DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear bis(triazacyclonane) copper complex

Article abstract of DOI:10.1016/j.jinorgbio.2015.01.013

The dinuclear copper(II) complex [Cu₂{bcmp(-H)}(μ-OH)](NO₃)₂·H₂O (1, bcmp = 2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol) has been synthesized and characterized by electrospray ionization mass spectrometry, potentiometric titration and cyclovoltammetry. The X-ray structure of the analogous perchlorate salt [Cu₂{bcmp(-H)}(μ-OH)](ClO₄)₂·2.5H₂O (2) was determined. Cytotoxicity studies showed very promising activity of 1 against various pancreatic tumor cell lines with IC₅₀ values comparable or even lower than those of cisplatin. The Cu complex displayed low toxicity against a human non-tumor cell line (HEK 293) demonstrating selectivity for cancer cells. 1 converts supercoiled pUC19 plasmid DNA into the nicked form at micromolar concentrations in the absence of added reductants. A detailed kinetic study on the hydrolysis of the DNA model bis(2,4-dinitrophenyl) phosphate (BDNPP) has been performed. 1 hydrolyses BDNPP with a second order rate constant of 0.047 M s^{- 1} at pH 8 and 40 °C. Finally, single cell electrophoresis (comet assay) and fluorescence microscopy analysis showed that 1 interacts with cellular DNA and induces apoptotic cell death of Capan-1 pancreatic cancer cells. Western blotting analysis indicated that the Cu complex activates the p53 dependent pathway of apoptosis.

Full text of DOI:10.1016/j.jinorgbio.2015.01.013

JIB-09657; No of Pages 7
Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex
Diego Montagner a,1, Valentina Gandin b,1, Cristina Marzano , Andrea Erxleben ⁎
b
a,
a
School of Chemistry, National University of Ireland, Galway, Ireland
b
Dipartimento di Scienze del Farmaco, Universita' degli Studi di Padova, Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The dinuclear copper(II) complex [Cu {bcmp(-H)}(μ-OH)](NO ) ·H O (1, bcmp = 2,6-bis(1,4,7-triazacyclonon-
2
3
2
2
Received 3 October 2014
Received in revised form 15 January 2015
Accepted 18 January 2015
Available online xxxx
1
-ylmethyl)-4-methylphenol) has been synthesized and characterized by electrospray ionization mass spec-
trometry, potentiometric titration and cyclovoltammetry. The X-ray structure of the analogous perchlorate salt
Cu {bcmp(-H)}(μ-OH)](ClO ·2.5H O (2) was determined. Cytotoxicity studies showed very promising activ-
[
2
)
4 2
2
ity of 1 against various pancreatic tumor cell lines with IC50 values comparable or even lower than those of cis-
platin. The Cu complex displayed low toxicity against a human non-tumor cell line (HEK 293) demonstrating
selectivity for cancer cells. 1 converts supercoiled pUC19 plasmid DNA into the nicked form at micromolar con-
centrations in the absence of added reductants. A detailed kinetic study on the hydrolysis of the DNA model
bis(2,4-dinitrophenyl) phosphate (BDNPP) has been performed. 1 hydrolyses BDNPP with a second order rate
constant of 0.047 M s− at pH 8 and 40 °C. Finally, single cell electrophoresis (comet assay) and fluorescence mi-
croscopy analysis showed that 1 interacts with cellular DNA and induces apoptotic cell death of Capan-1 pancre-
atic cancer cells. Western blotting analysis indicated that the Cu complex activates the p53 dependent pathway of
apoptosis.
Keywords:
Copper
Cytotoxicity
DNA damage
Metallodrugs
1
©
2015 Elsevier Inc. All rights reserved.
1
. Introduction
ascorbic acid. Metal-catalyzed hydrolysis of the phosphodiester backbone
of DNA, however, is also an attractive strategy for damaging tumor cell
DNA irreversibly, because the mechanism does not require the addition
of external agents. Furthermore, hydrolytic metallonucleases may find
additional applications in molecular biology as substitutes for restriction
enzymes or footprinting agents or as biomimetic systems helping to elu-
cidate the mechanism of action of natural nucleases [9]. While natural
enzymes have an active site with one or more metal ions surrounded
by amino acids that are essential for the catalytic processes, in artificial
nucleases the protein matrix is replaced by simple organic ligands.
In this paper we report the synthesis and full characterization of a
novel dicopper(II) complex with a binucleating ligand containing a phe-
nol scaffold and two triaza crown binding sites. The metallonuclease
properties of the Cu complex were investigated in isolated DNA and
the effects of DNA cleavage on cancer cell growth were assessed in
Capan-1 pancreatic cancer cells.
Copper is an essential element for most aerobic organisms. Employed
as a structural and catalytic cofactor it is involved in many biological
pathways [1–3]. In the search for new anticancer metallodrugs that can
overcome the limitations of routinely used Pt drugs such as severe side
effects, toxicity and inherent and acquired resistance, there is growing
interest in biometals like Cu, as they are biocompatible and less toxic
that non-endogenous heavy metals. Furthermore, Cu is an interesting
candidate for the treatment of cancers because of its bioavailability and
the observation of increased Cu levels in cancer tissue [4].
The importance of Cu in anticancer therapy is demonstrated by the
ever increasing group of cytotoxic Cu complexes described in the litera-
ture, with the number of papers reported in 2012 being eight times
greater than in 2008 [5,6]. Several synthetic Cu complexes have been
found to be active both in vitro and in vivo [4].
Cytotoxic Cu(II) complexes often show DNA cleavage activity [4,7,8],
suggesting a connection between these two properties. The mechanism
of Cu complex-induced DNA strand breaks often involves the formation
of reactive oxygen species (ROS) through the Fenton reaction following
the reduction of Cu(II) to Cu(I) by biological reducing agents such as
2. Experimental
2.1. Materials and instrumentation
Cu(NO
3 2 2
) ·2.5H O was purchased from TCI Europe and used as sup-
plied. All the other chemicals and solvents were of analytical or spectro-
scopic grade, obtained from commercial sources and used without
further purification. Deuterated solvents were obtained from Apollo
⁎
Corresponding author. Tel.: +353 91 492483; fax: +353 91 495576.
E-mail address: andrea.erxleben@nuigalway.ie (A. Erxleben).
Joint first authors.
1
http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013
162-0134/© 2015 Elsevier Inc. All rights reserved.
0
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

2
D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Scientific. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) and cisplatin were obtained from Sigma Chemical Co, St.
Louis, USA. Bis(2,4-dinitrophenyl) phosphate (BDNPP) was prepared ac-
cording to the literature [10]. 2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-
equivalent to 1.2 times those of the atom to which they were attached
(1.5 for methyl groups). The asymmetric unit of 2 that crystallized in
the triclinic space group P1 contains two crystallographically indepen-
dent complex cations. A MISSYM check [22,23] was carried out to rule
out any missed symmetry. The Flack's parameter [24] converged to
x = 0.65(2) indicating racemic twinning, which was taken into account
during the final cycles of least squares refinement using the twin law
−1 0 0 0 −1 0 0 0 −1 2. Graphics were produced with ORTEX [25].
Crystallographic data and details of refinement are reported in Table S1.
4
-methylphenol was synthesized by a modified literature procedure [11]
as recently described by us [12].
The pD values of D O solutions were adjusted using concentrated
DNO and NaOD. UV/Vis measurements were carried out on a Varian
2
3
Cary 50 scan spectrophotometer coupled to a Grant thermostated
water circulation bath. Elemental analyses (carbon, hydrogen and nitro-
gen) were performed with a PerkinElmer 2400 series II analyzer.
Electrospray ionization (ESI) mass spectra were recorded with a Waters
LCT Premier XE Spectrometer. Electrochemical measurements were
performed in 50 mM phosphate buffer (pH 7) at room temperature
using a CH Instruments 620 series potentiostat, coupled to an electro-
chemical cell containing an Ag/AgCl reference electrode (Bioanalytical
Systems), a platinum foil counter electrode (Goodfellow) and a 3 mm
glassy carbon working electrode (Goodfellow). Potentials are quoted
versus the Ag/AgCl reference electrode.
2.5. DNA plasmid cleavage
pUC19 DNA cleavage activity of compound 1 was evaluated by aga-
rose gel electrophoresis. Plasmid pUC19 DNA (1 μg/μL) was incubated
with increasing concentration (range 5–50 μM) of 1 in Tris buffer
(50 mM Tris, 18 mM NaCl, pH 8.2) at 37 °C for 3 h in the dark. The reac-
tion was quenched by the addition of 3 μL of loading buffer (0.25%
bromophenol blue and 30% glycerol) and samples were loaded onto a
1% agarose gel in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM
EDTA, pH 8.2). The gels were subjected to electrophoresis for 4 h at
50 V, followed by staining with 0.5 μg/mL ethidium bromide overnight.
Gel bands were visualized using a UV transilluminator and photographed
using an Olympus digital camera.
2
.2. Potentiometric titration
An aqueous solution of 1 (1 mM, 50 mL) was titrated with a stan-
dardized 0.1 M NaOH solution at 25 °C. The ionic strength was main-
tained at 0.1 M with KNO . pH values were measured with a Jenway
510 pH meter fitted with a Refex Sensor Ltd. EC-1910-11 glass elec-
3
2.6. Experiments with cultured human cells
3
trode. The program HYPERQUAD was used to calculate the deproton-
ation constant from the titration data [13].
Compound 1 was dissolved in DMSO just before the experiment and
a calculated amount of drug solution was added to the cell growth me-
dium to a final solvent concentration of 0.5%, which had no detectable
effect on cell killing. Cisplatin was dissolved in 0.9% NaCl solution.
2
.3. UV kinetic measurements
The hydrolysis rates of BDNPP were measured by monitoring the
2.7. Cell cultures
increase in the visible absorbance at 400 nm due to the release of
,4-dinitrophenolate. Rate constants were obtained by the initial rate
method (b5% conversion). Concentrations of 2,4-dinitrophenolate
2
Human breast (MCF-7), colon (HCT-15), and pancreatic (BxPC3, MIA
Paca-2, AsPC-1, Capan-1, PANC-1) carcinoma cell lines along with mel-
anoma (A375) cells and nontransformed embryonic kidney (HEK293)
were obtained from American Type Culture Collection (ATCC, Rockville,
MD). The human ovarian 2008 cancer cell line was kindly provided by
Prof. G. Marverti (Dept. of Biomedical Science of Modena University,
Italy). Human cervical carcinoma (A431) cells were kindly provided
by Prof. F. Zunino (Division of Experimental Oncology B, Istituto
Nazionale dei Tumori, Milan, Italy). Cell lines were maintained in the
logarithmic phase at 37 °C in a 5% carbon dioxide atmosphere using
the following culture media containing 10% fetal calf serum (Euroclone,
Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomy-
cin) and 2 mM L-glutamine: (i) RPMI-1640 medium (Euroclone) for
−
1
−1
were calculated from the extinction coefficients (12,100 M
for 2,4-dinitrophenolate). Concentrations were corrected for the degree
of ionization of 2,4-dinitrophenol at the respective pH value using pK
2,4-dinitrophenol) = 4.0 [14]. In a typical experiment 15 μL of a freshly
cm
a
(
prepared BDNPP stock solution (5 mM in DMSO) was added to a solu-
tion of the metal complex (1.5 mL, 0.25–5 mM) at 40 °C. The metal com-
plex solutions were buffered with 50 mM PIPBS (pH 3.5–5), MES
(
pH 5–6.7), HEPES (pH 6.8–8.5) and CHES (pH 8.5–11.0). The ionic
strength was maintained at 0.1 M with KNO . Cleavage rates were
corrected for the spontaneous hydrolysis of the substrate [10]. The
3
a
kinetic pK value was determined by fitting the data to
2
008, MCF-7, HCT-15, A431, AsPC-1 and BxPC3 cells; (ii) F-12 HAM'S
ꢀ
h
þi
ꢁ.ꢀ
h
þiꢁ
(Sigma Chemical Co.) for A549 cells; iii) DMEM (Sigma Chemical Co.)
for MIA Paca-2, PANC-1, A375 and HEK293 cells; IMDM (Sigma Chemi-
cal Co.) for Capan-1 cells.
kobs
¼
kH2O
H
þ kOHKa
K þ H
:
a
To determine the solvent deuterium isotope effect, analogous kinetic
experiments were performed in 99.9% D O. The correction pD =
2
2.8. MTT assay
pHmeasured + 0.4 was applied to the pH meter readings [15,16]. Kinetic
runs were run in duplicate to give a reproducibility of ± 15%.
The growth inhibitory effect towards tumor cells was evaluated by
means of the MTT assay [26]. Briefly, (3–8) × 10 cells/well, dependent
3
2
.4. X-ray analysis
upon the growth characteristics of the cell line, were seeded in 96-well
microplates in growth medium (100 μL). After 24 h, the medium was re-
moved and replaced with fresh media containing the compound to be
studied at the appropriate concentration. Triplicate cultures were
established for each treatment. After 72 h, each well was treated with
10 μL of a 5 mg/mL MTT saline solution, and following 5 h of incubation,
100 μL of a SDS solution in 0.01 M HCl was added. After an overnight in-
cubation, cell growth inhibition was detected by measuring the absor-
bance of each well at 570 nm using a Bio-Rad 680 microplate reader.
The mean absorbance for each drug dose was expressed as a percentage
of the control untreated well absorbance and plotted vs drug
Crystal data for compound 2 were collected at room temperature on
an Agilent (formerly OxfordDiffraction) Xcalibur CCD diffractometer
using graphite-monochromated Mo-K radiation (λ = 0.71069 Å)
17]. The structure was solved by direct methods and subsequent Fouri-
α
[
2
er syntheses and refined by full-matrix least squares on F using
SHELXS-97, SHELXL-97 [18–20] and Oscail [21]. The scattering factors
were those given in the SHELXL program. Hydrogen atoms except
those for the water molecules of crystallization were generated geomet-
rically and refined as riding atoms with isotropic displacement factors
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
concentration. IC50 values, the drug concentrations that reduce the
mean absorbance at 570 nm to 50% of those in the untreated control
wells, were calculated by a four parameter logistic (4-PL) model.
evaporated using a rotavapor. The residue was dissolved in 15 mL meth-
anol and filtered. Slow diffusion of diethyl ether into the alcoholic solution
afforded green crystals of formula [Cu
Yield: 480 mg (0.71 mmol, 69%). Anal. Calcd. (%) for C21
37.33; H, 5.96; N, 16.58; found: C, 37.15, H, 5.31; N, 16.88%. ESI MS
2 3 2 2
{bcmp(-H)}(μ-OH)](NO ) ·H O.
H
40
N
8
O
9
Cu : C,
2
2
.9. Comet assay
+
(
positive ion mode): m/z: 596.11 [M–NO
[Cu {bcmp(-H)}(μ-OH)](ClO ·2.5H O (2) suitable for X-ray
analysis were obtained by slow evaporation (5 weeks) of an aque-
ous solution of 30 mg of 1 in the presence of 10.8 mg NaClO . Cau-
3 2
–H O] . Crystals of
Single-cell gel electrophoresis for detection of DNA damage was per-
2
4
)
2
2
formed using the comet assay reagent kit purchased from Trevigen
Trevigen Inc., Gaithersburg, MD, US) according to the manufacturer's
(
4
5
2
instructions. Briefly, Capan-1 (2 × 10 ) cells were seeded in 25 cm
tion! Although no problems were encountered in this work, transition
metal perchlorate complexes are potentially explosive and should be
handled with proper precaution.
flasks in growth medium (6 mL). After 24 h, cells were incubated for dif-
ferent exposure times (6 and 12 h) with increasing concentrations of
the tested compound. Subsequently, cells were washed twice with
cold phosphate buffered saline (PBS), harvested, centrifuged, and resus-
3. Results and discussion
5
pended at 1 × 10 cell/mL in 1% low melting point agarose (LMPA,
Trevigen). Then 50 μL of the cells-LMPA mixture was layered onto frost-
ed microscope slides which were pre-coated with 1% normal agarose.
After the agar had been allowed to set at 4 °C, the slides were immersed
3.1. Synthesis and characterization of [Cu
2
{bcmp(-H)}(μ-OH)](NO
3 2 2
) ·H O
(1) and [Cu {bcmp(-H)}(μ-OH)](ClO ·2.5H
2
)
4 2
2
O (2)
in lysis buffer (100 mM Na
% Triton X-100) for 1 h at 4 °C. The slides were then incubated in an al-
kaline electrophoresis solution (1 mM EDTA, 300 mM NaOH, pH N 13) at
°C for 40 min, followed by electrophoresis (1 V/cm) at 4 °C for 30 min.
2
EDTA, 2.5 M NaCl, 10 mM Tris pH 10.0, and
In this study, the dinucleating ligand 2,6-bis(1,4,7-triazacyclonon-1-
ylmethyl)-4-methylphenol (bcmp) with two 9-membered aza-crown
binding sites was chosen because of the well-established high affinity
of Cu2 ions for 1,4,7-triazacyclonane (tacn). The logK value for mono-
1
+
4
2
+
The slides were washed three times with neutralization buffer before
immersion in absolute ethanol for 20 min. and air-dried at room tem-
perature. The DNA was stained with SYBR Green (1 μg/mL) for 5 min.
at 4 °C. A total of 50 comets per slide, randomly captured at a constant
depth of the gel, were examined at 40-fold magnification in a fluores-
cence microscope (Olympus BX41, Milano, Italy; excitation, 495 nm;
emission, 521 nm) connected through a black and white camera to a
computer-based image analysis system. Comets were randomly cap-
tured at a constant depth of the gel, avoiding the edges of the gel, occa-
sional dead cells, and superimposed comets. DNA damage was
measured as tail length (distance of DNA migration from the middle
of the body of the nuclear core) using Cell-F software (Olympus).
nuclear Cu(tacn) is 15.5, suggesting that a highly stable bcmp com-
plex is formed under physiological conditions [27]. Furthermore, Cu
complexes of tacn ligands are generally good catalysts for phosphate
ester hydrolysis [28,29]. The aromatic spacer provides a rigid scaffold
for the bimetallic site, while the bridging phenoxy group shields the
electrostatic repulsion between the metal ions and holds the two
2
+
Cu ions in close proximity. We have previously reported a modified
synthetic procedure for bcmp [12].
The dinuclear Cu(II) complex [Cu
(1) was prepared by reacting the ligand bcmp with two equivalents of
Cu(NO in a methanol/water mixture at pH 6.8 (Chart 1). The com-
2 3 2 2
{bcmp(-H)}(μ-OH)](NO ) ·H O
3 2
)
plex has been characterized by elemental analyses and ESI mass spec-
trometry. The latter shows a signal centered at m/z = 596.1 which
+
2
.10. Hoechst 33258 staining
can be assigned to the isotopomers of [Cu
Fig. S1). [Cu {bcmp(-H)}(μ-OH)](ClO ·2.5H
recrystallizing complex 1 from water in the presence of NaClO
2 3
{bcmp(-H)}(μ-OH)(NO )]
(
2
)
4 2
2
O (2) was obtained by
. X-ray
suitable crystals of 2 were grown by slow evaporation of an aqueous
solution at r.t. and the structure is described below.
Capan-1 cells were seeded into 8-well tissue-culture slides (BD
4
4
2
Falcon, Bedford, MA, USA) at 5 × 10 cells/well (0.8 cm ). After 24 h,
cells were washed twice with PBS and following 48 and 72 h of treat-
ment with IC50 doses of the tested compound, cells were stained for
The potentiometric titration of 1 revealed a single deprotonation
5
1
min with 10 μg/mL of Hoechst (2′-(4-hydroxyphenyl)-5-(4-methyl-
-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate,
step with a pK
a a
value of 4.69 ± 0.09 (Fig. S2). The pK value for the de-
protonation of the first of the two terminally bound water ligands in
mononuclear [Cu(tacn)(H
2
+
Sigma-Aldrich) in PBS before being examined by fluorescence microsco-
2
O)
2
]
a
is 7.3 [29]. Thus, the low pK value
py (Olympus).
found for 1 is in line with a strong binding of the resulting hydroxide
to two Cu ions and can be assigned to the formation of the μ-OH species.
2
.11. Western blot analysis
a
As expected, the pK value is lower than that of the analogous Zn com-
plex (pK = 5.37) [12]. The electrochemical properties of the dicopper
a
6
About 10 Capan-1 cells were treated for 24 and 48 h with IC50 doses
complex were investigated using cyclic voltammetry (CV). Fig. S3
shows the cyclovoltammogram of the complex in phosphate buffer
displaying oxidation and reduction peaks at −0.04 V and −0.62 V
(vs. Ag/AgCl), respectively. The redox process is irreversible which
of the tested compound. Afterwards, cells were harvested, lysed in RIPA
buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS), and centrifuged
at 13,000 × g for 15 min at 4 °C. β-actin was used as a loading control. An
equal amount of proteins for each sample was electrophoresed on a 12%
SDS-PAGE and blotted to a nitrocellulose membrane. The membrane
was incubated for 1 h in PBS-Tween 20 (0.05%) containing 5% nonfat
milk and then at 37 °C for 1 h with primary antibodies. The membranes
were stained with the corresponding peroxidase-conjugated secondary
antibodies for 1 h at room temperature and detected by ECL according
to the manufacturer's protocol (GE).
I
I
suggests a fast decomposition of the Cu –Cu species. This has also
been observed for analogous binuclear Cu(II) complexes where redox
peaks have been assigned to a Cu centered two-electron reduction
II
I
process Cu
2 2
→ Cu [30,31].
The X-ray structure of 2 is shown in Fig. 1, selected bond lengths and
angles are listed in Table 1. The asymmetric unit of the Cu complex
contains two crystallographically independent complex cations, four
perchlorate anions and five water molecules of crystallization. The two
complex cations have very similar geometric parameters and exhibit
the typical features of five-coordinate Cu complexes of triazacyclononane
and its derivatives [32–34]. Each Cu center adopts a distorted square
pyramidal coordination geometry with the bridgehead nitrogen, one of
the secondary nitrogens, the bridging phenolate oxygen and the bridging
hydroxo group defining the basal plane, while the apical site is occupied
2
.12. Synthesis of the Cu complexes
[
Cu
2
{bcmp(-H)}(μ-OH)](NO
·2.5H O (380 mg, 2.06 mmol), dissolved in 15 mL of water
and a solution of bcmp (402 mg, 1.03 mmol) in 5 mL methanol. After
adjusting the pH value to 6.8 with NaOH the green solution was
3 2 2
) ·H O (1) was prepared by mixing
Cu(NO
3
)
2
2
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

4
D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3 3 2 2 2 3 2
Chart 1. Synthetic route for the synthesis of the Cu complex 1: i) BOC-ON, CHCl ; ii) THF/CHCl ; iii) TFA/CH Cl /Et O; iv) Cu(NO ) .
by the other secondary nitrogen. The four Cu ions lie 0.205(6) (Cu(1)),
.197(5) (Cu(2)), 0.180(6) (Cu(3)), and 0.198(6) Å (Cu(4)) above their
respective basal planes. The τ values which indicate the degree of trigo-
nal distortion from an ideal square pyramidal coordination geometry
range from 0.137 to 0.252. According to the definition by Addison
(2008) cancers as well as melanoma. For comparison purposes, the cy-
totoxicity of cisplatin, the most widely used metal-based anticancer
drug, was evaluated under the same experimental conditions. IC50
values, calculated from the dose-survival curves obtained after 72 h of
drug treatment from the MTT test, are shown in Table S2. While the cy-
totoxic activity was considerably lower than that of cisplatin against
MCF-7, A431, HCT-15, A549 and 2008 tumor cell lines, the dimetallic
Cu(II) complex retained a significant in vitro antitumor activity against
pancreatic cancer cells. Tested on a panel of five different pancreatic
cancer cell lines (Table 2), compound 1 showed a cell killing activity
that was similar or even better than that exerted by cisplatin. Interest-
ingly, the greater efficacy was detected in metastatic site derived pan-
creatic Capan-1 and AsPC-1 cancer cell, on the other hand, the
cytotoxic potency was about two times lower than that of cisplatin
against p53 null Mia PaCa-2 cells.
0
(
τ = (α-β)/60, where α and β are the two largest bond angles) an
ideal square pyramid has a τ value of 0, while τ is 1 for a trigonal bipyr-
9
amid [35]. As expected for d Cu ions with square pyramidal coordination
geometry, the Cu\N bond distances are shorter for equatorial nitrogens
(
2.001 to 2.017 Å) than for the apical nitrogen (2.198 to 2.257 Å) [36,37].
The apical nitrogens of the Cu ions are anti to each other. The Cu units
are planar (maximum deviation from the best weighted plane 0.005 Å in
cation 1 and 0.002 Å in cation 2). The Cu plane and the bridging phe-
2 2
O
2 2
O
nyl ring form a dihedral angle of 29.5(8) and 29.0(5) ° in cation 1 and 2,
respectively.
3
3
.2. Biological studies
.2.1. Cytotoxicity
The antiproliferative effects of 1 were assessed on a panel of six
human tumor cell lines including examples of human breast (MCF-7),
cervical (A431), colorectal (HCT-15), pancreatic (BxPC3) and ovarian
NO2
NO2
N(4)
N(3)
NO2
N(1)
O
O(1)
O
Cu(1)
Cu(2)
P
NO2
O
O(2)
N(2)
N(5)
OH
O
N(6)
Cu
Cu
O
2 4 2 2
Fig. 1. View of the cation of [Cu {bcmp(-H)}(μ-OH)](ClO ) ·2.5H O (2) with the atom
numbering scheme. For the sake of clarity, only one of the two crystallographically
independent complex cations is shown. Thermal ellipsoids are drawn at the 40% level.
Chart 2. Possible mechanism of action for the hydrolysis of BDNPP by complex 1.
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
5
Table 1
Selected bond lengths [Å], angles [°] and hydrogen bonding interactions in compound 2.
12
5
2
1
0 µM
5 µM
Cu(1)–N(1)
2.009(6)
2.015(6)
Cu(3)–N(1′)
2.035(6)
2.223(7)
10
8
2.5 µM
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–O(1)
Cu(1)–O(2)
Cu(2)–N(4)
Cu(2)–N(5)
Cu(2)–N(6)
Cu(2)–O(1)
Cu(2)–O(2)
Cu(3)–N(2′)
Cu(3)–N(3′)
Cu(3)–O(1′)
Cu(3)–Cl(1′)
Cu(4)–N(4′)
Cu(4)–N(5′)
Cu(4)–N(6′)
Cu(4)–O(1′)
Cu(4)–O(2′)
2.198(8)
1.933(5)
1.980(5)
2.017(6)
2.015(6)
2.257(7)
1.922(5)
1.961(5)
3.015(1)
86.5(3)
2.013(6)
1.934(5)
1.944(5)
2.044(6)
2.001(6)
2.232(6)
1.941(5)
1.946(5)
2.981(1)
84.0(3)
6
4
…
…
Cu(1) Cu(2)
Cu(3) Cu(4)
2
N(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(3)
N(2)–Cu(1)–N(3)
N(1)–Cu(1)–O(2)
N(2)–Cu(1)–O(1)
N(4)–Cu(2)–N(5)
N(4)–Cu(2)–N(6)
N(5)–Cu(2)–N(6)
N(4)–Cu(2)–O(2)
N(5)–Cu(2)–O(1)
Cu(1)–O(1)–Cu(2)
Cu(1)–O(2)–Cu(2)
N(1′)–Cu(3)–N(2′)
N(1′)–Cu(3)–N(3′)
N(2′)–Cu(3)–N(3′)
N(1′)–Cu(3)–O(2′)
N(3′)–Cu(3)–O(1′)
N(4′)–Cu(4)–N(5′)
N(4′)–Cu(4)–N(6′)
N(5′)–Cu(4)–N(6′)
N(4′)–Cu(4)–O(2′)
N(5′)–Cu(4)–O(1′)
Cu(3)–O(1′)–Cu(4)
Cu(3)–O(2′)–Cu(4)
85.4(3)
86.9(3)
84.3(3)
83.3(3)
0
161.1(2)
173.1(3)
86.5(2)
161.4(2)
176.5(3)
86.7(2)
6
h
12 h
83.6(2)
83.2(2)
Fig. 2. Single cell gel electrophoresis. Capan-1 cells were treated for different incubation
times (6 or 12 h) with increasing concentration of 1 and then processed for comet
assay. The comet tail length was calculated from the center of the cell and measured in mi-
crometers with CellF software. The error bars indicate the S.D.
82.0(2)
82.8(2)
166.7(2)
176.4(3)
102.9(2)
99.8(2)
162.2(2)
172.9(3)
100.6(2)
100.0(2)
cells presented brightly stained nuclei and morphological features typi-
cal of apoptosis, such as chromatin condensation and fragmentation,
thus confirming the induction of an apoptotic cell death.
Complex 1 was also tested against a human non-tumor cell line,
namely human embryonic kidney HEK293 cells (human non-cancerous
cells in rapid proliferation). Notably, against noncancerous cells, complex
It has been shown that in response to treatment with a variety of
DNA-damaging agents an induction of tumor suppressor p53 occurs.
The activation of p53 which plays a pro-apoptotic role by inducing
and downregulating many fundamental cell death effectors, ultimately
leads to apoptotic cell death [38]. By taking into consideration that the
cytotoxic potency of 1 is about two times lower against p53 null pancre-
atic Mia PaCa-2 cells than towards pancreatic p53 wild-type cells
(BxPC3, AsPC-1, Capan-1 and PANC-1), we studied the effect of 1 on
p53. Notably, complex 1 provoked a concentration-dependent increase
in p53 activity (Fig. 3B). Taken together, cytotoxicity and p53 results in-
dicate that this complex can activate apoptotic cell death pathways by a
mechanism that is p53-dependent. Thus, cancer cell sensitivity to the
studied Cu complex strongly depends on the p53 status.
1
elicited a cytotoxic activity approximately 5-fold lower than that record-
ed after cisplatin treatment (Table 2). The selectivity index value, (i.e. the
average IC50 towards normal HEK293 cells divided by the average IC50
towards all malignant cells included in the panel) calculated for 1 was
approximately 1.5-fold higher than that obtained with the reference
metallodrug, thus suggesting a noticeable preferential cytotoxicity of 1
versus neoplastic cells.
3
.2.2. DNA damage
The ability of 1 to cause DNA damage in Capan-1 cells was investi-
gated. After treatment with increasing concentrations of 1 for 6 and
2 h, evidence for cleavage of cellular DNA was obtained by alkaline sin-
1
gle cell gel electrophoresis (Fig. 2). Cells treated with 1 displayed a sta-
tistically significant increase in electrophoretic migration of the DNA
fragments, evidenced by well-formed comets. As evident form the
comet tail lengths (length of DNA migration), which are directly related
to the DNA fragment size, complex 1 caused a marked time- and dose-
dependent increase in comet tail lengths confirming its ability to cleave
cellular DNA.
3.3. DNA cleavage and phosphate diester hydrolysis
To gain insight into the molecular interactions of complex 1 with
DNA, we performed in vitro experiments on supercoiled (SC) plasmid
pUC19 DNA. The cleavage ability of the Cu complex was measured by
monitoring the conversion of SC-DNA (form I) into the nicked-circular
form (NC, form II) using gel electrophoresis. SC pUC19 DNA (1 μg/mL)
was incubated with increasing complex concentrations (range
5–50 μM) for 3 h. Fig. 4 shows representative gel electrophoresis pat-
terns resulting from incubation of SC-DNA with complex 1. Treatment
with complex 1 resulted in a concentration-dependent pUC19 cleavage.
Notably, at 25 μM concentration (lane 3), complex 1 was able to convert
SC-DNA into the NC form and at 50 μM concentration (lane 4) complete
nicking of DNA was observed as evidenced by the absence of the form I.
It is worthy of note that no external reducing agent was added. This
does not necessarily suggest a hydrolytic cleavage mechanism, as sever-
al self-activating oxidative Cu metallonucleases have been described
3
.2.3. Mechanism of cell death induction
Cell death induction in Capan-1 pancreatic cancer cells treated for 48
and 72 h with IC50 doses of complex 1 was also studied by monitoring
the morphological changes of Hoechst 33258-stained cells via fluores-
cent microscopy analysis (Fig. 3A). Compared to control cells, 1-treated
Table 2
IC50 values (μM) of 1 and cisplatin for pancreatic cancer cell lines. Cells (3-8 × 10 ·mL−1)
were treated for 72 h with increasing concentrations of tested compounds. Cell viability
was evaluated by means of MTT test. IC50 values were calculated by the dose–response
curves by means of four parameter logistic model (p b 0.05). SD = standard deviation.
4
[39–45]. In fact, Cu complexes that actually cleave DNA by a hydrolytic
mechanism are less numerous [46–48]. In order to distinguish between
an oxidative and hydrolytic mechanism, ROS scavengers specific for sin-
glet oxygen (DABCO), hydroxyl radical (DMSO) or hydrogen peroxide
IC50 (μM) ± SD
Compound
1
Cisplatin
(
(
DMTU), have been added. The nuclease activity of complex 1
Fig. S4) was not influenced by the addition of these scavengers, thus
MIA PaCa-2
Panc-1
Capan-1
BxPC3
AsPC-1
HEK293
30.43 ± 3.15
21.19 ± 4.25
11.67 ± 6.52
18.77 ± 2.24
14.73 ± 2.52
89.76 ± 4.65
16.76 ± 1.89
8.54 ± 3.54
13.17 ± 0.97
11.13 ± 2.36
15.11 ± 1.94
17.62 ± 2.87
suggesting that DNA cleavage is hydrolytic. Notwithstanding, we
thought it of interest to explore the potential of 1 to mediate phosphate
ester hydrolysis using BDNPP (bis(2,4-dinitrophenyl) phosphate)
which is a widely used model for the phosphodiester linkages in DNA.
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

6
D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
A
Control
1, 48h
1, 72 h
B
Control 6.25 μM 12.5 μM 25 μM
Fig. 3. Apoptosis induction and p53 activation. A. Hoechst 33258 staining. Capan-1 cells were incubated for 48 and 72 h with IC50 doses of complex 1 and stained with the fluorescent dye
Hoechst 33258. B. Capan-1 cells were treated with increasing concentrations of 1 for 48 h. The amount of p53 was detected by Western blotting analysis as described in the Experimental
Section.
1
-mediated BDNPP hydrolysis was monitored by following the in-
crease in the visible absorbance at 400 nm because of the release of
,4-dinitrophenolate. The dependence of the reaction rate of BDNPP
BDNPP in the presence of 1 is 0.98 (Table S3) suggesting a direct nucle-
ophilic attack of the OH group to the phosphorous (Chart 2). This agrees
with work reported by Neves and co-workers who showed that the hy-
2
III
II
cleavage on the pH value in the presence of 1 was studied over the pH
range 3.5–12.0. The plot of the pseudo-first-order rate constants vs.
pH revealed a sigmoidal curve (Fig. 5). Fitting the data to
drolysis of BDNPP by similar binuclear complexes of type Fe (μ-OH)M
III
II
and Fe (μ-CH
that involves intramolecular attack of a terminally Fe -bound hydroxide
50–53]. Neves et al. concluded that μ-OH was a poorer nucleophile than
3 2
COO) M (M = Zn, Cu, Mn, Fe) proceeds via a mechanism
III
[
ꢀ
h
þi
ꢁ.ꢀ
h
þiꢁ
a terminal OH. The dependence of the hydrolysis rate on the concentra-
tion of the Cu complex (pH 8.0, 40 °C, Fig. S5) shows a linear increase of
the hydrolysis rate in the 0.5–2.5 mM concentration range for 1. Higher
concentrations could not be analysed, as the complex precipitated out of
solution. A linear fit of the data gave an apparent second-order rate con-
kobs
¼
kH2O
H
þ kOHKa
K þ H
a
gave kH2O = 5.75 × 10− s−1, kOH = 6.0 × 10−4 s−1 and a kinetic pK
6
a
value of pH 6.97. The kinetic pK
modynamic pK value for the bridging water in [Cu
determined by potentiometric titration (pK
a
value is 2.3 units higher than the ther-
{bcmp(-H)}(μ-
= 4.69). This
−
1
a
2
stant of 0.047 M s . This agrees favorably with the apparent second
3
+
−1
H
2
O)]
a
order rate constant of 0.021 M s reported for the closely related dinu-
II
II
might be explained by a weakening of one of the μ-OH\Cu bonds on
substrate binding (Chart 2). The hydroxide in the catalytically active
species can act as a nucleophile or a general base. To gain more insight
clear Cu (μ-OH)Cu complex with the Schiff base ligand 6-amino-6-
methylperhydro-1,4-diazepine [54,55]. However, DFT calculations for
H D
into the reaction mechanism, the solvent deuterium isotope effect k /k
was measured. The solvent deuterium isotope effect is the classical
method for distinguishing between a nucleophilic and a general base
mechanism. Generally, a k
a nucleophilic mechanism for phosphate ester hydrolysis, where no
proton transfer is involved in the rate-determining step, while a k /k
value greater than 2 supports a general base mechanism, where
M-OH deprotonates an external nucleophile [49]. The average k /k
H D
/k value ranging from 0.8 to 1.5 indicates
H
D
H
D
value obtained at different pL values (L = H, D) for the hydrolysis of
Fig. 5. Rate-pH profile for the cleavage of BDNPP (5 × 10− M) in the presence of 1 (1 mM)
5
Fig. 4. DNA cleavage. Agarose gel electrophoresis patterns of SC pUC19 DNA incubated
with complex 1 in Tris buffer at 37 °C for 3 h. Lane 1: DNA control; lane 2: DNA + 5 μM
at 40 °C. [buffer] = 50 mM (buffer = PIPBS (pH 3.5–5), MES (pH 5–6.7), HEPES (pH 6.8–
1; lane 3: DNA + 25 μM 1; lane 4: DNA + 50 μM 1.
3
8.5), and CHES (pH 8.5–9.5); I = 0.1 M (KNO ).
Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013

D. Montagner et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
7
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BDNPP bis(2,4-dinitrophenyl) phosphate
CHES N-cyclohexyl-2-aminoethanesulfonic acid
DABCO 1,4-diazabicyclo[2.2.2]octane
2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol
[
[
[
[
[
[
DMSO
DMTU
EDTA
ESI
dimethylsulfoxide
dimethylthiourea
ethylenediaminetetraacetic acid
Electrospray ionization
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PBS
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
2-(N-morpholino)ethanesulfonic acid
phosphate buffered saline
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Appendix A. Supplementary data
Crystallographic data for 2 have been deposited with the Cambridge
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Please cite this article as: D. Montagner, et al., DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear
bis(triazacyclonane) copper complex, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.01.013