DNA binding, crystal structure, molecular docking studies and anticancer activity evaluation of a copper(II) complex

Article abstract of DOI:10.1007/s11243-018-0211-y

A copper complex [Cu(HPBM)(l-Phe)(H₂O)]·ClO₄(1) (HPBM?=?5-methyl-2-(2′-pyridyl)benzimidazole, l-Phe?=?l-phenylalanine anion) was synthesized and characterized by elemental analysis, IR, ESI–MS, HR–ESI–MS, ESR spectroscopy, and by X-ray single-crystal analysis. The binding constant of the complex with calf thymus DNA (CT-DNA) was determined as 7.38 (±?0.57)?×?10⁴?M^?1. Further studies indicated that the complex interacts with CT-DNA through minor groove binding. The in vitro cytotoxic activities of both the free proligand and the complex against Eca-109, HeLa and A549 cancer cells and normal LO2 cells were evaluated by the MTT method. The IC₅₀ values range from 5.7?±?0.1 to 8.3?±?0.6?μM. Free HPBM displays no cytotoxic activity against the selected cancer cells, with IC₅₀ values more than 100?μM. Double staining analysis showed that the complex can induce apoptosis in Eca-109 cells. Comet assays demonstrated that the complex can damage DNA and cause apoptosis. The complex also induces an increase in intracellular reactive oxygen species and a reduction in mitochondrial membrane potential. The complex can also increase the intracellular Ca²⁺ level and induce release of cytochrome c. The cell cycle arrest was investigated by flow cytometry. The results demonstrate that the complex induces apoptosis in Eca-109 cells through DNA-binding and ROS-mediated mitochondrial dysfunctional pathways.

Full text of DOI:10.1007/s11243-018-0211-y

Transition Metal Chemistry
https://doi.org/10.1007/s11243-018-0211-y
DNA binding, crystal structure, molecular docking studies
and anticancer activity evaluation of a copper(II) complex
1
1
1
1
1
1
2
Ya‑Xian Liu · Hui‑Wen Mo · Zhen‑Yu Lv · Fang Shen · Chun‑Lian Zhang · Yong‑Yu Qi · Zong‑Wan Mao ·
Xue‑Yi Le1
Received: 13 November 2017 / Accepted: 26 January 2018
©
Springer International Publishing AG, part of Springer Nature 2018
Abstract
A copper complex [Cu(HPBM)(l-Phe)(H O)]·ClO (1) (HPBM = 5-methyl-2-(2′-pyridyl)benzimidazole, l-Phe = l-pheny-
2
4
lalanine anion) was synthesized and characterized by elemental analysis, IR, ESI–MS, HR–ESI–MS, ESR spectroscopy, and
by X-ray single-crystal analysis. The binding constant of the complex with calf thymus DNA (CT-DNA) was determined as
4
−1
7
.38 (± 0.57) × 10 M . Further studies indicated that the complex interacts with CT-DNA through minor groove binding.
The in vitro cytotoxic activities of both the free proligand and the complex against Eca-109, HeLa and A549 cancer cells and
normal LO2 cells were evaluated by the MTT method. The IC values range from 5.7 ± 0.1 to 8.3 ± 0.6 µM. Free HPBM
5
0
displays no cytotoxic activity against the selected cancer cells, with IC values more than 100 µM. Double staining analysis
5
0
showed that the complex can induce apoptosis in Eca-109 cells. Comet assays demonstrated that the complex can damage
DNA and cause apoptosis. The complex also induces an increase in intracellular reactive oxygen species and a reduction
2
+
in mitochondrial membrane potential. The complex can also increase the intracellular Ca level and induce release of
cytochrome c. The cell cycle arrest was investigated by ꢀow cytometry. The results demonstrate that the complex induces
apoptosis in Eca-109 cells through DNA-binding and ROS-mediated mitochondrial dysfunctional pathways.
Introduction
investigated [5–8]. In particular, the DNA interactions
and anticancer activities of copper complexes have been
extensively investigated [9–11]. As an essential trace ele-
ment, copper is known to be involved in both metabolism
and endogenous oxidative DNA damage associated with
aging and cancer [12–14]. Recent research has shown that
transition metal complexes of aromatic heterocyclic ligands
can recognize nucleic acid and can be applied as DNA
structure probes, footprinting agents, site-speciꢁc cleav-
age agents, DNA “molecular light switches” and anticancer
drugs [15–18]. It is also known that biological activities
can be higher when a small biomolecular ligand such as
an amino acid, dipeptide and so on is incorporated into the
compound [19–21]. In continuation of our interests in this
ꢁeld, we have synthesized and characterized a new aromatic
heterocycle-based copper(II) complex with an amino acid
co-ligand (Scheme 1). We have further explored the DNA
binding and cleavage properties of the complex by electronic
absorption titration, viscosity measurements, circular dichro-
ism spectroscopy, cyclic voltammetry, DNA cleavage and
molecular docking studies. In addition, the in vitro anti-
cancer activity of the complex was investigated by MTT
method, apoptosis, comet assay, reactive oxygen species
One of the deꢁning features of cancer is the rapid growth
of abnormal cells beyond their usual boundaries, which
can then invade adjoining parts of the body and spread to
other organs [1]. In recent decades, the clinical success of
cisplatin has provided an essential tool in cancer chemo-
therapy. However, many platinum drugs are also associated
with side eꢂects such as severe toxicity and drug resistance
[
2–4]. In an attempt to overcome such problems, a number
of complexes based on other transition metals have been
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11243-018-0211-y) contains
supplementary material, which is available to authorized users.
*
Xue-Yi Le
lexyfu@163.com
1
2
Department of Applied Chemistry, South China Agricultural
University, Guangzhou 510642, People’s Republic of China
MOE Key Laboratory of Bioinorganic and Synthetic
Chemistry, School of Chemistry and Chemical
Engineering, Sun Yat-Sen University, Guangzhou 510275,
People’s Republic of China
Vol.:(0
1
123456789)
3

Transition Metal Chemistry
Scheme 1 The synthetic route of ligand HPBM and the Cu(II) complex. (i) H O, NaOH, 60 °C, 30 min, (ii) HPBM, (iii) MeOH, 60 °C, 2 h
2
assay, mitochondrial membrane potential, intracellular ROS
on a Bruker EMX A300 spectrometer (Bruker, Germany)
equipped with 100 kHz magnetic ꢁeld modulation. IR spec-
levels, the release of cytochrome c and cell cycle arrest.
−
1
tra (KBr disks, 4000–400 cm ) were obtained with a VER-
TEX 70 FT-IR spectrometer (Bruker, Germany). UV–Vis
spectra were recorded on a Pharmacia 2550 spectropho-
tometer (Shimadzu, Japan). Circular dichroism spectra were
recorded using a Chirascan CD spectropolarimeter (Applied
Photophysics Ltd., UK).
Experimental
Materials and instrumentation
The reagents and chemicals were obtained from commercial
sources and used without further puriꢁcation. l-Phenyla-
lanine (l-Phe) was purchased from Aladdin. Calf thymus
DNA (CT-DNA) (stored at 4 °C) was purchased from Sigma.
pBR322 DNA was obtained from MBI Fermentas (Lithu-
ania). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and tetramethylethylenediamine (TEMED)
were purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). Cancer cell lines consisting of HeLa
Synthesis of complex 1
To a solution of l-Phe (0.083 g, 0.50 mmol) in water (5 mL),
plus NaOH (0.02 g, 0.50 mmol), was added a solution of
Cu(ClO ) ·6H O (0.185 g, 0.50 mmol) dissolved in water
4
2
2
(5 mL) with stirring. A solution of HPBM [24] (0.105 g,
0.50 mmol) in MeOH (20 mL) was then added dropwise,
with stirring continued for 2 h at 50 °C. The resulting solu-
tion was ꢁltered, and the ꢁltrate was slowly evaporated at
room temperature until a blue microcrystalline product
was obtained. This was then recrystallized from MeOH to
give X-ray quality single crystals. Yield: 70%. Anal calc
for C H N O ClCu: C, 47.66; H, 4.15, N, 10.11%. Found:
(
human cervical cancer cell line), A549 (human lung ade-
nocarcinoma cell line), Eca-109 (human esophagus cancer
cell line) and the normal human liver cell line LO2 were
obtained from the Laboratory Animal Center of Sun Yat-Sen
University (Guangzhou, China). Deionized water was used
in all experiments. All experiments involving CT-DNA were
performed in Tris–HCl buꢂer solution (pH 7.2) containing
2
2
23
4
7
−
1
2
−1
C, 47.62; H, 4.13; N, 10.07%. Λ /Ω cm mol : 87.8.
M
3
−1
−1
λ
, in MeOH (ε /dm mol cm ): 208 (49,936), 344
max
max/nm
−
1
5
mM Tris–HCl and 50 mM NaCl. Solutions of CT-DNA in
(19,017). IR (KBr, cm ): 3446m, 3321w, 3269w, 3138w,
Tris–HCl buꢂer gave a ratio of UV absorbance at 260 and
1589s, 1454m, 1404s, 1120s, 1093s, 758m, 625m. ESI–MS
+
2
80 nm (A /A ) of ca. 1.8–1.9, indicating that the DNA
(MeOH): m/z = 436 [Cu(HPBM)(l-Phe)] . HR-ESIMS
2
60 280
6
3
+
was suꢃciently free of protein [22]. The concentration of
DNA was determined by UV absorbance at 260 nm using the
m/z for C H CuN O (M + H ) calcd (found): 436.09605
22 21 4 2
(436.09464). ESR (MeOH): g = 2.276, g = 2.044,
‖
⊥
−
1
−1
molar absorption coeꢃcient of 6600 M cm [23]. DNA
solutions were used after no more than 4 days.
A = 171.1 G.
II
Elemental analyses (CHN) were obtained with a Vario EL
elemental analyzer (Elementar, Germany). ESI–MS spectra
were measured using an API4000 triple quadrupole mass
spectrometer (AB Sciex, USA). ESR spectra at liquid nitro-
gen temperature (90 K) were recorded in methanol solution
Crystal structure determination and reꢀnement
X-ray diffraction measurements were performed
with a Bruker Smart CCD area detector in the range
4.33 < θ < 75.33° with Cu K radiation (λ = 1.54178 Å)
α
1
3

Transition Metal Chemistry
at 293 K. Empirical absorption corrections were applied
using the SADABS program [25]. The structure was deter-
mined using pattern methods, which yielded the positions
of all non-hydrogen atoms. All the hydrogen atoms of the
complex were placed in calculated positions with ꢁxed iso-
tropic thermal parameters and the structure factor calcula-
tions were included in the ꢁnal stage of full-matrix least-
squares reꢁnement. All calculations were performed using
the SHELXTL-97 suite of computer programs [26].
where t and t are the observed ꢀow times in the presence
0
and absence of the samples, respectively [29, 30].
Circular dichroism studies
CD spectra of CT-DNA (100 µM) were recorded in 5 mM
Tris–HCl/50 mM NaCl buꢂer solution, pH 7.2 at room tem-
perature by incrementing the [complex]/[CT-DNA] ratio
(r = 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0). The experiments were
performed under a nitrogen atmosphere. The CD spectra
were measured as the average of three independent scans
from 220 to 320 nm.
Crystallographic data (excluding structure factors) for the
structures reported in this work have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-1560445. Copies of the data can be
obtained free of charge on application via www.ccdc.cam.
ac.uk/data_request/cif or e-mail (deposit@ccdc.cam.ac.uk).
Cyclic voltammetry
Cyclic voltammetric experiments were carried out with a
CHI660C electrochemical workstation, purging the sample
Electronic absorption titrations
with N prior to measurements. A standard three-electrode
2
Absorption spectra titration was carried out with a ꢁxed
concentration of the Cu(II) complex (50 µM) while gradu-
ally increasing the concentration of DNA. To eliminate the
impact of absorbance changes due to DNA, equal volumes
of DNA were added to both the sample and reference cells.
After addition of DNA to the metal complex, the resulting
solutions were equilibrated for 5 min at room temperature.
The absorption spectra were then recorded in the range of
system comprising a Pt-microcylinder working electrode, a
Pt-wire auxiliary electrode, and a saturated calomel refer-
ence electrode (SCE). The supporting electrolyte was com-
prised of 10 mM Tris–HCl/50 mM NaCl buꢂer solution (pH
7.2). The experiments were performed in 1.2 mM complex
solutions in the absence and presence of CT-DNA.
DNA cleavage experiments
2
00–500 nm. In order to quantitatively evaluate the DNA-
binding aꢃnity, the intrinsic binding constant K was deter-
The cleavage of supercoiled pBR322 plasmid DNA (250 ng)
was monitored using agarose gel electrophoresis. The sam-
ple was incubated for 1 h at 37 °C in the dark. A loading
buꢂer was then added and electrophoresis performed at
b
mined using the Wolfe–Shimmer equation [27]:
[
DNA]∕(ꢀ − ꢀ ) = [DNA]∕(ꢀ − ꢀ ) + 1∕K (ꢀ − ꢀ ) (1)
a
f
a
f
b
b
f
where [DNA] is the concentration of DNA in the base pairs,
1
00 V for 45 min in Tris–boric acid–EDTA (TBE) buꢂer
ε , ε and ε correspond to the apparent extinction coeꢃcient
a
A
f
b
(pH 8.3) using 0.8% agarose gel containing 5 mL GoldView.
After electrophoresis, the bands were visualized under UV
light and photographed using a Bio-Rad Laboratories-Seg-
rate Gel Imaging System.
(
/[Cu]), the extinction coeꢃcient of the free (unbound)
obsd
and fully bound complex, respectively. From the plot of
[
DNA]/(ε − ε ) versus [DNA], the binding constant K was
a f b
given by the ratio of the slope to the intercept.
Molecular docking studies
Viscosity experiments
The molecular docking studies were performed using the
AutoDock 4.2 program [31] to investigate the interac-
tions between the complex and DNA containing iodinated
Viscosity experiments were performed using an Ubbe-
lohde viscometer maintained at constant temperature
(
25.0 ± 0.1 °C) in a thermostatic water bath. The ꢀow time
1
2-mer oligonucleotides d(C-G-C-G-A-A-T-T-BrC-G-C-G)
2
was measured using a digital stopwatch. Each ꢀow time was
(
PDB ID: 6BNA). The B3LYP hybrid density functional
measured in triplicate, and an average ꢀow time was calcu-
was used to optimize the structure of the complex with the
Gaussian 09 program package. The LanL2DZ basis set was
used for Cu, and the 6-31G* basis set was used for other
atoms. Before docking, water molecules and substrate were
deleted, while polar hydrogen atoms and Gasteiger charges
were added. The docking was performed in a 60 × 60 × 60
grid box with 0.375 Å spacing. A total of 100 runs of the
Lamarckian genetic algorithm were performed. All other
parameters were left at their default settings. All calculations
1
/3
lated. The values of (η/η ) , where η and η represent the
0
0
viscosity of CT-DNA (200 µM) in the presence and absence
of the complexes, respectively, were plotted against ratio
(
[
r) of [complex]/[DNA] ([complex]/[DNA] = 0.0–0.30)
28]. The viscosity values were calculated according to the
relationship
0
= (t − t )∕t
0
ꢀ
1
3

Transition Metal Chemistry
were carried out on a Dell T7500 server with dual XEON
was applied quickly on top of the gel, coated over the
microslide, covered immediately with a coverslip, and then
kept at 4 °C for 10 min. The coverslip was again removed
after the gel had set. A third coating of 50 μL of 0.5% low-
melting agarose was placed on the gel and kept for 15 min at
4 °C. After solidiꢁcation of the agarose, the coverslips were
removed, and the slides were immersed in an ice-cold lysis
solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 90 mM
sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and
10% DMSO) and kept at 4 °C for 2 h. All of the above opera-
tions were performed under low-lighting conditions to avoid
additional DNA damage. Electrophoresis was then carried
out at 25 V and 300 mA for 20 min. After electrophoresis,
5
660 cores. The hydrogen bonds and visual results were han-
dled using PyMol software (The PyMOL Molecular Visuali-
zation System, Version 1.5, Schrödinger, LLC).
In vitro cytotoxicity assays
In vitro cytotoxicity tests were carried out using the MTT
assay [32]. Human tumor cell lines Eca-109, HeLa, A549,
and human normal LO2 cells were seeded into 96-well micr-
4
otiter plates (1 × 10 cells per well) and incubated overnight
in a humidiꢁed atmosphere containing 5% CO at 37 °C. The
2
test complex was dissolved in DMSO and diluted with RPMI
−
1
1
640 (10% fetal bovine serum (FBS), 100 units per mL peni-
the cells were stained with 20 μL of EB (20 μg mL ) for
20 min in the dark. The slides were washed in chilled dis-
tilled water for 10 min to neutralize the excess alkali, air-
dried and scored for comets by ꢀuorescence microscopy.
cillin and 50 units per mL streptomycin). The concentrations
of the complex ranged from 1.57 to 100 µM. Control wells
were prepared by the addition of culture medium (200 mL).
The plates were incubated in a 5% CO incubator at 37 °C
2
for 48 h. A 10 µL aliquot of stock MTT dye solution (20 mL,
Reactive oxygen species assays
−
1
5
mg mL ) was then added to each well. After incubation
at 37 °C for 4 h, buꢂer (100 mL) containing dimethylfor-
mamide (50%) and sodium dodecyl sulfate (20%) was added
to transform MTT to a purple formazan dye. The optical
density (OD) of each well was then measured at 595 nm on
a microplate spectrophotometer.
2
′,7′-Dichlorodihydroꢀuorescein diacetate (DCFH-DA) was
used as a ꢀuorescent probe to measure the ROS levels. Eca-
6
1
09 cells were seeded into six-well plates (1 × 10 cells).
After culturing for 24 h, the medium was replaced with fresh
medium containing diꢂerent concentrations of complex. The
cells were then cultured 6 h, before staining with 20 μM
DCFH-DA in PBS for 30 min in the dark. Finally, the cells
were harvested and washed twice with PBS. The DCF ꢀuo-
rescence intensity was also determined.
Apoptosis studies
5
Eca-109 cells (2 × 10 ) were seeded onto chamber slides
in six-well plates and incubated for 24 h. The cells were
cultured in RPMI 1640 (10%, FBS) and incubated at 37 °C
Measurement of mitochondrial membrane potential
under a 5% CO . The medium was removed and replaced
2
with fresh medium containing the complex (6.25 µM), then
the plates were incubated for 24 h. The cells were washed
with ice-cold PBS and ꢁxed with formalin (4%, w/v). The
5
Eca-109 cells (2 × 10 per well) were treated with the
complex for 24 h. JC-1 (1 mg/mL), which was used as a
ꢀuorescence probe to detect the changes of mitochondrial
membrane potential, was added to stain the cells for 30 min
at 37 °C. The cells were then washed twice with PBS and
the ꢀuorescence intensity of JC-1 was determined with a
−
1
cell nuclei were stained with AO/EB (100 mg mL AO,
−
1
1
00 mg mL EB) for 10 min, then imaged under a ꢀuores-
cence microscope (Nikon, Yokohama, Japan) with excitation
at 350 nm and emission at 460 nm.
ꢀ
ow cytometer.
Comet assays
2
+
Intracellular Ca levels
DNA damage was investigated using the comet assay
according to the literature procedure [5]. Brieꢀy, Eca-109
cells in culture medium were incubated with 6.25 μM of
the complex for 24 h at 37 °C. The cells were harvested by
trypsinization after 24 h. A total of 100 μL of 0.5% nor-
mal agarose in PBS was dropped gently onto a fully frosted
microslide, covered immediately with a coverslip, and then
kept at 4 °C for 10 min. The coverslip was removed after
the gel had set. A mixture of 50 μL of the cell suspension
Eca-109 cells were treated with diꢂerent concentrations of
the complex for 24 h. The cells were incubated with 2.5 μM
Fluo-3AM for 30 min at 37 °C, then the cells were washed
three times and incubated for 20 min at 37 °C to ensure
that Fluo-3AM had been completely transformed into Fluo-
3. The cells were then washed twice with PBS and stained
−
1
with 5 μg mL DAPI solution. Finally, an ImageXpress
Micro XLS system was used to observe ꢀuorescence, and
a multiwavelength cell scoring module was used to ana-
lyze the data. The ꢀuorescence intensity per cell, which is
(
200 cells/μL) mixed with 50 μL of 1% low-melting agarose
was incubated at 37 °C. A total of 100 μL of this mixture
1
3

Transition Metal Chemistry
2
+
proportional to the level of Ca , was calculated as the total
uorescence intensity divided by the number of cells.
The UV–Vis spectra of HPBM (10 µM) and the complex
(10 µM) were recorded in methanol. As shown in Fig. S1
ꢀ
(
supporting information), a shift of 27 nm in the maximum
Cytochrome c assays
absorption wavelength (300–400 nm) is observed for the
complex compared with free HPBM. In addition, the com-
plex (10 µM) was luminescent in methanol solution at ambi-
ent temperature, with the maximum appearing at 436 nm.
The complex was also characterized by elemental analy-
sis, IR, ESI–MS, and HR–ESIMS. The elemental analyses
were in good agreement with the calculated values. In the
Eca-109 cells were seeded in a 12-well plate and incubated
overnight. The cells were treated with 3.13 or 6.25 μM of
the complex for 24 h, and then ꢁxed with ice-cold immunol
staining ꢁx solution for 30 min at room temperature. After
blocking the cells with immunol staining blocking buꢂer
for 1 h, the cells were treated with the primary antibody
against cytochrome c (cyt-c, 1:50 dilution) overnight at
−1
assay of IR spectra of the complex 1, a band at 3446 cm is
attributed to O–H stretching vibrations of H O, and a band
2
−
1
4
°C. Next, the plate was washed three times with immunol
at 3321 cm is assigned to –NH stretching vibrations.
2
staining buꢂer and assayed with Alexa Fluor 488-Labeled
Goat Anti-Mouse IgG (1:500 dilution) in the dark for 1 h
at room temperature. Finally, the cells were washed three
times with immunol staining wash buꢂer, and the cell nuclei
were stained with DAPI. Images were obtained using an
ImageXpress Micro XLS system, and the multiwavelength
cell scoring module was used to analyze the data. The inte-
grated intensity/cell representing the ꢀuorescence intensity
of each cell was used to measure the release of cyt-c. The
These data are comparable with those for [Cu(Gly-l-val)
−
1
−1
(HPB)(H O)]ClO (O–H: 3420 cm , N–H: 3322 cm )
2
4
−
1
[20]. No characteristic peak for –COOH (1700–1750 cm )
was observed, consistent with coordination of this group
−
1
to the copper atom. Two peaks at 1589 and 1404 cm are
−
attributed to asymmetric ν (−COO ) and symmetric ν
as
s
−
(−COO ) stretching frequencies. The frequency separation
−
−
−1
Δν [= ν (−COO ) − ν (−COO )] of 18 S cm is consist-
as
s
ent with unidentate coordination of the carboxylate group.
In the ESI–MS and HR–ESIMS spectra of the complex, the
observed molecular weight matched exactly with that of
ꢀ
ꢀ
uorescence intensity per cell was calculated as the total
uorescence intensity divided by the number of cells.
+
the coordination cation [Cu(HPBM)(l-Phe)] . The X-band
Cell cycle arrest studies
ESR spectrum shows that g > g which indicates that the
‖
⊥
unpaired electron is located in the copper(II) d
orbital,
x2–y2
6
Eca-109 cells (10 cells per well) were seeded into six-well
with an approximate square–pyramidal geometry. Addition-
ally, according to Addison’s parameter (τ = (β − α)/60),
τ = 0.26, which is also consistent with a square–pyrami-
dal Cu(II) complex [33]. The distorted square–pyramidal
structure was conꢁrmed by single-crystal X-ray diꢂraction
analysis. An ORTEP drawing of the cation is depicted in
Fig. 1. Crystal data, selected bond lengths, and bond angles
are collected in Tables 1 and 2. The structure consists of a
plates (Costar, Corning Corp., New York, NY, USA) and
cultured in RPMI 1640 (10% of FBS), by incubation at 37 °C
and 5% CO . The medium was removed, and fresh medium
2
(
DMSO concentration 0.05% v/v) containing 6.25 μM of the
complex was added to the cells. After incubation for 24 h,
the cell layer was trypsinized, washed with cold PBS, and
ꢁ
xed with 70% ethanol. A 15-μL aliquot of RNAse (0.2 mg/
mL) and 15 μL of propidium iodide (0.02 mg/mL) were
added to the cell suspensions, which was then incubated for
3
0 min at 37 °C. The samples were analyzed with an FACS
Calibur ꢀow cytometry instrument.
Results and discussion
Synthesis and characterization
The complex [Cu(HPBM)(l-Phe)(H O)]·ClO (1) was syn-
2
4
thesized by the direct reaction of copper(II) perchlorate
hexahydrate with HPBM and l-Phenylalanine in equimolar
quantities using 80% methanol as solvent. The complex was
puriꢁed by slow evaporation of solvent at room temperature.
The molar conductivity of a 1 mM solution of the com-
2
−1
plex in methanol was 87.8 S cm mol , indicating that a
Fig. 1 An ORTEP drawing of the complex. The thermal ellipsoids
‒
1
:1 electrolyte in which the anion ClO is not coordinated.
probability level is 30%
4
1
3

Transition Metal Chemistry
Table 1 Selected
crystallographic data for the
complex
C H N O ClCu
2
2
23
4
7
Formula weight
Space group
552.43
Crystal size (mm3)
Temp (K)
0.21 × 0.19 × 0.16
293 (2) K
Orthorhombic,
P2(1)2(1)2(1)
a (Å)
b (Å)
c (Å)
10.9386(4)
13.8994(3)
15.3101(4)
θ range for data collection (°)
No. of data/parameters/restraints
Index ranges
4.30–75.33
6936/4316
− 12 ≤ h ≤ 13,
−
−
17 ≤ k ≤ 8,
14 ≤ l ≤ 19
V (Å3)
2327.75(12)
4
1.582
Goodness-of-ꢁt on F2
Final R indices [I > 2σ(I)]
R indices (all data)
1.050
Z
R = 0.0418, wR = 0.1106
1
2
Dcalc (g cm−3)
R = 0.0442, wR = 0.1141
1 2
μ (Mo Kα) (mm−1) 2.839
Largest diꢂ. peak and hole (e Å−3) 0.586 and − 0.470
F (000)
1140
Table 2 Selected bond lengths (Å) and angles (°) for complex
determined by monitoring the changes in the MLCT bands
with increasing DNA concentration, giving a value of 7.38
Bond length
(Å)
Bong angle
(°)
4
−1
(
± 0.57) × 10 M . This is smaller than that of [Cu(Gly-l-
5
−1
Cu(1)–O(1)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–O(3)
1.949(2)
1.975(3)
2.037(3)
2.023(3)
2.281(3)
O(1)–Cu(1)–N(1)
O(1)–Cu(1)–N(3)
N(1)–Cu(1)–N(3)
O(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
N(3)–Cu(1)–N(2)
O(1)–Cu(1)–O(3)
N(1)–Cu(1)–O(3)
N(3)–Cu(1)–O(3)
N(2)–Cu(1)–O(3)
173.05(11)
81.91(11)
102.47(12)
92.90(10)
81.00(11)
157.53(11)
94.42(10)
90.78(11)
92.68(12)
109.56(11)
val)(HPB)(H O)]·ClO (3.21 × 10 M ) [20], which may
2
4
be due to steric hindrance from the methyl group of HPBM,
and/or the diꢂerent amino acid coordination.
Viscosity measurements
Viscosity measurements that are sensitive to the change
in length of DNA are regarded as the least ambiguous and
the most critical test of the binding mode in solution in the
absence of crystal structure [30, 34]. In general, classical
intercalation results in lengthening of the DNA helix, as
the base pairs are separated to accommodate the binding
ligand, leading to an increase in DNA viscosity. In contrast,
a partial intercalation and/or minor groove binding of the
ligand can bend (or kink) the DNA helix, reducing its eꢂec-
tive length and hence its viscosity [29, 34]. The eꢂects of
the complex, together with those of ethidium bromide (EB)
on the viscosity of rod-like DNA are shown in Fig. S3 (sup-
porting information). It is well known that EB increases the
relative speciꢁc viscosity, due to the lengthening of the DNA
double helix by intercalation. However, with increasing the
concentration of the present complex, the relative viscosity
of DNA solution was decreased compared with that of the
control. Hence, the results suggest that the complex interacts
with DNA through a partial intercalative or minor groove
binding mode.
+
−
[
Cu(HPBM)(l-Phe)(H O)] cation and a ClO anion. The
2 4
Cu(II) center of the complex is chelated by HPBM and an
l-Phe ligands, and the axial position is occupied by a water
ligand. The average Cu–N bond length is 2.011 (3) Å, which
+
is smaller than that reported for [Cu(glygly)(PyTA)] (2.014
(
3) Å) [21].
DNA‑binding studies
Electronic absorption titration
Electronic absorption spectroscopy is a useful method to
determine the interactions of metal complexes with DNA.
The electronic spectrum of the complex titrated with CT-
DNA is shown in Fig. S2 (supporting information). As the
concentration of DNA is increased, the metal-to-ligand
charge transfer transition (MLCT) bands of the complex
at 340 nm exhibits hypochromism of about 20.1%. These
observations indicate that the complex interacts with DNA
most likely through a mode that involves a stacking interac-
tion between the aromatic chromophore and the base pairs
of DNA. The DNA-binding constant of the complex was
Circular dichroism
Circular dichroism (CD) spectroscopy is a very sensitive
technique, commonly used to monitor the possible con-
formational changes of DNA upon interaction with metal
complexes. In general, CD spectra of DNA consist of two
characteristic peaks (a positive band at 275 nm due to base
1
3

Transition Metal Chemistry
stacking and a negative band at 246 nm due to the right-
handed helicity of B-DNA). As shown in Fig. S4 (supporting
information), with increasing ratios of [Cu(II)/[DNA], the
intensities of both the positive and negative bands decrease
gradually (shifting to zero levels). The positive and negative
peaks exhibit red shifts of 2 and 1 nm, respectively. These
data indicate that the interaction of the Cu(II) complex with
DNA disturbs the right-handed helicity and base stacking,
inducing certain conformational changes in the B-DNA [35].
The small changes in the positive bands demonstrate that
the complex intercalates partially into the DNA base pairs,
and thereby unwinding the double helix [36, 37], which is
consistent with the results obtained by viscosity measure-
ments. Similar results have been observed in other Cu(II)
complexes [20, 21].
1) and the complex alone (10 µM, lane 2), no DNA cleav-
age was observed. However, in the presence of Vc, 5 (lane
3), 7.5 (lane 4) and 10 µM (lane 5) of the complex resulted
in concentration-dependent DNA cleavage, such that the
amount of Form I decreased gradually, whereas the amount
of Form II increased. Thus, we infer that Vc acts as an initia-
tor for the complex to cleave pBR322 DNA.
Molecular docking studies
Molecular docking calculation can be helpful for rational
drug design and mechanistic studies, by placing a small
molecule into the binding site of the DNA target region,
mainly in a non-covalent mode [30]. Hence, to explore the
interaction of the complex with B-DNA (PDB ID: 6BNA),
the system was modeled with AutoDock 4.2 [31]. As
shown in Fig. 2, we were able to obtain a model in which
the complex interacted with DNA through a minor groove
binding mode, in agreement with the results of the viscos-
ity measurements. The resulting relative binding energy
Cyclic voltammetry
Cyclic voltammetry (CV) can aꢂord useful information on
the binding of metal complexes to DNA, because of the
resemblance between electrochemical and biological reac-
tions. It is well known that if a metal complex binds with
DNA via electrostatic interactions, the electrochemical
potential of the complex will shift in the negative direction.
Conversely, intercalation to DNA results in a positive shift in
potential. The electrochemical behavior of the complex was
studied in methanol by CV in the absence or presence of CT-
DNA. The CV of the complex exhibited one oxidation and
one reduction peak in the sweep range from − 0.4 to 0.7 V,
with a scan rate of 100 mv/s. As shown in Fig. S5 (support-
ing information), no new peaks were observed upon addition
of CT-DNA to the complex, with R ([Cu(II)]/[DNA]) = 3.
In the absence of CT-DNA, the E value of Cu(I)/Cu(II)
−
1
of the docked complex with DNA was − 40.15 kJ mol .
Figure 2 also shows that two hydrogen bonds between the
complex and DNA bases. The lengths of these hydrogen
bonds are 2.576 Å (H1···6BNA:DT-19:O3′) and 2.215 Å
(O1···6BNA:DA-18:H3).
Cell viability assays by MTT method
The viabilities of cancer Eca-109, HeLa, A549, and nor-
mal LO2 cells in the presence of HPBM and the complex
was investigated by the 3-(4,5-dimethylthiazole)-2,5-diphe-
nyltetraazolium bromide (MTT) method. After incubation
1
/2
of the couple was 0.263 V, while in the presence of DNA
(
400 µM), the E value was 0.319 V. Hence, an increase of
1
/2
0
.056 V in the E value was observed, indicating that the
1/2
complex interacts with CT-DNA through an intercalative or
partially intercalative mode.
DNA cleavage studies
The cleavage of pBR322 DNA in the presence of the com-
plex was investigated by agarose gel electrophoresis. In gen-
eral, when circular plasmid DNA is subject to electrophore-
sis, relatively fast migration will be observed for the intact
supercoiled form (Form I). If scission occurs on one strand
(
nicking), the supercoil will relax to generate a slower-mov-
ing open circular form (Form II). If both strands are cleaved,
a linear form (Form III) that migrates between Form I and
Form II will be generated [38]. The eꢂect of the complex in
the presence of Vitamin C (Vc) on pBR322 DNA (250 ng)
is shown in Fig. S6 (supporting information). Evidently, for
the control (lane 0, DNA alone), DNA + Vc (0.5 mM, lane
Fig. 2 The molecular docked models of the complexes with DNA.
The hydrogen bonds between the complex and DNA are represented
using red dashed lines. (Color ꢁgure online)
1
3

Transition Metal Chemistry
of the cells with diꢂerent concentrations (1.57 → 100 µM)
of the complex for 48 h, the viabilities in the presence of
HPBM (a) and the complex (b) are depicted in Fig. S7
apoptotic cells were bright green, with obvious apoptotic
features such as nuclear shrinkage and chromatin conden-
sation. These results suggest that the complex can induce
apoptosis in Eca-7901 cells.
(
supporting information), and the IC values are listed
50
in Table 3. For free HPBM, when the concentration of
HPBM reaches 100 µM, the cell viability is still more
than 50%. The complex exhibits high cytotoxic activities
toward Eca-109, HeLa, and A549 cells, with IC values
Comet assays
Single cell gel electrophoresis (comet assay) in an agarose
gel matrix was used to study DNA fragmentation, which
is a hallmark of apoptosis [39]. As shown in Fig. 4, in the
control experiments (a), the cells failed to show a comet-
like appearance. When Eca-109 cells were treated with
3.13 or 6.25 µM of the complex (b and c) for 24 h, the cells
displayed well-formed comets. The extent of DNA dam-
age, which was estimated from the length of the comet tail,
increased in a concentration-dependent manner. Hence, this
complex can clearly induce DNA fragmentation, which will
result in apoptosis.
5
0
of 5.7 ± 0.1, 8.3 ± 0.6, and 7.1 ± 0.5 µM, respectively.
The cytotoxic activity of the complex against A549 cells
is however lower than that of [Cu(Gly-l-val)(HPB)(H O)]
2
ClO (IC = 2.84 ± 0.19 µM) [20], but comparable with
4
50
that of [Cu(dppt) (H O)](PF ) (dppt = 5,6-diphenyl-3-(2-
2
2
6 2
pyridyl)-1,2,4-triazine) (IC = 7.80 ± 0.25 µM) [11]. Since
5
0
Eca-109 cells were most sensitive to the complex, this cell
line was selected to undergo the following biological activ-
ity tests.
Apoptosis studies
Reactive oxygen species assay
Apoptosis was assayed using the acridine orange (AO)/
ethidium bromide (EB) double staining method. It is well
known that AO can pass through cell membranes, but EB
cannot. Hence, AO can stain the living and apoptotic cells
green, while EB stains necrotic cells red. As shown in Fig. 3,
in the control (a), living Eca-109 cells were stained bright
green in spots and kept nuclei integrity. After Eca-109 cells
were treated with 6.25 µM of the complex for 24 h (b), the
A number of potential anticancer agents induce apoptosis
through the generation of reactive oxygen species (ROS).
2′,7′-Dichlorodihydroꢀuorescein diacetate (DCFH-DA)
was used as ꢀuorescence probe to assay the cellular ROS
levels induced by the complex. In the presence of intra-
cellular esterases, DCHF-DA is transformed into a ꢀuo-
rescent product, namely, dichloroꢀuorescein (DCF). As
shown in Fig. 5a, in the control experiment (a), no obvious
ꢀ
uorescent points were observed. After the treatment of
Table 3 The IC50 (µM) values of the Cu(II) complex and ligand
SGC-7901 cells with either Rosup or the Cu(II) complex,
a large number of green ꢀuorescent points were found.
The DCF ꢀuorescence intensity was quantiꢁed with a
multiwavelength cell scoring module; this is proportional
to the amount of ROS produced by the cells. As shown
in Fig. 5b, in the negative control, the DCF ꢀuorescence
HPBM toward the selected cell lines
Compound
Eca-109
127.6 ± 3.3 102.6 ± 2.5 148.9 ± 7.6 111.5 ± 9.8
5.7 ± 0.1 8.3 ± 0.6 7.1 ± 0.5 7.9 ± 0.7
HeLa
A549
LO2
HPBM
Cu(II) complex
Fig. 3 Eca-109 (a) were exposed to 6.25 μM of the complex (b) for 24 h and stained with AO/EB and imaged under ꢀuorescence microscope
1
3

Transition Metal Chemistry
Fig. 4 Comet assay of EB-stained control (a) and 3.13 and 6.25 µM of the complex (b, c) treated Eca-109 cells at 24 h incubation
Fig. 5 A Intracellular ROS levels assay of Eca-109 cells (a) exposure
to Rosup (b, positive control) and 6.25 (c) and 12.5 µM (d) of the
complex for 6 h. B The DCF ꢀuorescence intensity was determined in
Eca-109 cells exposure to Rosup (50 µM, a positive control, 30 min)
and 6.25 and 12.5 µM of the complex for 6 h
intensity was 7.61. When Eca-109 cells were incubated
with either 6.25 or 12.5 µM of the complex for 6 h, the
DCF ꢀuorescence intensity was increased in a concen-
tration-dependent manner. These results indicate that the
complex can increase intracellular ROS levels.
Mitochondrial membrane potential
Mitochondria play an important role in apoptosis through
the release of pro-apoptotic factors, including cytochrome
c and the apoptosis-inducing factor [40, 41]. The changes
1
3

Transition Metal Chemistry
of mitochondrial membrane potential (MMP) in Eca-109
cells treated the complex were investigated using JC-1 as
of red/green ꢀuorescence intensity was determined by ꢀow
cytometry. As shown in Fig. 6, in the control (a, up and
down), the ratio of red/green was 4.17. When Eca-109 cells
were treated with carbonylcyanide-m-chlorophenylhydra-
zone (cccp), 6.25 or 12.5 µM of the complex for 24 h, the
ratio of red/green ꢀuorescence decreased. Hence, the com-
plex can induce a concentration-dependent decrease in the
mitochondrial membrane potential.
ꢀ
uorescent probe. JC-1 emits red ꢀuorescence at high MMP,
while at low MMP, JC-1 emits green ꢀuorescence. The ratio
Intracellular calcium levels
2
+
High intracellular levels of Ca are associated with the
2
+
induction of cell death. [42]. The intracellular Ca levels
in the presence of the complex were therefore assayed using
2+
Fluo-3AM (indicating cytosolic Ca ) as a ꢀuorescent probe,
2
+
which combines with Ca to give Fluo-3. The intracellular
2
+
Ca level in Eca-109 cells induced by the complex was
determined using on ImageXpress Micro XLS system and
multiwavelength cell scoring module. As shown in Fig. 7a,
in the control experiment (a), no obvious green ꢀuorescent
points were observed, consistent with a very low level of
Fig. 6 Assay of Eca-109 cells mitochondrial membrane potential
with JC-1 as ꢀuorescence probe staining method. The ratio of red/
green ꢀuorescence after Eca-109 cells exposed to cccp (positive con-
trol) and 6.25 and 12.5 µM of the complex for 24 h
2
+
Fig. 7 A Intracellular Ca levels were assayed after Eca-109 cells
were exposed to 3.13 and 6.25 µM of the complex for 24 h. B The
integrated ꢀuorescent intensity/cell was determined after Eca-109
cells were treated with diꢂerent concentration of the complex for
24 h. *P < 0.05 represents signiꢁcant diꢂerences compared with con-
trol
1
3

Transition Metal Chemistry
2
+
intracellular free Ca . After treatment of the Eca-109 cells
Cytochrome c assays
with diꢂerent concentrations of the complex (b and c) for
2
4 h, a number of bright green ꢀuorescence points were
Cytochrome c (cyt-c) can induce cell apoptosis by activating
caspase proteases, which are released from the mitochon-
drial membrane space under conditions of mitochondrial
dysfunction [43]. The high concentration of intracellular
observed, indicating that the complex can increase intracel-
2
+
lular Ca levels. To quantitatively determine this eꢂect, the
uorescence intensity of Flu-3 was determined as shown
ꢀ
2
+
in Fig. 7b. The results demonstrate that the complex can
Ca and reduction in MMP may lead to the release of
cytochrome c from the mitochondria. As shown in Fig. 8a, in
the control experiment, no obvious green ꢀuorescent points
2
+
enhance the level of intracellular Ca in a dose-dependent
manner, which further increases intracellular ROS levels.
Fig. 8 A The release of cyt-c was examined after Eca-109 cells
were exposed to diꢂerent concentration of the complex for 24 h. B
The integrated ꢀuorescent intensity/cell was determined after dif-
ferent concentration of the complex treated Eca-109 cells for 24 h.
*P < 0.05 represents signiꢁcant diꢂerences compared with control
1
3

Transition Metal Chemistry
synthesis of new copper(II) complexes as potent anticancer
drugs.
Acknowledgements We acknowledge the Program of Natural Science
Foundation of Guangdong Province (2015A030313423) and Guang-
dong Province College Students’ Innovation and Entrepreneurship
Training Program (201710564307) for ꢁnancial support.
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