Enhancing the copper(II) complexes cytotoxicity to cancer cells through bound to human serum albumin

Article abstract of DOI:10.1016/j.jinorgbio.2014.12.012

We use Schiff-base salicylaldehyde benzoylhydrazone (HL) as the ligand for copper(II), resulting in the complexes [CuCl(L)] · H₂O (C1), [CuNO₃(L)] · H₂O (C2) and [CuBr(L)]₂ (C3). We characterize the Cu(II) compo

Full text of DOI:10.1016/j.jinorgbio.2014.12.012

Enhancing the copper(II) complexes cytotoxicity to cancer cells through
bound to human serum albumin
Yi Gou, Yao Zhang, Jinxu Qi, Zuping Zhou, Feng Yang, Hong Liang
PII:
DOI:
Reference:
S0162-0134(14)00305-5
doi: 10.1016/j.jinorgbio.2014.12.012
JIB 9635
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
19 August 2014
10 December 2014
10 December 2014
Please cite this article as: Yi Gou, Yao Zhang, Jinxu Qi, Zuping Zhou, Feng
Yang, Hong Liang, Enhancing the copper(II) complexes cytotoxicity to cancer cells
through bound to human serum albumin, Journal of Inorganic Biochemistry (2014), doi:
10.1016/j.jinorgbio.2014.12.012
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Enhancing the copper(II) complexes cytotoxicity to cancer
cells through bound to human serum albumin
Yi Gou, Yao Zhang, Jinxu Qi, Zuping Zhou, Feng Yang*, Hong Liang*
State Key Laboratory Cultivation Base for the Chemistry and Molecular
Engineering of Medicinal Resources, Ministry of Science and
Technology of China, Guangxi Normal University, Guilin, Guangxi,
China
*Corresponding authors: (F. Yang) E-mail: jxyangfeng@gmail.com; (H.
Liang) E-mail: hliang@mailbox.gxnu.edu.cn; Phone/Fax: 86-773-212-
0958; Postal address: 15 Yucai Road, Guilin, Guangxi, China. Zip code:
541004
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Abstract
We use Schiff-base salicylaldehyde benzoylhydrazone (HL) as the ligand
for Copper(II), resulting in the complexes [CuCl(L)]·H2O (C1),
[CuNO3(L)]·H2O (C2) and [CuBr(L)]2 (C3). We characterize the Cu(II)
compounds’ interactions with human serum albumin (HSA) using
fluorescence spectroscopy and molecular docking. These studies revealed
that Cu(II) compounds propensity bound to IIA subdomain of HSA
possible by hydrophobic interactions and hydrogen bond. Cu(II)
compounds produce intracellular reactive oxygen species (ROS) in cancer
cells. Complexes of HSA and copper(II) compounds enhance about 2-
fold cytotoxicity in cancer cells but do not raise cytotoxicity levels in
normal cells in vitro. Compared with C3 alone, HSA–C3 complex
promotes HepG2 cell apoptosis and has a stronger capacity to promote
cell cycle arrest at the G2/M phase of HepG2.
Keywords: copper(II) compound; human serum albumin; cytotoxicity;
cell apoptosis
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1. Introduction
In the 1950s, salicylaldehyde benzoylhydrazone (HL) was found to
possess mild bacteriostatic activity when tested in vitro against
microorganisms such as Mycobacterium tuberculosis, Candida albicans
and Mycobacterium smegmatis [1]. It was noted that HL, in particular,
appeared to be an unusually potent inhibitor of DNA synthesis and cell
growth in a variety of human and rodent cell lines grown in culture [2−4].
Subsequently, a range of tridentate ONO hydrazones, including HL, were
evaluated as iron-chelating drugs in vivo and HL was found to mobilize
iron from iron-loaded reticulocytes in vitro [5, 6]. Interestingly, copper(II)
complexes showed significantly stronger anticancer activity than did free
ligands [7]. Due to biological interest in copper chelate systems, a
number of single-crystal x-ray structural studies have been carried out on
copper with HL and its analogues [8−12]. Unfortunately, their poor water
solubility and moderate anti-proliferative characteristics have hindered
their potential for application and, therefore, little is known about the
bioactivity mechanisms of these compounds [8, 13].
A promising approach to circumvent the poor water solubility of
metal compounds and to improve their efficacy towards malignant cells is
to couple anticancer drugs to suitable carrier macromolecules. Various
types of macromolecules have been used as carrier molecules, including
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poly (ethylene glycol) polymers, nanoparticles, nanotubes, liposomes,
dendrimers and protein biomolecules [14, 15]. Among them, human
serum albumin (HSA) is promising for drug carrier owing to its unique
biochemical and pharmacological characteristics [16−18]. Current studies
showed that HSA not only increases the solubility of metal compounds,
but also enhances their antitumour activity [19−24]. HSA is the most
abundant protein in blood plasma, which plays a central role in drug
pharmacokinetics and distribution [25]. In order to better develop drugs,
it is necessary to understand the interactions of lead drugs and HSA [26,
27]. To date, the interactions of HSA with metal compounds have been
extensively investigated [28]. The studies revealed that the modes of
metal compounds interacting with HSA are interesting. For example, the
axial maleimide of a Pt(IV) complex forms a covalent bond with the sole
free cysteine thiol of HSA [29]. The ruthenium compounds could bind to
HSA either through direct coordination to HSA or in a non-coordinate
fashion [19, 20, 30]. Meanwhile, many copper anticancer compounds
have also been synthesized and studied on their interactions with HSA
[31−36].
With this background in mind and considering that copper is a crucial
trace element in redox chemistry specific to the growth and development
in all living organisms [37], we attempted to synthesize copper(II)
compounds by reacting CuCl2/Cu(NO3)2/CuBr2 with HL in methanol. We
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were excited to obtain a novel phenolic oxygen-bridged binuclear
copper(II) compound C3 (Scheme 1). It is worth mentioning here that
many metalloenzymes and proteins contain in their active sites two
copper ions that operate cooperatively [38–41]. Thus, a great deal of
attention has been given to bimetallic copper compounds with two metal
ions in close proximity because they provide an opportunity to study
intramolecular binding, bimetallic catalysts, magnetic exchange
interactions, multi-electron redox reactions and an activity mimicking the
possible activation of small substrate molecules by enzymes [42–45].
While there have been a few reports about C1 and C2 in the literature, no
attempt has been made to explore the effect of compounds binding to
HSA [46–48]. In this study, we tested the antiproliferative activity of HL
and C1–C3 compounds in the human cancer cell line. Our results indicate
that these copper(II) compounds bound with HSA can improve their
bioactivity.
2. Experimental section
2.1 Material
HSA (fatty acid content < 0.05%) was purchased from the Sigma
Chemical Company and used without further purification. All other
chemicals and solvents used were of high purity and available from
commercial sources. Water used in the reactions was distilled prior to use.
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Elemental analyses (C, N, and H) were carried out on a Perkin-Elmer
2400 analyzer. Infrared (IR) spectra were recorded using KBr pellets
(4000–400 cm-1) on a Nexus 870 FT-IR spectrophotometer. UV–visible
spectra were measured on a Cary 1E UV-Visible spectrophotometer in
the 200–800 nm range.
2.2. Synthesis of copper compounds
Compounds HL, C1 and C2 were synthesized according to literature
procedures [46–48].
2.2.1. Synthesis of [CuBr(L)]2 (C3)
Benzoylhydrazine (0.27 g, 2 mmol) and salicylaldehyde (0.24 g, 2
mmol) were dissolved in an aqueous methanol solution (15 mL) and
stirred 1 h to give an orange solution, which was added to a methanol
solution (15 mL) of CuBr2 (0.44 g, 2 mmol). The mixture was stirred for
another 30 min at room temperature to give a celadon solution and then
filtered. The filtrate was kept in air for a week, forming blue block
crystals. The crystals were isolated, washed three times with distilled
water and dried in a vacuum desiccator containing anhydrous CaCl2.
Yield: 616 mg (65%). Anal. Calcd for C28H22Br2Cu2N4O4 (765.40): C,
43.94; H, 2.89 and N, 7.32. Found: C, 43.95; H, 2.89 and N, 7.33. IR
(KBr, cm-1): 1600 ν(C=N); 555, 511, 476, 446 ν(Cu–N/Cu–O) .
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2.3. Crystal structure determinations
X-ray crystallographic data were collected on a Bruker SMART Apex
II CCD diffractometer using graphite-monochromated Mo-Kα (λ =
0.71073 Å) radiation. Empirical adsorption corrections were applied to all
data using SADABS. The structures were solved by direct methods and
refined against F2 by full-matrix least-squares methods using the
SHELXTL version 5.1 [49]. All of the non-hydrogen atoms were refined
anisotropically. All other hydrogen atoms were placed in geometrically
ideal positions and constrained to ride on their parent atoms. The
crystallographic data for compound C3 are summarized in Table 1.
Selected bond lengths and angles are given in Table 2.
2.4. Density functional theory (DFT) calculations
All of the calculations were done with the GAMESS suite of codes
[50]. The DFT/B3LYP [51, 52] method was used for the electronic
structure determination. Considering both the calculation cost and the
accuracy, we used the 6-311+G with a set of ‘‘d’’ and ‘‘p’’ polarization
functions for Cu and Br and 6-31+G(d,p) for the rest atoms. The
contribution of a group to a molecular orbital was calculated using
Mulliken population analysis. GAUSSSUM 2.2 [53] was used to calculate
group contributions to the molecular orbitals. A natural bond orbital
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(NBO) analysis was also made for C3 using the NBO code [54]. A
detailed topological analysis of the electron density was made according
to atoms in molecules (AIM) theory, using the program Multiwfn [55].
The atomic coordinates were obtained from the x-ray structures.
2.5. Biology
2.5.1. Cell culture
Culture medium DMEM (with L-glutamin), Fetal bovine serum
(FBS), PBS (phosphate buffered saline) (pH = 7.2) and Antibiotice-
Antimycotic were from E.U Gibco BRL. Human cervical cancer cell lines
HeLa, human lung carcinoma cell lines A549, human liver hepatocellular
carcinoma cell lines HepG2 and human liver cell lines HL-7702
(purchased from the American Type Culture Collection and the German
Collection of Microorganisms and Cell Cultures) were maintained in
DMEM supplemented with 10% FBS, 50 U/mL penicillin, 50 mg/mL
streptomycin at 37 °C and 5% CO2.
2.5.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) cytotoxicity assay
To gain deeper insight into the effect of HSA interactions on the
activity of the compounds, two sets of experiments were performed: (1)
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all compounds were dissolved in PBS with 0.5% DMSO and incubated
with HSA for 24 h at room temperature, after which the HSA–complexes
incubated with above human cancer cell lines for 48 h, and (2) all
compounds were dissolved in PBS with 0.5% DMSO and then tested.
The MTT assay was performed according to the literature method [56].
Briefly, 100 μL of cell suspensions at a density of 5 × 104 cells/mL was
seeded in triplicate in 96-well plates and incubated for 24 h at 37 °C in
5% CO2. Then the medium was replaced with the respective medium with
10% FBS containing the compounds at various concentrations and
incubated at 37 °C under conditions of 5% CO2 for 48 h. The final
DMSO concentration in all experiments was < 0.5% in medium. The
absorbance of the converted dye in living cells was measured at a
wavelength of 570 nm. Values shown are the mean values of at least three
measurements. IC50 values (the drug concentrations that reduced the
number of viable cells to 50% of the control level) were determined by
the nonlinear multipurpose curve-fitting program GraphPad Prism.
2.5.3. Apoptosis by Flow Cytometry
The apoptotic events induced by the copper(II) compound (C3) and
HSA–copper(II) complex (HSA–C3) were determined by annexin V
staining and PI according to the manufacturer’s protocol for the Annexin
V-FITC Apoptosis Detection Kit (Abcam). For these analyses, we used 1
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× 105 cells/mL, which were incubated at 5% CO2 and 37 °C with the C3
and HSA–C3 for 12 h. The HepG2 cells were resuspended in 100 µL 1 ×
annexin V-binding buffer (10 mM Hepes/NaOH, 140 mM NaCl, 2.5 mM
CaCl2, pH 7.4), and 5 μL each of annexin V and PI were added to each
sample. Next, we incubated the cells for 15 min at room temperature and
then subjected them to flow cytometric analysis (FACScan, Bection
Dickinson, San Jose, CA). The rate of cell apoptosis was analyzed.
2.5.4. Cell cycle distribution analysis
HepG2 cells were cultured in 70-mm culture dishes, grown to 70%
confluence, and treated with concentration of C3 and HSA–C3
determined by their IC50 values. FACS analysis was performed after 24 h
of treatment as described [57]. For cell cycle analysis, the washed cells
were fixed with 75% ethanol, washed with PBS, stained with PI and
analyzed by flow cytometry using a 488 nm laser (FACScan, Bection
Dickinson, San Jose, CA). For each sample, 10000 events were recorded.
2.5.5. Intracellular reactive oxygen species (ROS) Measurements
Intracellular ROS generation was determined using 2’,7’-
dichlorodihydro-fluorescein diacetate (H2DCF-DA) [58] (Beyotime
Institute of Biotechnology, Haimen, China). HepG2 cells (1 × 105
cells/well) were incubated with either HL (0.25 μM), Cu(II) (0.25 μM),
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C1–C3 (0.25 μM) or their HSA–complexes (0.25 μM) for 48 h at 37 °C.
Cells were collected for flow cytometric assessment. The fluorescence
intensity was monitored with excitation wavelength at 488 nm and
emission wavelength at 525 nm.
2.5.6. Statistical Analysis
All experiments were repeated 3 to 5 times. Student's t test was applied
to evaluate the significance of differences measured. Results were
expressed as mean ± SD and considered to be significant when p < 0.05.
2.6. Protein binding studies
Protein-binding studies were performed by fluorescence quenching
experiments using HSA. HSA solution (1 µM) was titrated by successive
additions of test solutions C1–C3 using micropipettes for all of the
experiments. The fluorescence emission spectra were scanned from 300
to 420 nm after excitation at 280 nm (the maximum emission was
obtained at 347). The binding constant of HSA for compounds can be
analyzed according to the Scatchard equation [59]:
log[(F0－F)/F] = logK + n × log(Q) (1)
where F and F0 are the fluorescence intensities of protein in the presence
and absence of the quencher, respectively; n is the number of binding
sites; K is the binding constant and [Q] is quencher concentration. From
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the plot of log[(F0－F)/F] versus log [Q], the number of binding sites (n)
and the binding constant (K) was calculated.
The fluorescence quenching mechanism was usually analyzed using
the well-known Stern–Volmer equation [60]:
F0/F = 1 + Kq τ0 [Q] (2)
where Kq is the quenching rate constant of the biological macromolecule,
τ0 is the average lifetime of the protein without any quencher and the
fluorescence lifetime of the biopolymer is 10-8 s [61].
Thermodynamic parameters were calculated based on the temperature
dependence of the binding constant for compounds–HSA binding. The
temperatures used were 298, 310 and 320 K. The enthalpy change (ΔH0)
was calculated from the slope of the Van’t Hoff equation [62]:
lnK = – ΔH0/RT +ΔS0/R (3)
The value of ΔS0 was also obtained from the linear Van’t Hoff plot. The
free energy changes (ΔG0) at different temperatures (T) can be calculated
from the following relationship:
ΔG0= ΔH0 – TΔS0 (4)
In Eq. (3) and (4), R is gas constant and K is the binding constant at
temperature T. Owing to the temperature only changes a little, the
enthalpy change (ΔH0) can be regarded as a constant.
2.7. Molecular modeling
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AutoDock 4.2 (graphical user interface) [63] was used for protein–
ligand docking and the crystal structure of HSA was retrieved from the
Protein Data Bank (PDB code: 1BJ5). All the water molecules and the
bound ligands were removed prior to modeling experiments. Preparation
of the 1BJ5 protein with AutoDock Tools (ADT) involved the addition of
polar hydrogen atoms, partial charge correction and finally, Gasteiger
charges were calculated for each atom of the protein. Affinity grid maps
of 40 × 45 × 40 Å grid points and 0.375 Å spacing were generated using
AutoGrid program. Docking calculations consisted of 2.5 × 107 energy
evaluations using the Lamarckian genetic algorithm (LGA) method. The
obtained 100 docked structures were clustered according to 2.0 Å RMSD
(root-mean-square deviation) and were arranged according to their
binding energies and population criteria for each analysis as well as the
main interactions between the copper(II) compounds and the binding site
of the receptor.
3. Results and Discussion
3.1. Crystal structure description
The compound C3 crystallizes in the triclinic system, space group P-1.
It contains two Schiff-base ligands, two Cu atoms and two Br (Fig 1A).
Phenolic oxygen atoms from a neighbouring molecule act as bridging
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atoms to the phenolato-bridged dinuclear copper(II) centre, with a typical
Cu···Cu distance of about 3.026 Å. The centroid of the C3 dimer lies on
an inversion centre i.e. the second part of the dimer is generated by
symmetry. Both Cu atoms (Cu1 and Cu1i, symmetry code: (i) 1 − x, − y, 2
− z) are five-coordinated by three oxygen atoms from two different
Schiff-base ligands, a nitrogen atom from a Schiff base-ligand and one
terminal Br atom. The coordination geometry in this compound is very
similar to that in [CuCl(L1)]2 [12]; the only difference is the presence of a
Cl atom at the apical position. All Cu−X bond distances are very similar
to those reported in related copper(II) hydrazone compounds [12, 46–48].
The coordination polyhedron around the Cu1 centre could be described as
a slightly-distorted square pyramid (τ = 0.11) [64], with the metal
displaced from the O1/O2i/N1/O2 basal plane (maximum displacement
0.08 Å for the N1 atom), and the Br2 atom at the apex, with the metal
displaced by 0.299 Å toward Br2 from the mean basal plane. In the solid
state, the discrete copper(II) dimers of C3 were further linked into a one-
dimensional (1D) polymeric chain by the N–H···Br interactions involving
a nitrogen atom (N2) from a Schiff-base ligand and a bromine atom (Br2ii)
bonded to the Cu1ii (N2···Br2ii = 3.29 Å and the N2–H1N···Br2ii angle is
173.2º, symmetry code: (ii) –1 + x, y, z, Fig 1B). Interestingly, there is a
π···π stacking arising from the aromatic rings of the Schiff-base ligands
in the layers, as shown in Fig 1C (face-to-face distance: 3.35 Å and
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centre-to-centre distance: 4.04 Å) [65]. In this study, the resulting
structure of the dimeric C3 copper(II) compound displayed a 2D 4-
connected net with sql (44.62) topology as shown in Fig 1D.
3.2. Theoretical calculations
To gain insight into C3’s electronic structures and bonding properties,
we used the density functional theory (DFT) method for our calculations.
The formal charge of copper is + 2 in C3. However, the calculated charge
on the copper atom, obtained from natural population analysis, is
approximately 0.98 in C3. The charge on the Br ligands is equal to –0.72
and those on the O1, O2 and N1 are –0.63,–0.75 and –0.32, respectively.
This is a result of charge donation from the ligands to the copper centre.
The natural atomic orbital d occupancies are as follows: dxy 1.544, dxz
1.982, dyz 1.984, dx2-y2 1.758 and dz2 1.975 in C3. These data suggest that
the donation from the ligands to the dCu orbitals plays a key role in C3’s
electronic structure. In the frontier region, the highest occupied molecular
orbital (HOMO) and the HOMO–1 of C3 are localized on copper atoms
characterized by a significant contribution from Br ligands. The frontier
molecular orbitals of C3 are depicted in Fig. S1. The lowest unoccupied
molecular orbital (LUMO) is located on copper (75% β spin) with an
admixture of Schiff-base ligands (99% α spin). Similarly the LUMO+1 is
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primarily localized on copper (47% β spin) with an admixture of the
Schiff-base ligands (98% α spin).
In this study, we applied the quantum theory of atoms in molecules
(QTAIM) to analyse the strength of the metal–ligand interaction. Table 3
lists the (3, −1) bond critical points (BCPs) and (3, +1) ring critical points
(RCPs) of the charge density, and the BCP electron densities (ρBCP)
results indicate that the Cu–halogen bonds are weaker than the Cu–N/Cu–
O bonds. In C3, an electron charge density ρBCP bond order of Cu1–N1 <
Cu1–O2 < Cu–O1 < Cu–O2i is seen at the critical point of the Cu–N/O
bonds, which is in consistent with the inversely proportional decrease in
bond lengths from Cu1–N1 to Cu1–O2i. All BCPs are characterized by
ρBCP and ∇2ρBCP values in keeping with shared (C–O, C–N, C=N and C–
H bonds) and closed shell (Cu–O, Cu–N and Cu–Br bonds) behaviours.
The positive ∇2ρBCP, negative HBCP and the GBCP/VBCP ratio all indicate
that the Cu–O, Cu–N and Cu–Br bonds belonged to partially covalent
interactions. The ellipticity ε, defined as ε = λ1/λ2–1, is a measure of the
ratio of the rate of density decrease in the two directions perpendicular to
the bond path at the BCP, the degree of π-character and the general shape
of the bond. This value is zero for the standard single/triple bond because
the electron density distribution at this BCP is symmetric. The ε values of
all Cu–N, Cu–O and Cu–Br bonds are nonzero. This is attributable to the
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preferential accumulation in the direction parallel to the N/O/Br lone pair.
Interestingly, we found two additional intermolecular hydrogen bonds,
C13–H13···O1i and C13i–H13i···O1 (Fig 2 and S2). In Table 3 we report
the geometrical properties and topological parameters associated with
these hydrogen bonds. According to a hydrogen bond classification based
on geometrical parameters [66], their features (C···O = 3.05 Å, H···O =
2.26 Å and a C–H···O angle of 141.6º) correspond with those of
moderate hydrogen bonds, which also have relatively low interaction
energies (EHB = 13.01 kJ·mol−1 [67]).
3.3. Anticancer properties of copper(II) compounds and HSA-C3 complex
The results in Table 4 indicate that Cu(II) and HL show relatively low
activity against the cancer cells, but the C1–C3 compounds show high
cytotoxicity against cancer cells. This implies that the chelation of the
Cu(II) ion with HL is responsible for the observed high cytotoxic
properties of the C1–C3 compounds. Specifically, C3 has higher
cytotoxicity than either C1 or C2. While HSA can improve the efficiency
of anticancer agents such as chlorambucil, doxorubicin and some Ru-
based and Pt-based compounds [19, 20, 68–71], the next question is
whether or not HSA can influence the anticancer activity of Cu(II)
compounds? To answer this question, we performed MTT assays to
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evaluate its performance in vitro. It should be noted that in general, the
pre-incubation of copper(II) compounds with HSA has resulted in
increased activity in the mortality of cancer cell lines, while leaving
normal cells (HL-7702) no improving. HSA, by itself, has no cytotoxic
effect on cancer cells or normal cells (Table 4).
Does HSA affect the apoptosis and cell cycle arrest induced by Cu(II)
compounds? To investigate this question, we first looked at C3 and
HepG2 cells because the HSA–C3 complex has high cytotoxic activity in
HepG2 cells relative to other cancer cell lines. Results from Annexin V-
FITC/PI staining showed that the percentage of apoptosis in HepG2 cells
is 9.87% for C3 and 27.9% for HSA–C3, indicating that HSA–C3 can
promote apoptosis in cancer cells (Fig 3). Furthermore, the percentage of
cell numbers in the G2/M phase treated with C3 was 15.42%, but this
percentage increased to 28.69% when the G2/M phase was treated with
HSA–C3. In contrast, the percentage of untreated control cells in the
G2/M phase was 7.04% (Table 5). These results suggest that C3 may
cause an accumulation of cells in the G2/M phase of the cell cycle by
delaying or inhibiting cell cycle progression at the G2/M phase. Clearly,
HSA improves the C3’s capacity for cell cycle accumulation at the G2/M
phase. In addition, we found that the effects of C3 on cell cycle
distribution patterns were dose-dependent, as shown in Fig 4. According
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to our results, HSA has a significant influence on C3 anticancer
properties, improving its efficiency to a certain extent. The most likely
reason is that the complexes are more soluble when bound to HSA, which
results in (slightly) more toxicity over the 48 h period that they used.
3.4. Intracellular ROS Assay
–
Reactive oxygen species (ROS), including O2 , H2O2 and HO–, are
generated during aerobic metabolism. ROS play an important role in cell
signalling and homeostasis [72, 73]. Cancer cells exhibit greater ROS
stress than do normal cells. A cancer cell can die in one of three ways:
apoptosis, necrosis and autophagy. At low levels, ROS facilitate cancer
cell survival and proliferation [74, 75]. But excessive levels of ROS can
result in apoptosis, autophagy and necrosis in cancer cells [76, 77].
Previous results have demonstrated that copper-based compounds can
produce intracellular ROS in SK-N-MC cells [58]. Do cancer cells
produce intracellular ROS when induced by C1–C3 compounds? To
answer this question, we examined the ability of these compounds to
catalyse the production of intracellular ROS in HepG2 cells by using a
fluorescent 2’,7’-dichlorofluorescein (DCF) probe and flow cytometry.
The results showed that the incubation of HepG2 cells with C1–C3 and
their HSA complexes led to a shift of the DCF fluorescence peak to the
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right, relative to that of the control cells (Figs 5A–C), reflecting increased
oxidative damage to these cell populations by the Cu(II) compounds. We
quantified the DCF fluorescence peaks of these compounds and the
results are shown in Fig 5D. These results demonstrate that C3 caused a
significant (p < 0.01) increase in 2’,7’-dichlorodihydrofluorescein
(H2DCF) oxidation to 547 ± 18% in the control cells after 48 hours
incubation. The C3 was found to be 57% and 53% more effective than
C1 and C2, respectively, in inducing intracellular ROS. Importantly, the
high redox activity, 0.25 µM, of the HSA–C3 complex was significantly
(p < 0.01) greater (785 ± 31%) than the 0.25 µM activity of the C3 after
48 hours. Similar results were found for C1 and C2 (Fig 5). Hence, the
enhanced permeability of the Cu(II) compounds due to the presence of
HSA as a carrier likely plays an important role in the increased
intracellular redox activity observed. However, no significant increase in
H2DCF oxidation was observed with the same concentrations of HL,
HSA–HL and Cu(II) alone over this same incubation period. These
results suggest that these compounds’ marked antitumor activity may due
to their ability to generate cytotoxic ROS during a redox cycle [70].
3.5. Binding properties of three compounds to HSA
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HSA may increase the solubility of metal compounds [19]. To test
this idea, we added C1–C3 compounds to an HSA solution and incubated
the mixtures for 3 hours in the dark at room temperature. The resultant
dispersion was centrifuged for 10 min at 4000g to remove the precipitates.
As shown in Fig 6, the UV intensity of the supernatant was significantly
higher (6-fold for C1, 5.2-fold for C2 and 3-fold for C3) than that of the
identically treated C1–C3 solutions without HSA. This suggests that the
solubility of copper compounds may be changed by their binding with
HSA. Therefore, to better understand how copper compounds bind with
HSA, we investigated the binding properties of Cu(II) compounds with
respect to HSA using fluorescence spectroscopy and molecular docking.
The fluorescence of HSA at around 347 nm was gradually quenched
upon increasing the concentration of compounds C1–C3 with a little blue
shift (ca. 3 nm, 4 nm and 2 nm for C1, C2 and C3, respectively) (Fig 7A
and S3). These data indicated that copper(II) compounds interact with
HSA and quench its intrinsic fluorescence. We calculated the HSA
quenching rate constants (Kq) [Eq. (2)] to be 1.19 (± 0.02) × 1014 M−1 s−1
for C1, 1.37 (± 0.03) × 1014 M−1 s−1 for C2 and 1.87 (± 0.05) × 1014 M−1
s−1 for C3 at 298 K (Fig 7B). The calculated Kq values are much larger
than the maximum scattering collision quenching constant (2.0 × 1010
M−1 s−1), which implies that the fluorescence quenching of HSA is
initiated by the formation of the HSA and C1–C3 complexes rather than
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by dynamic collision. The binding constant K is calculated according to
Eq. (1) (Fig 7C). The K values of HSA for C1–C3 were found to be 0.41
(± 0.04) × 105 M−1, 1.51 (± 0.03) × 105 M−1 and 7.94 (± 0.07) × 105 M−1
at 298 K, respectively. The value of n (0.89 for C1, 0.91 for C2 and 1.01
for C3) indicates the existence of a single binding site in HSA for these
compounds (Table 6).
Meanwhile, we calculated the thermodynamic parameters for C1–C3
binding to HSA using Eqs. (3) and (4), and these parameters are
presented in Table 6. The binding process is spontaneous because ΔG0 <
0. A comparison of the binding affinity of C1–C3 for HSA at different
temperatures (298, 310 and 320 K) clearly shows that C3 has a relatively
low ΔG0 (ca. −34 kJ·mol−1) for HSA. That ΔH0 < 0 and ΔS0 > 0 reveals
that C1–C3 interact with HSA mainly by hydrophobic interactions and
hydrogen bonds [62].
We carried out molecular modelling using AutoDock software to
build possible mode of the interaction of copper(II) compounds and HSA.
The docking results reveal that Cu compounds are located within the
binding pocket of IIA subdomain of HSA (Fig 8). This region consists of
a modestly flexible hydrophobic cavity delimited by some polar and
hydrophobic residues. Besides hydrophobic interactions, Cu complexes
form possible hydrogen bond with polar residue (Fig 8).
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4. Conclusion
Since the chelation of copper(II) ion with a ligand results in a
synergistic effect, Cu(II) compounds derived from salicylaldehyde
benzoylhydrazone possess high anticancer activities. Furthermore, the
binuclear Cu(II) compound (C3) shows stronger anticancer activity than
mononuclear copper compounds (C1 and C2). The efficiency of
copper(II) compounds can be enhanced to some extent by their binding
with HSA. Our results may be helpful in the development of copper(II)
hydrazone compounds for lead drug applications.
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Acknowledgements
This work was supported by the Natural Science Foundation of China
(31060121, 31460232, 21171043 and 21431001), Natural Science
Foundation
of
Guangxi
(2012GXNSFCB053001,
2013GXNSFGA019010 and 2014GXNSFDA118016), Technology
division of Guilin (20130403-1) and Guangxi ‘Bagui’ scholar program to
ZF Chen and ZP Zhou.
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