Mixed-ligand Cu(II) hydrazone complexes designed to enhance anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2018.07.022

The ligand quantity, ligand type, and coordination geometry have important influences on the anticancer activity of metal-based complexes. On the basis of the structures of previously reported 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H₂O 1a, [C

Full text of DOI:10.1016/j.ejmech.2018.07.022

Accepted Manuscript
Mixed-ligand Cu(II) hydrazone complexes designed to enhance anticancer activity
QingYou Mo, JunGang Deng, Yani Liu, GuiDong Huang, ZuoWen Li, Ping Yu, Yi
Gou, Feng Yang
PII:
S0223-5234(18)30578-6
10.1016/j.ejmech.2018.07.022
EJMECH 10557
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2018
Revised Date: 5 July 2018
Accepted Date: 8 July 2018
Please cite this article as: Q. Mo, J. Deng, Y. Liu, G. Huang, Z. Li, P. Yu, Y. Gou, F. Yang, Mixed-ligand
Cu(II) hydrazone complexes designed to enhance anticancer activity, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.022.
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Mixed-ligand Cu(II) hydrazone complexes designed to enhance
anticancer activity
QingYou Mo a,b,1, JunGang Denga,1, Yani Liuc, GuiDong Huanga, ZuoWen Lib, Ping
Yud, Yi Goub*, Feng Yangd*
a Affiliated hospital, Guilin Medical University, Guilin, Guangxin, China
b School of Pharmacy, Nantong University, Nantong, Jiangsu, China
c
Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, China
d
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China, Guangxi Normal
University, Guilin, Guangxi, China
*Corresponding authors.
E-mail addresses: sanchuanok@126.com (Y. Gou), fyang@mailbox.gxnu.edu.cn (F.
Yang).
1 These authors have contributed equally to this work.
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Abstract
The ligand quantity, ligand type, and coordination geometry have important
influences on the anticancer activity of metal-based complexes. On the basis of the
structures of previously reported 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H2O 1a,
[Cu(L2)Cl]·H2O 2a, and [Cu(L2)NO3]·H2O 3a), we subsequently designed,
developed, and characterized a series of corresponding 1:1:1 Cu(II)/ligand/co-ligand
complexes ([Cu(L1)(Py)Cl]·H2O 1b, [Cu(L2)(Py)Cl] 2b, and [Cu(L2)(Py)NO3] 3b),
where L1 = (E)-N'-(2-hydroxybenzylidene)acetohydrazide, L2 = (E)-N'-(2-
hydroxybenzylidene)benzohydrazide, and Py = pyridine. All six Cu(II) complexes
were assessed for their in vitro anticancer properties against a panel of human cancer
cells, including cisplatin-resistant A549cisR cell lines. Interestingly, we observed that
the 1:1:1 Cu/ligand/co-ligand mixed-ligand Cu(II) complexes exhibited higher
anticancer activity than the corresponding 1:1 Cu(II)/ligand complexes. In particular,
the 1:1:1 Cu(II)/ligand/co-ligand complex 3b displayed the greatest toxicity toward
several cancer cells with better IC50 (1.12–3.77 µM) than cisplatin. Further
mechanistic explorations showed that the 3b complex induced DNA damage, thus
resulting in mitochondria-mediated apoptotic cell death. Furthermore, the 3b complex
displayed pronounced cytostatic effects in the MCF-7 3D spheroid model.
Keywords: Mixed-ligand copper(II) complexes; Anticancer activity; Spheroid assay;
Anticancer mechanism
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1. Introduction
Although highly effective in treating various solid tumors, platinum-based agents
(e.g., cisplatin and its second and third generation analogs) are still limited by
dose-limiting side effects and inherited or acquired drug resistance [1, 2]. These
drawbacks have galvanized chemists to discover alternative chemotherapeutic
strategies that are based on endogenous metals with improved pharmacological
properties [3–6]. Among the bio-essential metals, copper is found in all living
organisms and can be employed as a catalytic and structural cofactor; therefore, it has
a pivotal role in redox chemistry, development, and growth pathways [7]. Copper has
played a central role in angiogenesis, which is a critical process for tumor growth,
invasion, and metastasis [8–10]. Many investigations reported that numerous
cancerous tissues, such as breast, brain, and prostate cancer tissues, take up more
copper than normal tissues [5, 11, 12]. Therefore, copper complexes have been
considered promising alternatives to platinum-based agents, and a variety of
copper-based anticancer agents have been studied as potential anticancer drugs in
recent years [5].
Hydrazones are known to be an important class of Schiff base ligands with many
pharmacological activities, such as anticancer, antituberculostatic, and antimicrobial
activities [13–15]. Hydrazones can coordinate in either monoanionic, dianionic, or
neutral forms and can form various complexes with many transition metal cations,
such as Cu(II), Fe(III), and Zn(II) [16–18]. Many investigations demonstrated that the
biological activity of hydrazone copper complexes was higher than that of ligands
alone; this finding is particularly relevant for anticancer activity [19–23]. The
introduction of N-containing co-ligands such as imidazole, pyridine, quinoline,
phenanthroline, and their derivatives might affect the planarity, hydrophobicity, and
coordination geometry of copper complexes and the anticancer activity of
copper-based agents [24, 25]. A large number of mixed-ligand copper complexes
were recently reported, and many of them showed prominent anticancer activity [25–
33]. These studies provided opportunities for developing copper-based anticancer
agents that contain hydrazones ligands and N-containing co-ligands. In addition,
pyridine is a common prodrug and is one of the building blocks of many biologically
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important molecules. Recently, the application and chemistry of pyridine compounds
have attracted significant attention because of their biological importance [34].
The purpose of our study was to explore whether the introduction of an
N-containing co-ligand pyridine (Py) into aroylhydrazones copper(II) complexes
would result in a synergistic effect and whether this approach could consequently
represent an effective strategy for potentially developing copper-based anticancer
drugs. Given that 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H2O 1a,
[Cu(L2)Cl]·H2O 2a and [Cu(L2)NO3]·H2O 3a) showed potential anticancer activity
in vitro [22, 35], we synthesized the corresponding 1:1:1 Cu(II)/ligand/co-ligand
complexes ([Cu(L1)(Py)Cl]·H2O 1b, [Cu(L2)(Py)Cl] 2b and [Cu(L2)(Py)NO3] 3b) to
assess their anticancer activity, where L1 and L2 are the tridentate
(E)-N'-(2-hydroxybenzylidene)acetohydrazide
ligand
and
(E)-N'-(2-hydroxybenzylidene)benzohydrazide ligand, respectively (Scheme 1). Even
though there are a wide variety of mixed-ligand copper complexes that have been
proposed as promising cytotoxic agents, only a few studies have attempted to
investigate their working mechanisms [5]. Therefore, among the six complexes
proposed, we chose the 1:1:1 Cu(II)/ligand/co-ligand complex 3b to be further
investigated for its anticancer mechanism.
2. Experimental section
2.1. Materials
All reagents and solvents for synthesis and analysis were commercially available.
H2O used in all reactions was distilled prior to use. Elemental (C, H and N) analyses
were carried out on an Elementar Vario Micro Cube elemental analyzer. Infrared
spectra (IR) were measured using KBr pellets (400−4000 cm-1) on a Nexus 870 FT-IR
spectrophotometer. The high resolution electrospray ionization-mass spectroscopy
(HRMS-ESI) was recorded on Exactive liquid chromatography-MS mass
spectrometer (Thermofisher Scientific, USA). UV-Vis spectra were recorded on a
Cary 1E UV−Visible spectrophotometer. The fluorescence spectra were recorded on a
MPF-4 spectrofluorometer.
2.2. Synthesis of copper(II) complexes
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The
ligands
(E)-N'-(2-hydroxybenzylidene)acetohydrazide
(L1),
(E)-N'-(2-hydroxybenzylidene)benzohydrazide
(L2),
and
complexes
[Cu(L1)Cl]·2H2O (1a) [Cu(L2)Cl]·H2O (2a) and [Cu(L2)NO3]·H2O (3a) were
synthesized according to literature procedures (shown in Supplementary Material)
[36-38].
Synthesis of complex [Cu(L1)(Py)Cl]·H2O (1b). The complex was prepared by
adding L1 solution in methanol (0.17 g, 1 mmol) and pyridine (0.08 g, 1 mmol) into
CuCl2·2H2O solution in methanol (0.17 g, 1 mmol). The reaction mixture was stirred
for 1 h at ambient temperature to obtain a clear blue solution. Dark green block
crystals for X-ray structural characterization were obtained by a slow evaporation
process of the blue solution after five days. Yield: 61%. Anal. Calcd for
C14H16ClCuN3O3 (373.29): C 45.05, H 4.32 and N 11.25; found: C 45.27, H 4.41 and
N 11.55. IR (KBr, cm-1): 3357m, 3039w, 2731w, 1607vs, 1559vs, 1528s, 1497w,
1447m, 1385s, 1324m, 1251w, 1210m, 1149m, 1110w, 1080w, 1039w, 998w, 898w,
773m, 697m, 642w, 612w and 476w.
Synthesis of complexes [Cu(L2)(Py)Cl] (2b) and [Cu(L2)(Py)NO3] (3b). L2
(0.24 g, 1 mmol) and pyridine (0.08 g, 1 mmol) were dissolved in 15 mL of methanol.
The mixture was stirred for 10 min to produce a yellow solution, which was then
added to a methanol solution (10 mL) of the mixed copper(II) salts (CuCl2·2H2O:
0.17 g, 1 mmol for 2b; Cu(NO3)2·3H2O: 0.24 g, 1 mmol for 3b). The reaction mixture
was stirred for another 1 h at ambient temperature to obtain a clear blue solution. Dark
green block crystals were obtained after five days by evaporating the blue solution
slowly, which were then examined by X-ray diffraction spectroscopy.
Complex 2b (Yield: 76.8%): Elemental analysis calcd (%) for C19H16ClCuN3O2:
C 54.68, H 3.86 and N 10.07; found: C 54.85, H 3.72 and N 10.22. IR (KBr, cm-1):
3457m, 2946m, 2785m, 1608vs, 1552vs, 1445s, 1388s, 1347m, 1312m, 1201m,
1146m, 1068w, 892w, 796w, 756m, 693vs, 581w, 539w, 497w and 441w.
Complex 3b (Yield: 82.1%): Elemental analysis calcd (%) for C19H16CuN4O5:
C51.41, H 3.63. and N, 12.61; found: C 51.52, H 3.72 and N 12.75. IR (KBr, cm-1):
3038m, 1604vs, 1540w, 1499vs, 1444vs, 1379vs, 1348s, 1284m, 1197m, 1146m,
1067m, 1035m, 946w, 892m, 757m, 697vs, 634w, 587w, 552w, 500w and 453w.
2.3. Crystal structure determinations
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The single crystal data collection of complexes 1b-3b were carried out on a
SuperNova CCD Area Detector equipped with graphite monochromated Mo-Ka (λ =
0.71073 Å) radiation at room temperature. SADABS program were applied for
empirical adsorption corrections of all data. The structures of the Cu(II) complexes
were solved by direct methods and refined by full-matrix least-squares methods
(against F2) using the SHELXTL-2014 [39]. All non-H atoms were refined
anisotropically. The crystallographic data for complexes 1b-3b are summarized in
Table S1 and Table 1. Crystallographic data for the structural analyses have been
deposited at the Cambridge Crystallographic Data Centre (CCDC), reference numbers
1587786 for 1b, 1033106 for 2b and 1011406 for 3b.
2.4. Cell lines
The human lung carcinoma A549 cell lines, A549cisR (cisplatin-resistant cell
lines), human hepatocellular Bel-7402 cell lines, human breast cancer line MCF-7 cell
lines and normal lung fibroblast WI-38 cells were obtained from American Type
Culture Collection (ATCC, Manassas, VA). These cells were cultured in DMEM
(with −glutamin) culture media supplemented with 1% antibiotic-antimycotic
L
solution and 10% fetal bovine serum (FBS) at 37 °C and 5% CO2.
2.5. Cytotoxicity assay
The cytotoxicity of all the test compounds was examined by a previously reported
method using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. In brief, 100 µL of the cell line was seeded into each well of 96-well plates at 5
× 104 cells/mL and allowed to adhere for 24 h at 37 °C under 5% CO2. Thereafter, the
test compounds in a concentration range of 0−50 µM were added to the plates and
incubated for 48 h at 37 °C under 5% CO2. A total of 10 µL MTT (5 mg/mL) saline
solution was added into each well and then incubated for 5 h at 37 °C under 5% CO2.
The medium was then discarded, and 100 µL of DMSO (dimethyl sulfoxide) was
added into each well. The absorbance of each well was recorded at 570 nm by using
an enzyme-labeling instrument (SpectraMax M5, Molecular Devices).
2.6. Cell morphology analysis
The assay was performed with MCF-7 cells. The MCF-7 cells (1 × 105 cells/mL)
were seeded in six-well plates for 24 h at 37 °C under 5% CO2 and then incubated
with Cu(II) complexes (2 µM) at 37 °C for another 24 h. Thereafter, the cells were
visualized under a bright-field microscope.
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2.7. Cellular uptake study
The intracellular uptake of Cu(II) complexes was performed with MCF-7 cells.
Briefly, 2 × 106 cells were seeded in 10 cm culture dishes at 37 °C under 5% CO2.
After 24 h, the medium was replaced, and the cells were incubated with the copper(II)
complexes (4 µM) for 6 h. The cells were then carefully washed with phosphate
buffered saline (PBS) several times and harvested by trypsinization. Cell digestion
was performed using 3 mL of HNO3 for 2 h at 60 °C. The copper content in the cells
was analyzed using a graphite furnace atomic absorption spectrometer (GF-AAS).
2.8. Anticancer mechanism of the 3b complex
2.8.1. DNA binding and cleavage studies
Electronic absorption spectra in the range of 200–700 nm were recorded by
increasing the amounts of calf-thymus DNA (CT-DNA) introduced to the test
complex (35 µM) in pH 7.2 buffer (5 mM Tris-HCl/50 mM NaCl) at room
temperature. For each addition, the samples were allowed to equilibrate for 10 min,
and any changes in the absorption spectra were recorded.
Emission spectra in the range of 530–750 nm were determined by setting the
excitation wavelength to 510 nm. The test complex was added to a mixture containing
10 µM CT-DNA and 8 µM ethidium bromide (EB) in pH 7.2 Tris-HCl buffer. The
apparent binding constant (Kapp) of the 3b complex was measured using the following
equation [40]:
ꢃ
ꢆ
ꢃ
ꢆ
ꢀꢁꢂ ꢄꢅ = ꢀꢇꢈꢈ complex
(1)
where KEB = 1 × 107 M−1, and [complex] is the concentration of the 3b complex, thus
causing a one-half reduction of the fluorescence intensity of the EB-CT-DNA system.
Viscosity experiments were conducted using Micro Ubbelohde viscometer at 25 ±
0.5 °C. The concentration of CT-DNA was fixed at 200 µM, whereas 3b and EB
concentrations were varied from 0 to 100 µM in pH 7.2 Tris-HCl buffer. Data were
presented as (η/ηo)1/3 vs. [compound]/[DNA], where η and ηo represent the viscosity of
the CT-DNA solution in the presence and absence of the test compounds,
respectively.
The efficiency of the 3b complex to cleave DNA was monitored using agarose gel
electrophoresis. In this study, a total volume of 20 µL in each reaction mixture
contained supercoiled plasmid pBR322 DNA (200 ng), Tris-HCl buffer pH 7.0, and
different concentrations (25, 50, and 100 µM) of the 3b complex. The mixtures were
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then incubated at 37 °C. After 3 h, 4 µL of 5X loading buffer was added.
Subsequently, the reaction mixtures were loaded into 1% agarose gel (1 mg/mL in
TAE) containing 1% (v/v) GoldView II and then analyzed using gel electrophoresis in
1X TAE buffer at 100 V for 90 min.
2.8.2. Topoisomerase I inhibition assay
The human topoisomerase I (Topo I) inhibitory activity by the 3b complex was
studied in the agarose gel electrophoresis experiments. One unit of Topo I was
incubated with 0.25 µg supercoiled pBR322 plasmid DNA in the presence of each test
complex (0–25 µM) in a buffer containing the following: 35 mM Tris-HCl (pH 8.0),
72 mM KCl, 0.1% BSA, 5 mM DTT (dithiothreitol), 5 mM spermidine, and 5 mM
MgCl2. The final volume of each sample was 20 µL. The mixtures were then
incubated at 37 °C for 30 min, and the reaction was stopped by adding a loading
buffer solution (4.5% sodium dodecyl sulfate, 45% glycerol, and 0.25% bromophenol
blue) into each mixture. Electrophoresis was performed in a 0.8% agarose gel in 1X
TAE buffer at 70 V for 120 min.
2.8.3. Molecular docking studies
To obtain the binding information of the 3b complex toward DNA and Topo I,
docking studies were performed using Autodock Vina [41]. The crystal structures of
the DNA (d(CGCGAATTCGCG)2) and human Topo I were obtained from the protein
data bank (PDB ID: 1BNA and 1T8I, respectively). AutoDock program tools were
utilized to delete the water molecules, add polar hydrogen atoms, and assign other
parameters. In the docking studies, the single bonds of the Cu(II) complex were
allowed to rotate, but the human Topo I and DNA structures were set to remain rigid.
2.8.4. Cell apoptosis
For the apoptosis experiments, Annexin V FITC kit (Sigma Aldrich, Missouri,
USA) was used. MCF-7 cells were seeded in six-well plates and then incubated with
the 3b complex at different concentrations (0–2 µM). After 24 h, the cells were
harvested and then resuspended in 1X annexin binding buffer (200 µL). Subsequently,
5 µL annexin V and 5 µL propidium iodide (PI) were added to each sample, and the
mixtures were incubated on ice in the dark. An additional amount of binding buffer
(300 µL) was added after 20 min, and the cells were analyzed immediately by flow
cytometry.
2.8.5. Change in mitochondrial membrane potential assay
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The mitochondrial membrane potential was analyzed in the presence of JC-1
probe (Beyotime Jiangsu China). MCF-7 cells were exposed to the 3b complex (0–2
µM) for 24 h at 37 °C. Subsequently, the cells were harvested and then resuspended in
a prewarmed JC-1 solution (5 µg/mL in PBS). After incubating for 30 min at 37 °C in
the dark, the samples were washed three times with PBS and immediately measured
by flow cytometry.
2.8.6. Detection of intracellular reactive oxygen species
Intracellular reactive oxygen species (ROS) production was measured with
2’,7’-dichlorofluorescein diacetate probe (DCFH-DA) (Beyotime Jiangsu China).
MCF-7 cells were incubated with the 3b complex (0–2 µM) for 24 h. The cells were
then harvested and washed three times with PBS, followed by resuspension in 500 µL
of a serum-free medium containing 10 µM DCFH-DA probe. After 30 min of
incubation at 37 °C in the dark, the samples were rewashed three times and suspended
in 1 mL of PBS. The ROS generation in the MCF-7 cells was immediately measured
by flow cytometry.
2.8.7. Western blot analysis.
Immunoblotting analysis was carried out as previously described [26, 42]. Primary
antibodies: mouse anti-β-actin (Abcam), rabbit anti-γH2AX (Abcam), rabbit
anti-phos-CHK1 (Abcam), rabbit anti-phos-CHK2 (Abcam), rabbit anti-phos-p53
(Abcam), mouse anti-cleaved caspase-9 (Abcam), mouse anti-cytochrome c (Abcam),
mouse anti-cleaved caspase-3 (Abcam) and mouse anticleaved PARP-1 (Abcam).
Secondary antibodies: horseradish peroxidase-conjugated sheep anti-mouse and
horseradish peroxidase-conjugated donkey anti-rabbit (Cell Signalling Technology).
2.9. Inhibition of tumor spheroid growth
Three-dimensional spheroids were prepared according to a previously reported
method [43]. Briefly, 1.5% (wt/v) agarose DMEM solution was heated to 80 °C for 30
min and then autoclaved for 30 min. Subsequently, 50 µL of agarose solution was
pipetted into each well of a 96-well microtiter plate under sterile condition. After the
agarose solidified, the agarose-coated plates were stored at ambient temperature and
covered until use. Each 20 µL drop of MCF-7 cell (5000 cells/drop) was prepared on
the upper lip of a cell culture petri dish and incubated in an inverted position at 37 °C
under 5% CO2. After 72 h, one multicellular spheroid was transferred to each
agarose-coated well, and 200 µL of DMEM media was added into each well.
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Thereafter, MCF-7 spheroids were allowed to settle for 24 h in a humidified CO2
incubator followed by incubation with the test Cu(II) complexes at various
concentrations.
2.10. Statistical analysis
All biological experiments have been carried out at least thrice with triplicatesin
each experiment. Student's t test was performed to evaluate the significance of the
measurement differences. Results were expressed as mean ± SD (standard deviations)
and considered to be significant when p< 0.05.
3. Results and discussion
3.1. Description of structures
The structure of 1b together with the atom labels scheme is shown in Fig. 1A. In
complex 1b, the monoanionic ONO tridentate L1− ligand is coordinated to copper(II)
through the azomethine nitrogen (N2), deprotonated phenolic oxygen (O2) and the
ketoamide oxygen (O1) forming respectively six- and five-membered chelate rings. In
1b, the fourth coordination site is occupied by the pyridine nitrogen atom (N3), and
completing the coordination sphere in the apical site is a Cl– anion. In 1b, the
coordination geometry around the Cu(II) could be described as distorted square
pyramid as evident from the trigonality parameter [44], τ = (β − α)/60 = 0.091, where
β and α are the N2−Cu1−N3 (171.6°) and O2−Cu1−O1 (166.1°) angles, respectively.
In the crystal packing of 1b, the discrete copper(II) units were linked into a 1D
polymeric chain by the N1−H1···O3 hydrogen bonds and bifurcated O3−H3A···Cl1i
and O3−H3B···Cl1iii interactions (Fig. 1B).
Views of the asymmetric unit of 2b and 3b with atom labels are shown in Fig. 1C.
The asymmetric units contain one tridentate L2− ligand, one Cu(II) center, a
−
coordinated pyridine molecule, and one Cl/NO3 anion. In 2b, the copper atoms
exhibits a five-coordinate environment with a nitrogen atom and two oxygen atoms
from a Schiff base-ligand, a nitrogen atom from the pyridine ligand, and one terminal
Cl atom (Fig. 1C). In 2b the coordination polyhedron around the Cu1 center could be
described as a slightly distorted square pyramid (τ = 0.0001), with the metal displaced
from the O1/N2/O2/N1 basal plane (maximum displacement 0.005 Å for the N2
atom), and the Cl atom at the apex, with the metal displaced by 0.22 Å toward Cl
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from the mean basal plane. In 3b, the coordination geometry around copper(II) in the
−
complex is very similar to that in 2b; the only difference is the presence of a NO3
anion at the apical position (Fig. 1E). In 3b the index of the degree of trigonality, τ,
has a value 0.004, indicating a much more distorted structure compared to 2b. All
Cu−X bond distances of 2b and 3b are in good agreement with those found in the 1b
complex (Table S1). In solid state of 2b, the dimers are formed via N−H···Cl
interactions involving a nitrogen atom (N3) from a Schiff-base ligand and a chloride
atom (Cl1i) bonded to the Cu1i from the adjacent molecule (N3···Cl1i = 3.11 Å and
the N1−H1···Cl1i angle is 156.2°, symmetry code: (i) 1 − x, 1 − y, 1 − z, Fig. 1D).
Furthermore, a small extent of π···π stacking (Fig. 1D) between pyridine rings is
noted in 2b, a fact that could provide some stability to the dimeric structure. In 3b, the
−
monomeric units are interlinked by the NO3 anions through the N−H···O hydrogen
bonds (Fig. 1F), which leads to a dimer fashion, and then a self-assembly of dimers by
means of π···π stacking affords a one-dimensional (1D) polymeric chain (Fig. 1G).
3.2. Mass spectrometry
In order to confirm the structures found in the solid state and the stability of the
ligand/co-ligand of these complexes in solution, these Cu(II) complexes were
dissolved in methanol containing 10% (v/v) DMSO and then characterized by
electrospray ionization mass spectrometry (ESI-MS) (Fig. 2). When the in-source
energy was 0 eV, the ESI-MS spectrum of the 1a complex showed the parent signal at
m/z = 239.99, which could be assigned as isotopic envelopes corresponding to
[(L)Cu(L1)]+ (fit: 240.00), thus suggesting that the solvent H2O molecules and the
coordinated Cl− anion were lost under electrospray ionization conditions. The parent
cluster ions of 2a and 3a complexes were both found at m/z = 334.04, which could
correspond to the [Cu(L2) + CH3OH]+ structure (fit: 334.04), thus suggesting that the
coordinated anions were replaced by CH3OH (methanol). Furthermore, some lower
m/z species were found at 380.03 and 302.01, which could be assigned as [Cu(L2) +
DMSO]+ (fit: 380.03) and [Cu(L2)]+ (fit: 302.01), respectively.
In the ESI-MS spectrum of 1b, the ion peak at m/z 319.04 was assignable to
[Cu(L1)(Py)]+ (fit: 319.04), which was formed by losing the coordinated Cl− anion
and the solvent H2O molecules. The ESI-MS spectra of 2b and 3b displayed similar
fragmentation patterns and showed molecular parent ion peaks at m/z = 381.05 for
[Cu(L2)(Py)]+ (fit: 381.05), thus suggesting that 2b and 3b have the same structures
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in the solution. These results are in good agreement with the proposed structures. The
results also indicated that the ligand/co-ligand of these Cu(II) complexes were
essentially inert in the solution.
3.3. In vitro cytotoxicity
The in vitro anticancer activity of the 1a–3a and 1b–3b complexes, the free Schiff
base ligands (L1 and L2), and the co-ligand was evaluated in a panel of human cancer
cell lines by the MTT test. For comparison, the anticancer activity of cisplatin was
also examined under identical conditions.
Table 2 shows the IC50 values. All Cu(II) complexes showed micromolar
toxicities (1.12–47.36 µM), which were comparable to and in many cases
significantly better than that of cisplatin. The IC50 values of the 1:1:1
Cu(II)/ligand/co-ligand complexes 1b–3b were significantly lower (p < 0.01−0.05)
than those of the corresponding 1:1 Cu(II)/ligand complexes 1a–3a, thus indicating
that the introduction of the co-ligand pyridine moiety into 1a–3a enhanced the
anticancer activity of 1a–3a. The 1b complex was 2.76-, 2.95-, 7.75-, and 7.74-fold
more cytotoxic (p < 0.01) than the 1a in Bel-7402, MCF-7, A549, and A549cisR cells,
respectively. The IC50 values of 2b were 1.91-, 3.12-, 1.40-, and 1.48-fold lower (p <
0.05) than those of 2a in Bel-7402, MCF-7, A549, and A549cisR cells, respectively.
Similarly, the IC50 values of 3b in Bel-7402, MCF-7, A549, and A549cisR cells were
2.09-, 3.43-, 1.46-, and 1.47-fold lower (p < 0.05) (i.e., more cytotoxic) than those of
3a, respectively. These results suggested that the introduction of pyridine co-ligand
affected the type and number of ligands, as well as the coordination geometry in the
complexes, thus leading to a change in the hydrophobicity of the Cu(II) complexes, an
increase in cellular uptake (vide infra), and an improvement in the anticancer activity
of the Cu(II) complexes.
As revealed by the observed mean of IC50 values, the potency of the 1a–3a to kill
cancer cells followed the order 3a ≈ 2a > 1a and that of 1b–3b followed the order 3b
≈ 2b > 1b. None of the Cu(II) complexes displayed any cross-resistance with cisplatin,
as ascertained by their ability to kill the A549cisR and A549 cell lines
indiscriminately. The co-ligand, the free Schiff base ligands, and the Cu(II) salts
showed relatively low activity against cancer cells, thus implying that the anticancer
activities by these Cu(II) complexes were derived from themselves altogether and not
merely from the free Cu(II) chaperoned into the cancer cells by these ligands.
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Furthermore, the cytotoxicities of these complexes were also evaluated in normal
human WI-38 cells. In general, 2a, 3a, and 1b–3b complexes were less potent toward
WI-38, thus suggesting selective toxicity toward cancer cells over normal cells.
3.4. Cell morphology analysis
Fig. 3A depicts the effects of these Cu(II) complex (2 µM) treatments on the
morphology of MCF-7 cells. The cells under treatment with 1a and 1b complexes
exhibited similar cell morphologies (spindle shaped and anchorage dependent) to that
of the control cells. However, the cells treated with other Cu(II) complexes,
particularly 2b or 3b, were found to be mainly spherically shaped and lucent, thus
indicating that the cell bodies were damaged. These results suggested that the
apparent change in MCF-7 cell morphology was induced by the 2a, 3a, 2b, and 3b
cytotoxic Cu(II) complexes.
3.5. Cellular uptake
Most previous studies reported that the cytotoxicity of metal complexes was
correlated with their cellular uptake [45, 46]. Thus, the cellular uptake was
investigated by treating MCF-7 cancer cells with 4 µM of 1a–3a and 1b–3b for 6 h.
The intracellular Cu amount was then quantified by GF-AAS, and the results are
shown in Fig. 3B. As expected, the results showed that the uptake of the 1b, 2b, and
3b complexes were 1.51-, 1.57-, and 1.56- fold greater (p < 0.05) than that of the 1a,
2a, and 3a complexes, respectively, thus corroborating their cytotoxicity results. It
can be speculated that the internalization of these Cu(II) complexes might play a vital
role in their cellular cytotoxicity.
3.6. Possible anticancer mechanism of the 3b complex
3.6.1. DNA binding and cleavage studies
Considering that 3b or 2b showed the most potent cytotoxic effects compared
with the other analogs, the 3b complex was selected for further investigation. DNA is
the important intracellular anticancer target of many metallodrugs [5, 47, 48].
Therefore, metallodrug–DNA interactions are important for understanding the
anticancer mechanisms of metal-based drugs.
To investigate the binding of the 3b complex to CT-DNA, multiple spectroscopic
techniques were used. In this study, the UV-Vis absorption spectra of 3b in the
presence and absence of CT-DNA were recorded (Fig. 4A). The observed peaks in the
UV region are characteristics of π−π* for ligand transitions. Owing to the addition of
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CT-DNA to the test complex, the absorption bands of the 3b complex at 308 and 377
nm exhibited hypochromisms of approximately 14.1% and 8.4%, respectively, with a
hypochromic shift of ca. 2 nm in the above band positions. The results indicated a
strong interaction between the 3b complex and CT-DNA, and it is possible that the 3b
complex bound to the CT-DNA helix via intercalation.
To further study the binding mode of the 3b complex to CT-DNA, EB
displacement studies were performed. As shown in Fig. 4B, the emission intensity
decreased by approximately 63.8% with the addition of 25 µM of the 3b complex to
the EB-CT-DNA system, thus showing that the 3b complex efficiently competed with
EB and that the 3b complex could interact with CT-DNA possibly by intercalative
mode [49]. The binding constant (Kapp) values were obtained for the 3b complex by
using Equation (1). The Kapp value (2.67 ± 0.15 × 106 M−1) of 3b was less than the
binding constant value (107 M−1) of classical intercalators and metallointercalators,
thus suggesting that the interaction between CT-DNA and the 3b complex occurred
via a moderate intercalative mode.
The viscosity of CT-DNA, which is sensitive to changes in DNA length, was
measured to analyze the binding nature of the 3b complex with CT-DNA. As depicted
in Fig. 4C, the viscosity of CT-DNA increased with increasing 3b complex
concentration, thus proving that the binding of the 3b complex to CT-DNA was via
intercalation.
Docking studies were performed to provide conclusive results and complement
the spectroscopic results into the insight of complex−DNA interaction. The B-DNA
scaffold from 1BNA was used in a broad search for the complex–DNA interactions of
the 3b complex. Fig. 4D shows the energetically favorable docked poses obtained
from the molecular docking of the 3b complex with the B-DNA. The results showed
that the Cu(II) complex fitted well into the curved contour of the B-DNA into the
minor groove region situated within the A−T rich region. The energy minimized
structure suggested that the Cu(II) complex was stabilized by hydrophobic contacts
and van der Waals forces with the B-DNA functional groups. In this model, the L2
ligand of the 3b complex was inside the minor groove, whereas the pyridine group
(co-ligand) of the 3b complex pointed outside of the minor groove. Moreover, the
nitrogen atoms (N2 and N3) of the Cu(II) complex might be engaged by
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hydrogen-bonding interactions with the DNA nucleobases DA-6 (NHDA···N3 = 3.13
Å) and DT-20 (N2−H2···ODT = 2.54 Å).
The ability of the 3b complex to cleave supercoiled pBR322 plasmid DNA was
assessed by gel electrophoresis. Fig. 4E shows the results of the agarose gel
electrophoretic separations of the supercoiled pBR322 plasmid DNA with increasing
concentrations of the 3b complex. The Cu(II) complex could relax the Form I
(supercoiled form) of DNA into Form II (open circular form) in a dose-dependent
manner. The aforementioned results collectively implied that the 3b complex could
interact with the DNA target and might lead to DNA damage in cancer cells.
3.6.2. Topoisomerase I inhibition
DNA Topo I is an essential nuclear enzyme and is vital for the topological change
of DNA [50]. Topo I poisoning can disturb the topoisomerase in cancer cells by
inducing DNA damage that ultimately leads to cell apoptosis [51]. Topo I is currently
considered a significant intracellular target for anticancer drugs [5]. Therefore, the
effects of the 3b complex on the activity of human Topo I by agarose gel
electrophoresis were investigated. The result showed that the Cu(II) complex
significantly prevented the unwinding of DNA by the action of human Topo I at
concentrations as low as 12.5 µM, thus indicating that the complex was the inhibitor
of human Topo I (Fig. 4F).
Docking studies were further performed to explore the potential interaction mode
of the 3b complex with Topo I. As depicted in Fig. 4G, the phenyl group of the Cu(II)
complex was intercalated onto the DNA cleavage site of Topo I and formed
base-stacking interactions with both T10 (‒1) and G11 (+1) base pairs. Furthermore,
the O1 atom and N3 atom of the Cu(II) complex formed hydrogen-bonding
interactions with Thr718 and Arg364, respectively. The pyridine group pointed
outside of the major groove and formed a hydrophobic interaction with IIe535. These
results suggested that the Cu(II) complex fitted well into the DNA cleavage site and
might suppress the association of the Topo I enzyme with DNA, thereby influencing
the Topo I inhibition activity or the ability to form a stable complex with DNA.
3.6.3. Biomarkers of DNA damage
The results of the DNA binding, DNA cleavage studies, and Topo I inhibition assay
suggested that the 3b complex was likely to induce DNA damage. To further
investigate whether the 3b complex could induce DNA damage in cancer cells,
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western blotting analyses were used to examine the expression of the biomarkers
related to the DNA damage pathway. Accordingly, MCF-7 cells were incubated with
the 3b complex for 24 h, and the untreated cells were used as controls. As shown in
Fig. 4H, the phosphorylated forms of p53 (Ser15), CHK1, CHK2, and H2AX
(γH2AX) expressions were upregulated by the 3b complex compared with the
untreated controls, thus indicating DNA damage in MCF-7 cancer cells [52–54].
3.6.4. Analysis of apoptosis
Many anticancer agents evidently exert their cytotoxic effects by activating
apoptosis [55]; hence, we investigated the features related to these pathways. To
evaluate cell apoptosis in the presence of the 3b complex, FITC-Annexin V/PI dual
staining was performed in MCF-7 cells. Flow cytometry analysis showed that the 3b
complex could efficiently induce concentration-dependent apoptosis in MCF-7 cells
(Fig. 5A).
3.6.5. Induction of mitochondrial damage
Mitochondria damage is tightly linked to apoptosis pathways [56, 57]. Interestingly,
recent studies provided evidence for the differences in the structure and function of
mitochondria in cancer and normal cells, thus providing us the opportunities to target
mitochondria when designing anticancer agents [42, 58]. To establish whether the 3b
complex–induced apoptosis was related to mitochondrial damage, we examined the
influence of the complex on the mitochondrial membrane potential (∆ψm) of MCF-7
cells
by
using
the
lipophilic
fluorescent
probe
JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide) staining
method. JC-1 staining indicated a loss of ∆ψm, as evidenced by a few JC-1 aggregates
(indicating hyperpolarized mitochondria and showing red fluorescence) and increased
JC-1 monomers (indicating depolarized mitochondria and showing green
fluorescence). As shown in Fig. 5B, treating MCF-7 cells with the 3b complex caused
the loss of ∆ψm in a dose-dependent fashion, thus illustrating the activation of
mitochondrial-mediated apoptosis pathway.
Another confirmation that the 3b complex exerted apoptosis via the mitochondrial
pathway was obtained from the western blotting analyses, which were conducted to
monitor the expression levels of cytochrome c, cleaved caspase-3, cleaved caspase-9,
and cleaved PARP-1 (poly ADP ribose polymerase 1). The cytochrome c in the
mitochondria fraction was downregulated, whereas its amount in the cytoplasm was
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upregulated (Fig. 5C), thus confirming that the treatment with the 3b complex
resulted in the loss of ∆ψm and the mitochondrial release of cytochrome c. As a result,
the levels of cleaved caspase-3 and cleaved caspase-9 were upregulated. Subsequently,
cleaved PARP-1 was also upregulated, thus regulating the apoptosis program of
MCF-7 cells.
3.6.6. Elevation of ROS levels
Cell apoptosis induced by metal-based complexes is closely related to ROS
generation [59]. Increased ROS can damage intracellular macromolecules and induce
apoptosis or alter cell function [60–62]. Thus, we monitored how the treatment of
MCF-7 cells with the 3b complex could affect the production of intracellular ROS by
using a fluorescent H2DCFDA probe (2’,7’-dichlorofluorescein diacetate). As shown
in Fig. 5D, the cells treated with the 3b complex had a right-shifted fluorescence peak
relative to the control cells, thus confirming that the 3b complex indeed induced ROS
production. At a concentration of 1 µM, the 3b complex increased the intracellular
ROS level by ca. 40% compared to the control group (Fig. S1).
3.7. Inhibition of tumor spheroid growth
Multicellular tumor spheroids are superior to monolayer tumor cells in simulating
internal solid tumor tissue because multicellular tumor spheroids mimic the tight cell–
cell interactions, intricate cellular heterogeneity, and tumor microenvironment
observed in vivo more closely [63, 64]. Therefore, multicellular 3D MCF-7 tumor
spheroids were used in the current study to evaluate the potential of the 3b complex to
prevent the growth of solid tumors.
The inhibition of MCF-7 tumor spheroid growth was investigated after the
treatment with the 3a or 3b complex at a concentration of 4 µM for seven days. As
shown in Fig. 6, the MCF-7 tumor spheroids kept increasing in volume in the absence
of Cu(II) complexes. The obvious volume reduction of the MCF-7 tumor spheroids
was observed for the 3a complex after a seven-day treatment, but the obtained 3D
spheroids were compact. However, the tumor cells on the surface of MCF-7 tumor
spheroids treated with the 3b complex were more disorganized. Moreover, the cell
membrane ruptured and the cell leaked. These results confirmed that the 3b complex
was more effective than the 3a complex as an antitumor agent.
4. Conclusions
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We presented a Cu-based platform for a possible anticancer agent by introducing a
pyridine co-ligand into 1:1 Cu(II)/ligand complexes to form 1:1:1
Cu(II)/ligand/co-ligand complexes with a minor synthetic effort; this platform
resulted in changes in biochemical properties. We demonstrated that the 1:1:1
Cu(II)/ligand/co-ligand complexes exhibited excellent in vitro anticancer activity and
showed two- to eightfold better activity than the corresponding 1:1 Cu(II)/ligand
complexes in Bel-7402, MCF-7, A549, and A549cisR cancer cell lines. Further
mechanistic studies provided that the 3b complex induces the DNA damage, resulting
in an ROS-mediated mitochondrial dysfunction apoptosis pathway. In conclusion, the
results of our study might potentially represent a powerful strategy for enhancing the
anticancer activity of metal-based agents.
Acknowledgements
This work is supported by the Natural Science Foundation of Guangxi
(2017GXNSFEA198002), High-level innovation team and distinguished scholar
program of Guangxi universities to F. Yang, the National Natural Science Foundation
of China (grant No.81503161) and the Technology Division of Nantong
(MS12016022).
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Figure captions
Scheme 1. Synthesis of the 1:1 Cu(II)/ligand complexes (1a-3a) and the 1:1:1
Cu(II)/ligand/co-ligand complexes (1b-3b).
Figure 1. (A) The local coordination environment of 1b, H atoms are omitted for
clarity. (B) Hydrogen bonding interactions (black dashed lines) of the 1b complex
with the surrounding moieties (Symmetry code: i = – x, – y, 1 – z; ii = 1 – x, – y, 1 – z;
iii = 1 + x, y, z). (C) The local coordination environment of 2b, H atoms are omitted
for clarity. (D) Perspective view of a dimeric structure formed by hydrogen bonds
(black dashed lines) and π···π stacking (blue dashed lines) in 2b (Symmetry code: i =
1 – x, 1 – y, 1 – z). (E) The local coordination environment of 3b, H atoms are omitted
for clarity. (F) Perspective view of a dimeric structure formed by hydrogen bonds
(black dashed lines) in 3b (Symmetry code: i = 1 – x, – y, 2 – z). (G) Perspective view
of a 1D chain formed by π···π stacking (blue dashed lines) and hydrogen bonds (black
dashed lines) in 3b.
Figure 2. ESI-MS spectra of 1a-3a and 1b-3b in MeOH containing DMSO (10% v/v)
when the in-source energy was 0 eV. The red line simulated m/z envelope and black
line is observed spectra.
Figure 3. (A) Change in morphology of MCF-7 cells in the presence of 1a-3a and
1b-3b (2.0 µM and 24 h). Scale bar: 500 µm. (B) MCF-7 cell uptake of 1a-3a and
1b-3b (*p < 0.05).
Figure 4. (A) Absorption spectra of the 3b complex (35 µM) upon the titration of
CT-DNA (0−32 µM). (B) Effect of addition of the 3b complex on the emission
intensity of EB (8 µM) bound to CT-DNA (10 µM) in a pH 7.2 Tris-HCl buffer. (C)
Effect of increasing amounts of the 3b complex or EB on the relative viscosity of
CT-DNA (200 µM). (D) Molecular docked model of the 3b complex with B-DNA
(PDB ID: 1BNA). (E) Agarose gel electrophoresis patterns for the cleavage of
pBR322 plasmid DNA by the 3b complex at pH 7.2 at 37 °C for 5 h. Lane 1: DNA
alone (control); Lanes 2−4: DNA with the 3b complex at the concentrations of 25, 50,
100 µM, respectively. (F) Effect of the 3b complex on Topo I mediated DNA
relaxation; lane 1, DNA control; lane 2, Topo I + DNA; Lanes 3 and 4: DNA + Topo
I with the 3b complex at the concentrations of 12.5 and 25 µM, respectively. (G)
Molecular docked model of the 3b complex with the human-DNA Topo I (PDB ID:
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1T8I). (H) Western blot analysis of proteins related to the DNA damage pathway in
MCF-7 cells.
Figure 5. (A) Apoptotic effects of the 3b complex on MCF-7 cells. MCF-7 cells were
treated with the 3b complex at the indicated concentrations for 24 h and then analyzed
by flow cytometry after annexin V-FITC/PI staining. (B) Effects of the 3b complex
on mitochondrial membrane potential (∆ψm) analyzed by flow cytometry and JC-1
staining. (C) Effects of the 3b complex on cytochrome c (in the cytoplasm and
mitochondria, respectively), cleaved caspase-9, cleaved caspase-3, and cleaved
PARP-1. MCF-7 cells were treated with the 3b complex at the indicated
concentrations for 24 h and then analyzed by Western blotting. (D) Analysis of ROS
production by flow cytometry after MCF-7 cells was treated with vehicle or the 3b
complex for 24 h.
Figure 6. Morphology of 3D MCF-7 tumor spheroids treated with vehicle, 3a or 3b
on at the indicated concentrations for 7 days. Scale bar: 200 µm.
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Table 1 Crystal data for complexes 1b-3b.
complex
1b
2b
3b
Empirical formula
C14H16ClCuN3O3 C19H16ClCuN3O2 C19H16CuN4O5
Molecular weight
373.29
triclinic
P−1
417.34
monoclinic
P21/c
443.91
triclinic
P−1
Crystal system
Space group
a/Å
8.8844(11)
9.6621(11)
9.8950(11)
83.903(6)
68.477(5)
88.055(6)
785.70(15)
2
7.627(4)
20.335(11)
11.502(6)
90.00
8.4027(6)
11.1825(11)
11.5411(9)
62.413(9)
88.709(6)
79.905(7)
944.13(14)
2
b/Å
c/Å
α/°
β/°
95.388(9)
90.00
γ/°
Volume/Å3
1775.9(17)
4
Z
ρcalcg/cm3
µ(Mo−Kα) (mm−1)
F(000)
1.578
1.561
1.561
1.574
1.399
1.197
382
852.0
454.0
Data/restraints/parameters 3589/0/203
Goodness−of−fit on F2
1.093
3623/0/235
1.054
3857/0/262
1.056
Final R1, wR2 [I > 2σ(I)] 0.0476, 0.1113 0.0496, 0.1443 0.0505, 0.1405