1. 1 3
J Biol Inorg Chem
DOI 10.1007/s00775-015-1307-x
ORIGINAL PAPER
SOD mimetic activity and antiproliferative properties of a novel
tetra nuclear copper (II) complex
Sagiv Weintraub1
· Yoni Moskovitz1
· Ohad Fleker1
· Ariel R. Levy1
· Aviv Meir1
·
Sharon Ruthstein1
· Laurent Benisvy1
· Arie Gruzman1
Received: 24 June 2015 / Accepted: 21 October 2015
© SBIC 2015
Introduction
The DNA crosslinker, cisplatin, is widely used as an anti-
cancer drug by more than 50 % of patients suffering from
different types of cancer [1, 2]. During the last 30 years,
encouraged by cisplatin’s success as a first-choice drug for
many human cancers, medicinal and coordination chem-
ists have worked in concert to introduce novel metal-based
anticancer drugs [1, 3–6]. Such drugs aim to improve tox-
icity and the drug-resistance problems associated with cis-
platin. For instance, cisplatin is known to have neurotox-
icity and nephrotoxicity side effects, especially in children
[7–9]. In this context, novel chemotherapeutic metal-based
compounds containing metals such as titanium, ruthenium,
tin, and rhodium have been recently reported [10, 11].
Additionally, a number of copper complexes have recently
been shown to have anticancer properties [12–16].
The metal-based anticancer drugs’ mode of action, for
example, that of cisplatin, involves DNA interactions that
inhibit cell replication. In addition to this anti-DNA activ-
ity, the mechanism underlying the SOD mimetic effect has
been considered as a possible means of anticancer activ-
ity for metal-containing anticancer drugs. Superoxide
dismutases (SODs) are vital metalloenzymes (e.g., cyto-
plasmic CuZnSOD-1, mitochondrial manganese SOD-2
(MnSOD), and extracellular CuZnSOD-3) that catalyze
the dismutation of superoxide radical anion (O2
·−
), thereby
protecting the cells against oxidative damage and apopto-
sis [17–19]. The majority of cancer cells have increased
rates of metabolism as well as an increased production of
radical oxidative species (ROS) such as O2
·−
[20–22], as
compared with normal cells. Moreover, it has been shown
that in cancer cells all of the three above-mentioned SOD
enzymes are depleted, resulting in an excess of O2
·−
, which
Abstract The search for novel anticancer therapeu-
tic agents is an urgent and important issue in medicinal
chemistry. Here, we report on the biological activity of the
copper-based bioinorganic complex Cu4 (2,4-di-tert-butyl-
6-(1H-imidazo- [1, 10] phenanthrolin-2-yl)phenol)4]·10
CH3CN (2), which was tested in rat L6 myotubes, mouse
NSC-34 motor neurone-like cells, and HepG-2 human liver
carcinoma. Upon 96 h incubation, 2 exhibited a significant
cytotoxic effect on all three types of cells via activation of
two cell death mechanisms (apoptosis and necrosis). Com-
plex 2 exhibited better potency and efficacy than the canon-
ical cytotoxic drug cisplatin. Moreover, during shorter
incubations, complex 2 demonstrated a significant SOD
mimetic activity, and it was more effective and more potent
than the well-known SOD mimetic TEMPOL. In addition,
complex 2 was able to interact with DNA and, cleave DNA
in the presence of sodium ascorbate. This study shows the
potential of using polynuclear redox active compounds for
developing novel anticancer drugs through SOD-mimetic
redox pathways.
Keywords Tetra nuclear copper (II) complex ·
Apoptosis · SOD mimetic · Cytotoxicity · Anticancer drugs
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1307-x) contains supplementary
material, which is available to authorized users.
* Laurent Benisvy
benisvy@gmail.com
* Arie Gruzman
gruzmaa@biu.ac.il
1
Department of Chemistry, Bar-Ilan University,
5290002 Ramat Gan, Israel

2. J Biol Inorg Chem
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after conversion to the perhydroxy radical (HO2
·
), induces
tumor formation and progression [23]. On the other hand,
it has been shown that artificial over-expression of the
SOD enzymes in cancer cells leads to a significant anti-
tumor effect [24–26]. Though the pharmacological use of
SOD enzymes would constitute a therapeutic modality, it
is limited by classical protein pharmacokinetic problems
such as instability in oral administration, short plasma
half-life, insignificant membrane permeability, and immu-
nological reactions [27]. Therefore, to overcome these
drawbacks, small molecules capable of mimicking SOD
activities have been developed [19, 28]. Most of these
SOD mimetics possess at least one redox-active metal ion,
as in the active site of the natural SODs [e.g., copper (II),
ferrous and manganese (II)]. For example, metalloporphy-
rin complexes of iron, manganese, and copper have been
shown to possess antitumor activity by converting superox-
ide to hydrogen peroxide and by a subsequent Fenton-like
reaction, consequently, generating a hydroxyl radical that
kills cells [3, 29]. Dozens of mono- and dinuclear com-
plexes, predominantly with a copper (II) ion, have shown
an excellent ability to mimic SOD activity [16, 30, 31].
These include complexes of amino acid residues, peptides,
salicylate derivatives, as well as macrocyclic and tetraden-
tate Schiff-bases [32–36]. It is important to mention that
among all reported SOD mimetics, DNA-targeting copper
(II)-based complexes, in particular, have shown many
promising characteristics that made them suitable for anti-
cancer drug development. One of the important reasons for
that phenomenon is related to the fact that one of the most
active anticancer drugs, bleomycin, is a copper-activated,
natural chemotherapeutic medicine [37]. For example,
based on this molecule, and using Cu(II) and 1,10-phen-
anthroline as a ligand, Kellet and co-authors developed a
complex (SOD and catalase mimetic) that could initiate
free radical DNA scission [38]. Importantly, among many
copper (II)-based complexes [38, 39], compound [Cu
(3-methoxysalicylic acid) (1,10-phenanthroline)], which
was reported by O’Connor et al. [40] (Scheme 1), exhib-
ited an antiproliferative effect even in cisplatin-resistant
cancer cells [40].
Recently, we have designed a hetero-ditopic N,O-/N,N-
pro-ligand (LH2, Scheme 1) that has a redox-active N,O-
phenolimidazole chelating unit fused with a N2-phenan-
throline chelating unit [41]. The pro-ligand LH2 can bind
metal ions, either in their neutral form via the N2-phenan-
throline binding mode, as in the octahedral mono-nuclear
iron complex [Fe(LH2)3][ClO4]2 (1) (Scheme 1), or in the
dianionic N,O-/N,N bridging mode, as in the neutral tetra-
nuclear copper(II) complex Cu4L4 (2) [41] (Scheme 1).
Herein, we report the first investigation of these compounds
regarding their possible anticancer effects.
Scheme 1 Structure of [Cu
(3-methoxysalicylic acid)
(1,10-phenanthroline)], LH2,
Complex 1, and Complex 2

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Our data show that complex 2 is highly cytotoxic
towards NSC-34 motor neuron-like cells and HepG-2 car-
cinoma cells and that it is more potent than cisplatin. Com-
plex 2 possesses no appreciable catalase activity, but it has
exhibited a remarkable SOD-like activity, higher than that
of the well-known SOD mimetic agent TEMPOL. To the
best of our knowledge, this is the first time that a tetra-
nuclear complex was reported to have a significant SOD
mimetic-dependent cytotoxic effect in vitro, combined with
the ability to cleave DNA in a therapeutic concentration
range.
Materials and methods
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide, pBR322 DNA, amsacrine, cisplatin, and gel elec-
trophoresis reagents were purchased from Sigma Aldrich
(Rehovot, Israel). Santa Cruz (Dallas, TX, USA) supplied
TEMPOL. All tissue culture reagents, PBS tablets, and
medium were purchased from Biological Industries (Beit
Haemek, Israel). The SOD and catalase assay kits were
supplied by Cayman Chemicals (Ann Arbor, MI, USA).
The xanthine oxidase kit was purchased from BioVision
(Milpitas, CA, USA). The apoptosis detection kit was sup-
plied by Medical and Biological Laboratories (Nagoya,
Japan). DNA plasmid and HindIII restriction endonuclease
were purchased from New England Biolabs (Ipswich, MT,
USA). All materials were analytical grade and were used
without purification unless otherwise noted. The ligand
LH2 and complex 2 were synthesized as recently reported
by Mathias et al. [41].
Synthesis and characterization of 1
A solution of Fe(ClO4)2∙6H2O (0.0712 g, 0.1965 mmol,
1 eq.) in DMF (5 mL) was added to a solution of LH2
(0.25 g, 0.589 mmol, 3 eq.) and dissolved in a minimum
volume of DMF. The reaction mixture was stirred at room
temperature for ca. 1 h. The red solution was then evapo-
rated under vacuum to yield a red oil. The latter was dis-
solved in a minimum amount of methanol and the solu-
tion was left in the open air for 2–3 days, after which
time red crystals had formed. These were filtered with
a Büchner funnel, washed with Et2O, and dried under
vacuum to yield 0.260 g (95 %) of fine powder of [FeL3]
(ClO4)2 (1). Elemental analysis: Calc. for [C81H84N12O3Fe]
[ClO4]2·3DMF·6H2O: C 58.25, H 6.35, N 11.32, O 17.24;
Found: C 58.33, H 6.76, N 11.70, O 17.52 %. NMR (1H,
300 MHz, DMSO): δ 14.34 (s, 1H, OH/NH), 13.21 (s, 1H,
OH/NH), 9.23 (m, 2H, ArH), 8.19 (s, 1H, PhOH), 7.85 (m,
2H, ArH), 7.49 (m, 2H, ArH), 7.46 (s, 1H, PhOH), 1.49 (s,
9H, tBu), 1.42 (s, 9H, tBu). MS ES(+): m/z 664 {M2+}.
UV/vis (CH2Cl2): λmax/nm (ε/M-1 cm−1
): 524 (1930),
486sh (1370), 411sh (1600), 343 (6050), 300sh (10,700),
285 (15,000). IR (ZnSe): ν (cm−1
): 2963, 2923, 2881, 1660
(DMF), 1607 (C=C), 1549, 1508, 1486, 1476, 1466, 1441,
1409, 1389, 1372, 1311, 1286, 1251, 1226, 1203, 1144,
1089, 1028, 994, 936, 888, 862, 818, 815, 778, 729(CH),
666, 639, 620, 580.
Cell culture
NSC-34 cells were cultured in Dulbecco’s modified eagle
medium (DMEM) supplemented with 20 % heat inacti-
vated foetal bovine serum (FBS), 1 % l-glutamine, and 1 %
penicillin–streptomycin solution. HepG-2 was also cultured
in DMEM supplemented with 10 % FBS, 1 % l-glutamine,
and 1 % penicillin–streptomycin. In addition, a solution of
0.2 % amphotericin B was added [42]. The skeletal muscle
cell line (L6) was cultured in minimum essential medium
(Alpha-MEM) containing 2.2 gm/L sodium bicarbonate,
supplemented with 10 % FBS, 1 % l-glutamine, and 1 %
penicillin–streptomycin solution [43]. All cells were grown
at 37 °C in a humidified atmosphere, in the presence of 5 %
CO2.
Cytotoxicity assay
All test compounds were dissolved in DMSO and the
maximum percentage of DMSO present in all wells was
0.1 % (v/v). Each compound solution was added to the
wells in triplicate in the concentration range indicated in
the legends. Following a 96-h cell exposure to each com-
pound, the cells underwent an MTT assay [44]. Briefly,
cells were incubated with 200 μL of MT (5 mg/mL) in
0.1 M PBS, at 37 °C in a humid atmosphere with 5 % CO2
for 2 h. The media was then gently aspirated from test cul-
tures, and 200 μL of DMSO was added. The plates were
then shaken for 2 min, and the absorbance was detected
at 570 nm using a BioTek plate reader (Biotek, Winooski,
VT, USA).
UV/Vis spectrophotometry
The stability of complex 2 and the free ligand were inves-
tigated under biologically relevant conditions (37 °C, pH
7.4, PBS). Briefly, 2.5 µL of complex 2 (0.1 mM) solution
in DMSO and 10 µL of free ligand (0.2 mM) solution in
DMSO were added to 1 mL of PBS, and after the indicated
incubation times, each sample was transferred to a 2 mm
quartz cuvette and the spectra were recorded between

4. J Biol Inorg Chem
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wavelengths 200 and 2500 nm using a Varian Cary 5000
UV–Vis-NIR Spectrophotometer (Santa Clara, CA, USA).
EPR measurement
X-band CW-EPR (continuous wave EPR) spectra of com-
plex 2 were recorded using an E500 Elexsys Bruker spec-
trometer (Billerica, MA, USA) operating at 9.0–9.5 GHz.
The spectra were recorded at room temperature using a
microwave power of 20.0 mW, a modulation amplitude of
3.0 G, a time constant of 120 ms, and a receiver gain of
60.0 dB. The samples were measured in 0.8 mm capillary
quartz tubes (VitroCom, Mountain Lakes, NJ, USA).
DNA binding study (CD measurement)
The DNA binding experiments were performed in distilled
water (pH 7.0) using a pBER322 DNA plasmid (20 μg/
mL). DNA linearization was conducted as previously
described [45]. Solutions of complex 2 (final concentration,
15 μM), LH2 (final concentration, 60 μM), and amsacrine
(25 μM) were added separately to the DNA sample and
incubated for 30 min at room temperature. Each spectrum
was averaged from two successive accumulations at a scan
rate of 50 nm/min, keeping a bandwidth of 1.0 nm. Spectra
were recorded between wavelengths 180 and 400 nm in a
1 mm optical path length, using a Chirascan spectrometer
(Applied Photophysics, Surrey, UK).
Agarose gel electrophoresis
A DMSO solution containing the different compounds in
an Eppendorf tube was treated with the pBR322 plasmid
DNA (5 µL of 20 µg/mL) in Tris–HCl buffer (10 mM, pH
8.0). The content was incubated for 5 h at 37 °C and then
loaded after being mixed with 1 µL loading buffer onto a
1 % agarose gel. The electrophoresis was performed at a
constant voltage (80 V) until the bromophenol blue trav-
elled through 75 % of the gel. The plasmid bands were vis-
ualized by viewing the gel under a UV transilluminator and
then photographed.
DNA cleavage
Plasmid pBER322 DNA was incubated with sodium-l
ascorbate (at twice the complex concentration) and with
DMSO, complex 2, or LH2 for 5 h in Tris–HCl buffer at
37 °C. The electrophoresis was performed at a constant
voltage (80 V) until the bromophenol blue travelled through
75 % of the gel (1 % agarose). The plasmid bands were vis-
ualized by viewing the gel under a UV transilluminator and
then were photographed.
SOD activity assay
The O2
·−
dismutase activities of the metal complexes were
assessed using a modified nitro-blue-tetrazolium (NBT)
assay with a xanthine–xanthine oxidase system as the
source of O2
·−
. The quantitative reduction of NBT to blue
formazan by O2
·−
was followed spectrophotometrically at
450 nm using a BioTek plate reader (Biotek, Winooski,
VT, USA). Assays were run in 230 µL of buffer. Results are
graphed as the percentage inhibition of NBT reduction for
three concentrations. Tabulated results were derived from
linear regression analyses and are given as the concentra-
tion (μM) equivalent of 1 unit of bovine erythrocyte SOD
activity (supplied with the kit). A unit of SOD activity is
the concentration of the complex or enzyme that induces
50 % inhibition in the reduction of NBT (referred to as the
IC50 value).
Catalase activity assay
The ability of the complexes to disproportionate hydrogen
peroxide was assayed using a Cayman Catalase Assay Kit.
Measurements were taken in 240 µL of buffer. The activ-
ity of the samples was calculated from the average absorb-
ance of each sample at 540 nm using a BioTek plate reader
(Biotek, Winooski, VT, USA).
Xanthine oxidase activity assay
The possible inhibition of xanthine oxidase by complex 2
was investigated using a xanthine oxidase activity assay kit
that was purchased from BioVision (Milpitas, CA, USA).
Measurements were taken according to the manufacturer’s
instructions.
Apoptosis/necrosis assay
Apoptosis/necrosis analysis was determined using FACS
analysis (Becton–Dickinson, Franklin Lakes, USA). Hep-
G2 cells were treated with complex 2 (5 µM) or the free
ligand (20 µM) for 48 h. Results of the cell sorting were
analysed using Cell Quest software (Becton–Dickinson,
Franklin Lakes, NJ, USA). A total of 10,000 events were
acquired. The cell populations were displayed as a dot
plot divided into four quadrants with Annexin V-FITC
fluorescence (x-axis) versus propidium iodide fluorescence
(y-axis). Both reagents were supplied with the kit.
Statistical analysis
Results are given as mean ± SEM, n = 3. Statistical sig-
nificance (p < 0.05) was calculated among experimental

5. J Biol Inorg Chem
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groups using the two-tailed Student’s t test using the web
resource: http://www.graphpad.com/quickcalcs/ttest1.cfm.
Results
In vitro cytotoxic activity
The cytotoxic effect of LH2, as “free” or coordinated in
neutral and dianionic forms in complexes 1 and 2, respec-
tively, has been investigated in the following cancer cells:
NSC-34 (mouse neuroblastoma/mouse motor neurons)
that represents a central neural system tumor [46], HepG-2
(human liver hepatocellular carcinoma), which represents
a somatic tumor, and rat L6 skeletal muscle myotubes as
non-cancerous control cells. These cells spontaneously dif-
ferentiate at near confluency in low serum concentrations
from myoblasts to myotubes, which are multinuclear cellu-
lar clusters with many morphological similarities to native
muscle myofibrils. Such a transformation interrupts the
cellular proliferation and therefore L6 myotubes are in use
as a control for highly differentiated non-dividing cells in
in vitro experiments.
Figure 1a, b shows that both LH2, and complex 2 have
significant cytotoxic effects in both types of cancer cells.
Within the determined optimal conditions (an incubation
time of 96 h and a compound concentration of 5 μM) about
80 % of cells were killed by the cytotoxic effect that was
induced in an almost identical manner by 2 and LH2. In
contrast, when the ligand was bound to a Fe(II) center, as
in complex 1, no significant cytotoxic effect was observed
(Fig. 1a, b).
In addition, as shown in Fig. 1c, the elevated cytotoxic
effects of LH2 and 2 appear to be non-discriminatory,
which was also observed under similar conditions in L6
myotubes. For example, L6 cell viability was dramatically
reduced to 8.14 ± 0.15 % for 2 (Fig. 1c). It is important
to mention that under these experimental conditions free
Fig. 1 Cytotoxicity activity
(MTT) of test compounds after
96 h incubation in: a NSC-34
cells. b Hep-G2 cells. c L6
myotubes. (Pt cisplatin; CN
acetonitrile); n = 3

6. J Biol Inorg Chem
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copper ions (e.g., CuCl2), acetonitrile, and cisplatin did not
exhibit a cytotoxic effect towards cancer cells (see ESM,
Figure 1). Thus, both the free ligand and the neutral tetra-
copper (II) complex 2 appear to be highly cytotoxic to
both cancerous as well as non-cancerous cells, showing no
selectivity. In contrast, the strong N2-binding mode of LH2
to the Fe(II) ion in complex 1 appears to prevent the cyto-
toxicity of the ligand.
To better understand the cytotoxic activity of LH2 and
2, additional in vitro assays were conducted. The inactive
complex 1 was not tested in additional experiments.
Stability of complex 2 in aqueous medium
To rule out the possibility that the cytotoxic effect of com-
plex 2 is attributed to the free ligand activity, resulting
from the dissociation of the complex under physiological
conditions, the complex stability was monitored by UV/
Vis and EPR spectroscopies. As shown in Fig. 2, the UV/
vis spectra of complex 2 (0.1 mM, in PBS buffer at pH 7.4,
37 °C) remains identical for at least 96 h, and no decrease
in the intensity of the complex characteristic absorption
bands (at 286 and 345 nm [41]) was observed. Further-
more, X-band CW-EPR measurements were carried out
on aqueous solutions of complex 2 at room temperature at
0, 24, and 96 h incubation times. As shown in Fig. 3, the
characteristic isotropic single signal of complex 2 [41] at
ca. g = 2.1 remained identical during the entire incubation
time, up to 96 h. Moreover, no additional signals of free
Cu(II) ions were observed, indicating that the structure of
the tetracopper(II) complex 2 remained unchanged (Fig. 3).
These findings support the hypothesis that the cytotoxic
effect of complex 2 is not related to the cytotoxic activity
of the free ligand and that the cytotoxicity pathways of both
compounds have distinct mechanisms.
DNA binding and cleavage studies
The potential ability of LH2 (60 μM) and complex 2
(15 μM) to interact with DNA was investigated with super-
coiled pBR322 plasmid DNA and was monitored by circu-
lar dichroism (CD). The significant differences in the bio-
logical activity between complex 2 and cisplatin indicate
that both compounds have different cytotoxic mechanisms.
Therefore, instead of cisplatin, the well-known intercala-
tion agent amsacrine was used as a positive control for the
experiment. The CD DNA absorption rate of amsacrine
has been well studied in the 180–400 nm region [47–50].
Thus, the DNA and test compounds were run at this spe-
cific wavelength range, as shown in Fig. 4. The CD spec-
tra of pure DNA (20 μg/mL) were recorded as a standard
value (Fig. 4b). All bands obtained by this measurement
corresponded to DNA bands that were reported previously
in the literature. As shown in Fig. 4a, all three compounds
interact with DNA. Amsacrine, as predicted, binds to
DNA and markedly changes the native DNA structure; as
indicated by a blue shift in the DNA peak from 225 nm
(Fig. 4b) to 209 nm (Fig. 4a). This specific band repre-
sents the helicity of DNA under native conditions [50].
In addition, the intensity of the amsacrine signal in the
range of 200–220 nm increased dramatically compared
with pure DNA. Moreover, both compounds (complex 2
and LH2) also interacted with DNA. However, the mode
of interaction was slightly different. Complex 2 led to a
Fig. 2 UV/Vis spectra of complex 2 (10 µM) in phosphate-buffered
saline (pH 7.4) at 37 °C recorded at 24 and 96 h incubation time
Fig. 3 X-band CW-EPR spectra of complex 2 in fluid solution [5 µM
in 80 % PBS, pH 7.4/20 % DMSO] recorded at RT with incubation
times of 0 h (A), 24 h (B) and 96 h (C). The measurement was con-
ducted as described in “Materials and methods”

7. J Biol Inorg Chem
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stronger blue shift than did amsacrine (202 nm compared
with 209 nm) and to a lower signal intensity (Fig. 4a).
LH2 induced the most significant blue shift in the corre-
sponding band (195 nm), but the signal intensity values
were much lower than the corresponding amsacrine and
complex 2 values (Fig. 4a). Both molecules changed the
intensity of the CD signal. An additional band (275 nm) in
the DNA CD measurement occurred because of the stack-
ing interactions of the DNA bases [50]. This band is very
sensitive to any interactions between DNA and the differ-
ent ligands. For example, the intensity and wavelength of
the absorption of this band correlate with the aggregation
effect of amsacrine on the DNA duplex [50]. As shown in
Fig. 4c, amsacrine indeed increased the intensity and red-
shifted this band as compared with native DNA. Interest-
ingly, complex 2 did not affect the position of the band but
decreased the intensity of the signal as compared to DNA.
Conversely, LH2 did not affect the intensity of the band but
similarly to amsacrine, it induced a red shift of the band
(to 283 nm).
In addition to the binding activity, the possible effect
of compound 2 on DNA relaxation was tested by aga-
rose gel electrophoresis. As shown in Fig. 4d, complex 2
(15 µM) and LH2 (60 µM) did not appreciably affect the
DNA structure and more than 70 % of the DNA remained
supercoiled. However, in the presence of sodium ascorbate
reducing agent, the supercoiled DNA was totally cleaved
by complex 2, as shown in Fig. 4e. This observation fur-
ther suggests that complex 2 can interact with supercoiled
DNA and that the presence of redox-active Cu(II) ions [that
can be reduced to Cu(I)] is essential for supercoiled DNA
cleavage.
A B
C
wavelength
CD(mdeg)CD(mdeg)
wavelength
CD(mdeg)
wavelength
E
D
NC
LC
SC
NC
LC
SC
1 2 3 4 5 6
1 2 3 4 5 6
Sodium Ascorbate [120 µM]
180 200 220 240 260 280 300 320 340 360
230 240 250 260 270 280 290 300 310 320 350340330
200 220 240 260 280 300 320 340 360 380
Fig. 4 DNA binding and cleavage analysis determined by CD and
gel electrophoresis. a CD signal spectra of the pBR322 plasmid DNA
(20 μg/mL) in the presence of complex 2 (15 μM), LH2 (60 μM),
and amsacrine (25 μM). b CD signal spectra zoom in of the pBR322
plasmid DNA (20 μg/mL). c Zoom in on the CD signal spectra of
pBR322 plasmid DNA (20 μg/mL) in the presence of complex 2
(15 μM), LH2 (60 μM), and amsacrine (25 μM) at the 230–350 nM
range. d DNA binding determined by gel electrophoresis. DNA bands
designation: supercoiled DNA (SC), linear circular (LC) nicked cir-
cular (NC). Plasmid DNA was linearized with Hind III restriction
endonuclease and recovered by phenol extraction and ethanol precipi-
tation. Lane 1 marker; lane 2 DNA; lane 3 treated by Hind III DNA;
lane 4 DNA + 1 % DMSO; lane 5, DNA + complex 2 (25 µM); lane
6 DNA + LH2 (60 µM). e Plasmid pBER322 DNA (20 μg/mL) was
incubated without and with complex 2, DMSO, LH2 and sodium
ascorbate for 1 h in Tris–HCl buffer (pH = 7.4) at 37 °C. The plas-
mid bands were visualized by viewing the gel under a UV transillu-
minator. Lane 1 marker; lane 2 DNA; lane 3 DNA + 1 % DMSO;
lane 4 DNA; lane 5 DNA + complex 2 (25 µM); lane 6 DNA + LH2
(60 µM)

8. J Biol Inorg Chem
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Cell death pathways
The classical cell death pathways of apoptosis and necro-
sis [51] were investigated for both the ligand and com-
plex 2 using fluorescence-activated cell sorting (FACS)
analysis. To discriminate between apoptotic and necrotic
cells, dual cell staining was performed with fluorescent
Annexin V and propidium iodide (PI), respectively. Such
a method can show the levels of apoptosis and necrosis
detected by flow cytometry [52, 53]. Experiments were
conducted using HepG-2 cells after 48 h of incubation
because after incubating for 72 h (and obviously similarly
for 96 h, the time we used for MTT) the amount of live
cells would be insufficient to conduct the FACS analysis.
For complex 2 (Fig. 5a), out of the total amount of dead
cells, 38.5 % were detected as late apoptosis, 19.5 % were
detected as necrotic, and 42.0 % exhibited both late apop-
totic and necrotic signs. Regarding the ligand (in a concen-
tration chosen to be four times higher than 2, i.e. 20 µM,
as described for MTT viability assays), the apoptotic cell
death pathway is clearly dominant, with 30.67 % for early
and 59.6 % for late apoptosis, respectively. Only 9.72 % of
the dead cells were necrotic (Fig. 5b).
It is important to mention that the free ligand alone,
LH2, induced apoptosis more effectively than did com-
plex 2. The percentage of only apoptotic cells that was
detected after the cells were exposed to the free ligand
was around 79.0 %, compared with ~59 % for complex 2.
Such a difference in the amount of dead cells may indi-
cate that the two compounds have different mechanisms
of action.
SOD activity
The potential SOD mimetic activities of both compound
2 and LH2 were examined using the xanthine/xanthine-
oxidase-based method, which is used for generating the
superoxide radical [54]. The cell membrane-permeable
SOD mimetic TEMPOL was used as a positive control
[55, 56]. The obtained results are reported in SOD equiva-
lent activity units and are shown in Fig. 6a. The calculated
EC50 values are presented in Fig. 6b. The experimental data
indicated that ligand LH2 displayed no apparent SOD-like
activity, whereas compound 2 indeed exhibited SOD-like
activity with an EC50 of 1.9 µM. This value is almost iden-
tical to the value reported by O’Connor et al. [40] where
EC50 = 1.72 µM for the [Cu (3-methoxysalicylic acid)
(1,10-phenanthroline)]2+
complex. This evidence also
revealed that complex 2 is approximately 2.5-fold more
potent than TEMPOL (EC50 = 5.48 µM).
In order to rule out the possibility that the SOD-like
activity of complex 2 was not related to the direct inhibi-
tion of xanthine oxidase by the complex, the xanthine oxi-
dase activity of complex 2 was investigated using a com-
mercially available xanthine activity kit, which measures
the generation of hydrogen peroxide as a result of the
conversion of xanthine to uric acid by the enzyme. Such
a test was a crucial since Cu (II)-containing complexes
such as [Cu(II)(β-citryl-l-glutamate)] and even Cu(II) by
itself have shown significant xanthine oxidase inhibition
[57, 58]. The ESM Figure 2 shows that complex 2 did not
have any inhibitory effect on xanthine oxidase. Moreover,
as expected, the amount of hydrogen peroxide that was
Fig. 5 Determination of apoptosis/necrosis using flow cytometry
analysis. The apoptotic effect of complex 2 (a 5 µM) and LH2 (b
20 µM) after a 48 h incubation in Hep-G2 cells was determined by
the Annexin V/PI staining method. Each panel shows negative (via-
ble) cells (lower left quadrant), annexin V-positive (early apoptotic)
cells (lower right quadrant), PI-positive (necrotic) cells (upper left
quadrant), or annexin V and PI double-positive (late apoptotic) cells
(upper right quadrant)

9. J Biol Inorg Chem
1 3
detected in treatment using the complex 2 samples was
dose-dependently higher compared with the control meas-
urements, suggesting that hydrogen peroxide was produced
both by the xanthine oxidase reaction and by dismutation
of the superoxide radical (which was synthesized in the
process of oxidizing xanthine) by complex 2. Hydrogen
peroxide, which came from both sources, was determined
by the kit.
Catalase activity
Although the catalytic site of the catalase enzyme is occu-
pied by an iron metal ion, several copper complexes have
exhibited catalase activity [59, 60]. The ability of complex
2 to disproportionate hydrogen peroxide was investigated
using an H2O2 colorimetric detection method (Cayman
Catalase Assay Kit). This method is based on the fact that
catalase exhibits peroxidase activity in which low molec-
ular weight alcohols (in our case, methanol) can serve as
electron donors for its own oxidation towards formalde-
hyde. The obtained formaldehyde reacts with chromogen
(4-amino-3-hydrazino-5-mercapto-1,2,4-trizole) to pro-
duce a purple bicyclic heterocycle(5,6,7,8-tetrahydro- [1,
2, 4] triazolo[4,3b] [1, 2, 4, 5] tetrazine-3-thiol), which
is detected by a colorimeter [61, 62]. As shown in ESM
Figure 3, complex 2 displays significant catalase-like activ-
ity (5 µM of compound 2 converted 0.2 ± 0.02 µM of
hydrogen peroxide in 1 min), as compared with the ligand,
which is negligible. However, the catalase-like activity of
complex 2, calculated to be 0.0244 U/mg (by extrapolat-
ing a standard curve of bovine liver catalase), is extremely
low, compared with a well-known SOD/catalase mimetic
manganese-based complex (EUK-134) [56], with an activ-
ity equal to 26 U/mg.
Discussion
The potential antiproliferative effect of two (Fe- and Cu-
based) complexes bearing the phenol-phenanthroimidazole
ligand LH2 was tested in two cancer cell lines: NSC-34 and
in HepG-2. As a control for normal cells, non-cancer rat L6
skeletal muscle cells were used.
Cell viability assays showed that the octahedral mono-
nuclear iron complex 1 was inactive. In contrast, both the
neutral tetra-nuclear copper(II) complex Cu4L4 (2) and
the free ligand LH2 induce cytotoxic effect. In addition,
cell death pathway, DNA binding, and SOD activity stud-
ies indicate that the cytotoxic activity of the free ligand
and complex 2 operate through different mechanisms.
In particular, the complex 2 exhibits a remarkable SOD-
like activity, in combination with DNA cleavage ability;
whereas the ligand LH2 lacks both of these properties. Both
SOD-like activity and DNA cleavage ability of complex 2
may therefore play a key role in explaining the high cyto-
toxicity of the latter towards cancer cells.
It is known that SOD enzymes play a critical role in
cancer pathogenesis. The first reported work by L. Ober-
ley et al. in 1978 showed that H6 hepatoma cells do not
express Mn SOD [63] and that the loss of Mn SOD activ-
ity seems to be an important characteristic of tumor cells
in general [64]. Additionally, the transfection of MnSOD
gene and overexpression of the protein, in hamster cheek
pouch carcinoma cells, has shown to lead to a significant
cell growth rate decrease of 50 %. [25]. Such antiprolifera-
tive and anticancer effects of SOD overexpression in vitro
and in vivo were also obtained in many different types of
cancer cells and tumors [65]. For example, the volume of
human oral squamous carcinoma cell xenografts in mice
was reduced by 70 % after MnSOD overexpression [24].
Similar positive results were obtained with human breast
cancer cells in vitro [24]. In addition, the overexpression
of the CuZnSOD in cancer cell has been shown to induce
a similar antiproliferative effect than that of MnSOD [66].
Fig. 6 Measurement of SOD activity. a The SOD mimetic dose
response effect of test compounds. b EC50 values. The concentra-
tions were calculated as an equivalent for the effect of 1unit of SOD
supplied by the kit. The assay was conducted using a commercially
available SOD activity kit according to the manufacturer’s provided
protocol

10. J Biol Inorg Chem
1 3
Interestingly, in contrast to cancer cells, in normal cells, the
overexpression of SOD is believed to have a cytoprotective
effect owing to normal endogenous antioxidant protein lev-
els [66].
The molecular basis for the cytotoxic effect of overex-
pressed SODs in cancer cells has been explained by the
increased dismutation rate of superoxide anion to hydro-
gen peroxide (approximately by four orders of magnitude
over spontaneous dismutation) [3]. By increasing the rate
of H2O2 production over that of spontaneous superoxide
dismutation, the effect of SOD on cell growth could be due
perturbation of normal signaling pathways; or cell dam-
age caused through hydroxyl radical production formed
by Fenton-like reaction with metal cations such as Fe2+
or
Cu1+
[66–68].
Based on the knowledge obtained from SOD overex-
pression studies in cancer cells, it is believed that molecular
systems that have SOD-like activity may have potential use
as anticancer agents [24]. In that respect the herein reported
highly cytotoxic and SOD-mimetic complex 2 may have a
similar effect to that of overexpressed SOD in cancer cells.
Considering the tetranuclear nature of the complex 2, and
its ability to bind exogeneous ligand at labile positions of
each copper(II) ions [41], it is most likely that compound
2 acts according to the known SOD enzymatic mechanism
(Scheme 2) [38, 69]. Thus, presumably, the Cu(II) centers
in complex 2 bind and oxidises superoxide anions to oxy-
gen. The resulting reduced Cu(I) form of the complex, in
turn, may reduce superoxide anions, which, in the presence
of protons, produces hydrogen peroxide, although this
remains to be determined (Scheme 2).
Recently, the hydroxyl radical, which is an extremely
toxic substance for cells (inducing DNA cleavage or non-
specific reactions with other macromolecules), has been
generated from hydrogen peroxide in a Cu(I)/Cu(II) cata-
lytic system, as was shown by several researchers [38, 69].
According to the DNA binding assay results, we also
hypothesized that the cytotoxic effect of complex 2 can
be related to interruptions in the cell division cycle (DNA-
related cytotoxicity). Complex 2 binds to DNA as moni-
tored by the drastic changes induced in the CD spectrum
of DNA. The oxidative DNA cleavage ability of 1,10-phen-
anthroline (one of the structural domains in the ligand) in
the presence of Cu(II), was demonstrated in the 1970s by
Sigman et al. [70]; and contributed to the cytotoxic effect
of the copper complexes [71]. Recently, diverse types of
different copper-containing complexes with oxidative DNA
cleavage ability have been synthesized [72–77]. Most of
them exhibited a biological effect only in the presence of
some reductant. However, several complexes among them
were also able to cleave DNA without the presence of
reductant agents [39, 70]. As presented in this work, com-
plex 2 exhibited significant DNA cleavage in the presence
of sodium ascorbate as a reducing agent. Combining these
data with the fact that complex 2 has very efficient super-
oxide dismutase mimetic activity, we can conclude that
complex 2 can cleave DNA through free radical generation
and by these two effects it can induce the observed cyto-
toxic results.
Regarding catalase activity, it is commonly believed
that complexes exhibiting such activity may have a cell
protective effect (with cancer cells such an effect is not
wanted) due to their ability to catalytically disproportion-
ate H2O2 (generated by the SOD activity) to harmless water
and molecular oxygen; preventing thus the formation of
cytotoxic OH•
by Fenton chemistry [40]. The complex 2
has a low catalase activity compared with the potent SOD
mimetic [Cu(ph)(2,2′-bipy)]2
.
2H2O, reported by Kel-
let et al. [38]. This complex, which also has strong cata-
lase activity, was still extremely toxic for cancer cells [38].
Such results have validated the concept that even if catalase
activity is exhibited by a complex, the ability of hydrogen
peroxide directly or via the formation of the hydroxyl radi-
cal to consequently lead to cell damage is high. Moreover,
the generation of hydrogen peroxide is sufficient for this
effect, as was shown by Kasugai et al. for water-soluble Fe-
porphyrin-based SOD mimetics [29].
Based on the data presented here, we can conclude that
complex 2 is a potent SOD mimetic, which may explain its
cytotoxic activity in a similar manner to the antiprolifera-
tive and anticancer effects observed by SOD-overexpressed
SOD
Cu(II)
=
Cu(II) + O2
- Cu(I) + O2
O2
-2H+
H2O2
Cell proliferaƟon
X
Scheme 2 The proposed mechanism of Complex 2 action

11. J Biol Inorg Chem
1 3
cancer cells. However, we cannot rule out the possibility
that complex 2 cytotoxic activity is also mediated by DNA
binding and oxidative cleavage. Interestingly, our experi-
ments have indicated that for the specific cell lines used,
cisplatin displayed only a minor cytotoxic effect compared
with the significant activity of complex 2. Alternatively, the
free ligand (LH2), which was ineffective as a SOD mimetic
but binds DNA, induces its biological activity mainly by
a DNA-related mechanism of action, which is now under
investigation. In addition, the distribution of necrotic/apop-
totic cells, which was observed in the free ligand and in the
complex 2 treatments, was clearly different. This fact also
points out that both compounds have different mechanisms
of action.
In summary, we described here a potent and effective
cytotoxic copper-based complex 2 with SOD mimetic
properties. Complex 2 exhibited a highly potent cytotoxic
effect in two types of cancer cells (NSC-34 and HepG-
2). Interestingly, it was more potent than the well-known
chemotherapeutic drug cisplatin. Moreover, complex 2
was also more potent in the SOD activity assay than was
the well-known SOD mimetic molecule TEMPOL. Addi-
tionally, complex 2 exhibited very low catalase activity.
Taken together, such a combination of biological activi-
ties could be used to develop novel metal-based anti-
cancer drugs, which might provide an additional option
for inorganic chemistry-related therapeutics besides
cisplatin.
Acknowledgments This study was partly supported by Bar-Ilan
University‘s new faculty Grants for AG and LB. We thank Dr. M.
Kanovsky and S. Manch for editing the manuscript.
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