticity at 278 nm (Δε decrease from 1.82 to 1.55 M-1cm-1) and at 248 nm is observed, for the
ratio r = 0.2, as shown in Table 1, whereas there is no observable change at the wavelength of the
two bands.
The results suggest that the complex cannot change the conformation of the B-form of
DNA, and this is a first clue, that its mode of binding to DNA is not intercalative in nature, in
accordance
with
the
Tm
measurements.
Recently,
molecular
modelling
studies
of
[Co(phendione)2Cl2]+, shows that although it covalently binds to N7 of guanine, the ligand fits into
the major groove without disrupting the helical structure of the B-DNA [50].
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3.2.3. CV studies
Electrochemical investigation of complex-DNA interactions can provide a useful
complement to other methods and yield information about the mechanism of the interaction [51,52].
Thus, Fig. 3 shows the cyclic voltammograms of the complex 1 (100 μM) at the absence and
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presence of CT-DNA (200 μΜ). After addition of DNA to the complex no new redox peaks
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appeared, but the current intensity of all the peaks decreased, suggesting the existence of an
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interaction between 1 and CT-DNA, and can be explained in terms of an equilibrium mixture of free
and DNA-bound complex to the electrode surface [52]. From Fig. 3, is observed that in the presence
of DNA, the cathodic potential Epc shows a negative shift up to -313 mV (ΔΕpc= -44 mV), while the
anodic peak Epa presents a decrease of the current intensity with no shift to its potential. The
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negative shift of the cathodic potential may suggest the external binding of the complex to DNA.
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Please insert here Fig 3
3.2.4. Absorption titration
The interaction of small molecules with DNA can be investigated by absorption titration.
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Complexes that bound to DNA by intercalation usually cause a decrease in absorbance
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(hypochromism) and a red shift (bathochromism) to the wavelength at 310nm involving a strong ππ* stacking interaction between aromatic chromophore and the base pairs of DNA. The extent of
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P
hypochromism and bathochromism depends on intercalative binding strength [53,54]
Electronic absorption spectra of the complex 1 (constant concentration at 20 μM) in
phosphate buffer pH=7 is indicated in Fig. 4. Upon addition of DNA, complex exhibits decrease in
absorption at 310 nm (hypochromism) around to 30%. Despite the observable hypochromism, the
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interaction is not intercalative in nature, as suggest the negligible shift in the absorption maximum
wavelength. This behaviour agrees with a groove binding mode.
To analyze quantitatively the binding strength of complex 1, the intrinsic binding constant
Kb of 1 with DNA was obtained by monitoring the intense of the absorbance at 310 nm of 1 with
increasing concentration of DNA and by using eq 1. The value of the Kb, which is given as a ratio of
slope to y intercept obtained from the plot of [DNA]/(εa-εf) vs. [DNA] (inset Fig. 4), was determined
as 7.95(±1.89)x104 M-1. As was expected, the binding affinity with DNA is smaller than classic
intercalating
complexes
with
dppz
ligand,
such
as
[Ru(bpy)2dppz]2+
(5.0x106 M-1),
[Ru(phen)2dppz]2+ (5.1x106 M-1) [54]. Complex 1 has a smaller Kb value than the related Re(I)
complexes with an intercalative ligand (such as dppz) [8,16,20], due to the non-intercalative mode
of the phendione ligand.
Complex 1 exhibits a weak luminescence in phosphate buffer. Binding of 1 to CT-DNA did
not lead to any change in its emission intensities. Thus, emission spectroscopy could not be used to
examine the binding properties of the complex.
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13
Please insert here Fig 4
3.2.5. Viscosity measurements
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Optical photophysical probe provides necessary but not sufficient clues to support binding
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modes, whereas hydrodynamic measurements that are sensitive to the length change are regarded as
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the most critical tests of a binding model in solution. For example, a classical intercalator, such as
ethidium bromide, causes a significant increase in viscosity of a DNA solution because of the
separation of the base pairs at the intercalation site, and hence, the expansion of the DNA molecular
length. In contrast, a ligand that binds in the DNA grooves causes less-pronounced changes or no
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change in viscosity of a DNA solution [55,56]. Fig. 5 indicates the relative viscosity of DNA
([DNA]=100 μM) in the presence of varying amounts of complex 1. The flow time of each
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sample was recorded in triplicate, and each value did not differ from each other more than
0.4 s. The observed decrease of the relative viscosity (up to 0.92) reveals that complex 1 interacts
with groove or electrostatic binding mode, which can bent (or kink) the DNA helix reducing its
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effective length and concomitantly its viscosity. The Δ isomer of the groove binder [Ru(phen)3]2+
CE
P
Please insert here Fig 5.
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causes a similar decrease of the relative viscosity of DNA [55].
3.2.6. Gel Electrophoresis of Re(I) complex
The ability of complex 1 to cleave DNA upon irradiation was determined by agarose gel
AC
electrophoresis. Thus, supercoiled pBR322 DNA was incubated with different concentration of 1
(0-100 μM) and the samples was irradiated with λir> 335 nm for 2 h under aerobic conditions. The
results indicate (Fig. 6) that complex 1 promotes the single strand cleavage of the supercoiled (Form
I) to nicked DNA (Form II). The untreated plasmid DNA (lane 1) does not show any cleavage upon
irradiation. However, with increasing concentration of 1 (lanes 2-4) the amount of Form II increases
gradually, whereas that of Form I diminishes. No DNA cleavage was observed upon incubation of
DNA with 1 in the dark (lane 5).
The precursor mechanistic studies revealed that complex 1 can photocleave DNA under
both aerobic (lane 2-4) and anaerobic conditions (lane 7), with somewhat less cleavage yields
without O2 and under argon atmosphere (lane 7). In the anaerobic experiment, the sample was
subjected to three consecutive freeze-pump-thaw cycles, incubated for 24h under argon atmosphere
and then irradiated. The data in lane 7 reveal that an oxygen independent pathway exists but the
enhanced
photocleavage
activity
of
1
in
the
presence
of
oxygen
indicates
that
[Re(CO)3(phendione)Cl] can also induced DNA damage by the generation of ROS. Since DMSO is
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14
known to quench hydroxyl radicals, experiments in the presence of it were performed (lane 6)
indicating no attenuation of the photocleavage reaction. As a consequence and taking into
consideration the anaerobic photoinduced cleavage of DNA a mechanism via a proton coupled
electron transfer could be suggested, similar to the well documented mechanism of the cleavage of
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the complex [Ru(phen)2phendione]2+ [44].
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Please insert here Fig 6.
3.3. Cytotoxicity studies
In an attempt to elucidate the role of the metal centre when coordinated with the ligand
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phendione, we determined the cytotoxicity properties of complex 1, the precursor molecule
Re(CO)5Cl and the phendione ligand, against three human cancer cell lines, glioblastoma (T98G),
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prostate cancer (PC3) and breast cancer (MCF-7) cells using the MTT assay. As a positive control
was used cisplatin.
The IC50 values shown in Table 2, indicate that phendione is toxic against T98G (IC50=4.91
μΜ), PC3 (IC50=3.01 μΜ) and MCF-7 (IC50=2.45 μΜ) cell lines even more than cisplatin and
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confirm previous published results referring the toxicity of phendione in various cell lines
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[23,26,27]. The limited solubility of the complex 1 in aqueous medium did not allow the evaluation
of the IC50 values [57]. Complex 1 is not active in 10 μΜ (data not shown). However, in 50 μM
CE
P
concentration of 1 (Fig. 7) the cell viability decreases with respect to the control at least 45%, 40%
and 27% for MCF-7, T98G and PC3 cells, respectively. It is worth noting that the starting material
Re(CO)5Cl reduces the cell viability about 50% only at 200 μM (Fig. S4). The high cytotoxicity of
phendione ligand is reduced when the ligand coordinates with Re(I) core for all cell lines tested.
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Similar to our results, a significant decrease in cytotoxicity observed when phendione
coordinate with platinum. Roy et al. [27] have proved that coordination of this ligand led to a
decrease in cytotoxicity, reducing it from 1.4-fold against A2780R, to almost 7-fold against the lung
cancer cell line (H226). Nevertheless, according to our results, the Re(I) centre seems to reduce the
cytotoxic activity of the free ligand more than the Pt(II) centre, since Pt(phendione)Cl2 has
comparable IC50 values to cisplatin. Recently, it was referred that the Re(I) complex with appt
(appt=2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine]), which interacts to DNA with minor
groove bindings, has moderate cytotoxicity against several cancer cells (IC50= 50 μΜ against
HeLa), while it is non toxic against noncancerous normal lung fibroblasts [58]. Taking into account
all the above, we could conclude that the metal centre seems to play an important role and the
rhenium(I) centre reduces the toxic effect, at least with that ligand.
Please insert here Table 2 and Fig. 7
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3.4 PAF assays
3.4.1. Inhibitory effect of Re(I) metal complexes and phendione towards the PAF induced WRP’s
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aggregation.
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All compounds under investigation inhibited the PAF-induced aggregation of WRPs. This
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inhibitory effect is expressed by their IC50, since a low IC50 value reveals stronger inhibition of the
PAF-induced aggregation for a given compound concentration. The IC50 values of the samples
tested against PAF induced platelet aggregation are presented in Table 2. The IC50 values are
comparable to the ones achieved from the best specific PAF receptor inhibitors like BN 52021 [59].
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The interesting observation from these data is that the initial complex [Re(CO) 5Cl] with an IC50
value of 170 ± 90 nΜ is better PAF inhibitor than both the phendione ligand and complex 1 with
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IC50 values of 925 ± 130 nΜ and 862 ± 150 nΜ, respectively. These observations indicate that
coordination of phendione to metal core tricarbonyl-Re(I) enhances its inhibitory effect towards the
PAF-induced rabbit PRP aggregation but at the same time the phendione-metal coordination
reduces the inhibitory action of the [Re(CO)5Cl] complex. The later is in contrast with previous data
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where coordination of a ligand to a metal ion enhances the inhibitory activity towards PAF-
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aggregation of both the initial starting compounds –ligand and metallic moiety-e.g when
CE
P
organometallic moieties of Re coordinates to tamoxifen [60].
Please insert here Fig. 7
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3.4.2. Effect of Re(I) metal complexes towards the PAF-basic metabolic enzymes.
In order to determine the possible interactions between the two metal complexes under
study and PAF metabolism, the in vitro effect of these compounds on the activities of PAF-basic
metabolic enzymes lyso-PAF-AT and PAF-CPT was also studied (Fig. 8 and 9). The choice to
study these effects on the PAF-main enzyme activities on rabbit leukocytes homogenates depends
on a) the ease of these cells to be handled and separated b) the finding that they are the main
contributors of PAF-synthesis in blood in comparison to platelets or red blood cells and c) their
participation in inflammatory procedures and manifestations in different diseases, also through their
ability to produce PAF. We have found that both the Re (I) complexes affect PAF biosynthesis (Fig.
8 and 9), namely PAF-CPT and lyso-PAF-AT, and also its main catabolic enzyme, Lp-PLA2, (Fig.
10). Interestingly, the effects of the two complexes differed between the two PAF biosynthetic
enzymes. The [Re(CO)5Cl] complex greatly inhibits PAF-CPT activity while it increases lyso-PAF
AT activity as shown in Figure 8. Actually, it exhibits a very potent inhibitory effect towards PAFCPT (Fig 8) achieved even from the lowest concentration tested and remained stable for the full
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concentrations’ range. Taking into account that this complex also exhibits one of the most potent
anti-PAF effects in platelets, its additional inhibitory effect towards PAF-biosynthesis provide new
anti-inflammatory potentials for this compound.
On the other hand, complex 1 exhibits a small inhibitory effect against lyso-PAF AT that
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remained stable for all the concentrations tested. This complex increases PAF-CPT activity at low
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concentrations, an effect that is reduced at the higher concentrations as shown in Figure 9.
Please insert here Fig. 8 and 9.
Both of the complexes increase Lp-PLA2 activity, with a more potent increment in the case
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of 1 which maintained a higher activity even in the higher concentration tested compared to
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[Re(CO)5Cl]. The latter at a similar concentration returns to its basal level of activity (Figure 10).
Please insert here Fig. 10
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4. Conclusions
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In summary, from viscosity measurements, thermal denaturation and CV measurements it is
observed that fac-[Re(CO)3(phendione)Cl] binds with the grooves of the DNA double helix
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retaining its overall structure, whereas the un-coordinated ligand phendione interacts by
intercalation to the DNA. The different way of the interaction of complex 1 and the corresponding
ligand, namely phendione seems that reflects on their efficacy on various cancerous cell lines as
glioblastoma (T98G), prostate cancer (PC3) and breast cancer (MCF-7). Actually, from the IC50
AC
values it is evident that phendione is extremely toxic against T98G (4.91 μΜ), PC3 (3.01 μΜ) and
MCF-7 (2.45 μΜ) cell lines even more than cisplatin, whereas this cytotoxicity against all cell lines
tested is almost completely lost when phendione ligand coordinates to Re(I). Complex 1 is active in
50 μM since in this concentration almost 45% human breast (MCF-7), 40% glioblastoma (T98G)
and 27% prostate (PC3) cells are killed. It is worth noting that the starting material [Re(CO)5Cl] is
inactive at all tested cell lines even in very high concentrations (200 μM). On the other side, the
coordination of phendione to the tricarbonyl Re(I) core has different results on the inhibitory effect
towards PAF-induced aggregation of WRPs. Thus the initial complex [Re(CO)5Cl] is better PAF
inhibitor than both the phendione ligand and complex 1 indicating IC50 values comparable to the
ones achieved from the best specific PAF receptor inhibitors like BN 52021. Both these metal
complexes exhibit also the ability to modulate in vitro the activities of PAF-basic biosynthetic
enzymes, PAF-CPT and Lyso-PAF-AT, in the direction of down regulating PAF-levels, while only
complex 1 seems to affect PAF-catabolism. Taking in consideration that PAF is key-mediator of the
inflammatory malignant processes in different diseases the ability of the Re(I) complexes under
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study to affect and directly interact with both the PAF/PAF-R and the key biosynthetic enzymes of
PAF, provides a new perspective in the anti-malignant properties of these compounds. However,
several in vivo studies must follow in order to confirm this in vitro interesting outcome. Moreover,
complex 1 was found to promote the photocleavage of plasmid pBR322 DNA from supercoiled
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form I to nicked form II via a proton coupled electron transfer mechanism. This result according
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with its high affinity for DNA [K=7.95(±1.89)*104 M-1] and its accessible photoexcited states in the
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visible region makes complex 1 a potential photodynamic therapy agent.
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5. Abbreviations
1-O-alkyl- 2-sn-acetyl-glycerol
BN 5201
ginkolide
BSA
bovine serum albumin
appt
2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine]
bpy
2,2’-bipyridine
CD
circular dichroism
CDP
cytidine diphosphate-choline
CV
cyclic voltammetry
CT-DNA
Calf Thymus DNA
dppz
dipyrido[2,3-a:2’,3’-c]phenazine
DTT
dithiothreitol
ELISA
enzyme- linked immunosorbent assay
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CE
P
TE
D
MA
AAG
MCF-7
breast cancer
MTT
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromidePAF
Platelet activating factor
PAF-AH
PAF-acetylhydrolase
PAF-AT
PAF-acetyletransferase
PAF-CPT
PAF-cholinephosphotransferase
Lp-PLA2
isoform of PAF-AH
PC3
prostate cancer
phen
1,10-phenanthroline
phendione
1,10-phenanthroline-5,6-dione
PRP
Rabbit platelet plasma
TBE
Tris-borate-EDTA
TCA
trichloroacetic acid
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18
glioblastoma
THF
tetrahydrofuran
WEB 2170
hetrazepines
WRPs
Washed rabbit platelets
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T
T98G
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Acknowledgement
This research has been co-financed by the European Union (European Social Fund – ESF) and
Greek national funds through the Operational Program "Education and Lifelong Learning" of the
National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II
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investing in knowledge society through the European Social Fund. C.A.M. thanks the Special
Appendix A: Supplementary data
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Research Account of UoA for partial financial support.
UV-Vis of 1 in methanolic buffer solution (Figure S1), Circular dichroism measurements of the
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CE
P
[Re(CO)5Cl] (Figure S3).
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interaction of 1 with DNA (Figure S2), and % Cell Viability values of the precursor molecule
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Sonawane, R.R. Joshi, J. Inorg. Biochem. 100 (2006) 331-343.
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973742, 9 pages.
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[55] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319-9324.
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53 (2010) 4633-4641.
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[60] A. Vessiéres, S. Top, W. Beck, E. Hillard, G. Jaouen, Dalton Trans. 28 (2006) 529-541.
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Table 1. Thermal Denaturation and circular dichroism measurements of DNA binding
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studies of complex 1.
0
27.6
r=0.05
-1.62
27.8
r=0.1
-1.72
27.0
r=0.2
-0.76
r=0.5
-2.92
19.0
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r=0
λ (nm)
Δε (M-1cm-1)
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1.82
246
-1.86
278
1.82
245
-1.97
278
1.70
247
-1.50
279
1.55
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-1.97
278
1.73
245
1.93
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%hyp
Circular dichroism data
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ΔTm (oC)
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Tm measurements
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Table 2. Cytotoxicity studies and inhibition of PAF induced platelet aggregation for
cisplatin, phendione, complex (1) and the precursor molecule Re(CO)5Cl. The IC50 values
PC3
phendione
4.91 ± (2.06)
3.01 ± (1.72)
complex 1
> 50
> 50
Re(CO)5Cl
> 200
> 200
cisplatin
6.45 ± (1.64)
2.19 ± (0.11)
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a. from ref 59
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of PAF
2.45 ± (0.71)
0.925 ± (0.13)
>50
0.862 ± (0.15)
>200
0.170 ± (0.09)
11.06 ± (2.49)
0.55 ± (0.22)a
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(μΜ) are represented as mean ± standard deviation.
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Caption of Figures
Fig. 1. Structure of complex 1 and the corresponding phendione ligand.
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Fig. 2. UV-Vis spectra of complex 1 in CH2Cl2, MeCN, MeOH
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Fig. 3. Cyclic voltagramm of 1 (100 μM) in the absence (solid line) and in the presence of
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DNA (200 μM) (dashed line).
Fig. 4. Changes in the UV-Vis absorption spectra of complex 1 (20 μΜ) with increasing
DNA concentrations. Arrow indicates the decreasing intensity of the 300nm band upon
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fit linear for the titration of DNA with 1.
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increasing [DNA]. The inset shows the plot of –[DNA]/ (εa-εf)x108 vs. [DNA]x105 and the
Fig. 5. Effect of increasing amounts of complex 1 (0–50 μM) on the relative viscosity of
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CT-DNA (100 μM).
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Fig. 6. Agarose gel showing cleavage of pBR322 DNA (0.15 μg) incubated with different
concentrations of complex 1 after irradiation at λ>335 nm. Lane 1: plasmid DNA only,
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Lane 2: DNA+10 μΜ of 1, Lane 3: DNA+50 μΜ of 1, Lane 4: DNA+100 μΜ of 1, Lane 5:
DNA+100 μΜ of 1 in the dark, Lane 6: DNA+100 μΜ of 1+DMSO (0.9 M), Lane 7:
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DNA+100 μΜ of 1 deaerated.
Fig. 7. Effect of 50 μΜ of the tested compounds (Re(CO)5Cl, complex 1, ligand phendione,
cisplatin) on % cell viability. Red, green and blue bars represent human glioblastoma,
prostate and breast cancer cell lines respectively. The data are presented as mean (of three
independent experiments) ± standard deviation. The errors of the compound Re(CO)5Cl and
the complex 1 were about 3.5% and 5% at all tested cell lines, respectively. The error of the
other compounds was lower than 1%.
Fig. 8. The effect of Re(CO)5Cl on specific activity of lyso PAF-AT (■) and specific
activity of PAF-CPT (♦).
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Fig. 9. The effect of complex 1 on specific activity of lyso PAF-AT (■) and specific
activity of PAF-CPT (♦).
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Fig. 10. The effect of [Re(CO)5Cl] (■) and complex 1 (♦) on the specific activity of Lp-
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Graphical abstract (synopsis):
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The affinity and mode of complex fac-[Re(CO)3(phendione)Cl] and its constituents
[Re(CO)5Cl] and phendione binding to DNA, as well as their ability to cleave single-strand
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DNA were determined by physico-chemical techniques, whereas biological activities were
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derived from the cytotoxicity, platelet aggregation and enzyme inhibition assays.
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Fig. 5
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Fig. 7
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Highlights:
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A Re(I) complex of 1,10-phendione (1) was synthesized and fully characterized
This complex binds to the major groove of DNA
The [Re(CO)5Cl] is potential inhibitor of PAF whereas 1 reduces PAF catabolism.
The ground state of 1exhibits medium anticancer activity
Complex 1 photocleaves DNA via a proton coupled electron transfer mechanism
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