Zinc(ii) complexes containing bis-benzimidazole derivatives as a new class of apoptosis inducers that trigger DNA damage-mediated p53 phosphorylation in cancer cells

Article abstract of DOI:10.1039/c3dt33077j

In the present study, two zinc(ii) complexes containing bis-benzimidazole derivatives, Zn(bpbp)Cl₂ (1) and [Zn(bpbp)₂](ClO ₄)₂·CH₃CH₂OH·H ₂O (2) (bpbp = 2,6-bis(1-phenyl-1H-benzo[d]imidazol-2-yl)pyridine), have been designed, synthesized and evaluated for their in vitro anticancer activities. The underlying molecular mechanisms through which they caused the cancer cell death were also elucidated. The complexes were identified as potent antiproliferative agents against a panel of five human cancer cell lines by comparing with cisplatin. Complex 2 demonstrated dose-dependent growth inhibition on MCF-7 human breast carcinoma cells with IC₅₀ at 2.9 μM. Despite this potency, the complexes possessed great selectivity between human cancer cells and normal cells. Induction of apoptosis in MCF-7 cells by complex 2 was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Further investigation on intracellular mechanisms revealed that complex 2 was able to induce p53-dependent apoptosis in cancer cells by triggering DNA damage. On the basis of this evidence, we suggest that Zn(ii) complexes containing bis-benzimidazole derivatives may be candidates for further evaluation as chemotherapeutic agents for human cancers.

Full text of DOI:10.1039/c3dt33077j

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PAPER
Zinc(II) complexes containing bis-benzimidazole
derivatives as a new class of apoptosis inducers that
trigger DNA damage-mediated p53 phosphorylation in
cancer cells†
Cite this: Dalton Trans., 2013, 42, 5932
Shenggui Liu,‡a,b Wenqiang Cao,‡a Lianling Yu,a Wenjie Zheng,*a Linlin Li,a
Cundong Fana and Tianfeng Chen*a
In the present study, two zinc(II) complexes containing bis-benzimidazole derivatives, Zn(bpbp)Cl2 (1) and
[Zn(bpbp)2](ClO4)2·CH3CH2OH·H2O (2) (bpbp = 2,6-bis(1-phenyl-1H-benzo[d]imidazol-2-yl)pyridine), have
been designed, synthesized and evaluated for their in vitro anticancer activities. The underlying molecular
mechanisms through which they caused the cancer cell death were also elucidated. The complexes were
identified as potent antiproliferative agents against a panel of five human cancer cell lines by comparing
with cisplatin. Complex 2 demonstrated dose-dependent growth inhibition on MCF-7 human breast carci-
noma cells with IC50 at 2.9 μM. Despite this potency, the complexes possessed great selectivity between
human cancer cells and normal cells. Induction of apoptosis in MCF-7 cells by complex 2 was evidenced
by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Further inves-
tigation on intracellular mechanisms revealed that complex 2 was able to induce p53-dependent apopto-
sis in cancer cells by triggering DNA damage. On the basis of this evidence, we suggest that Zn(II)
complexes containing bis-benzimidazole derivatives may be candidates for further evaluation as chemo-
therapeutic agents for human cancers.
Received 22nd December 2012,
Accepted 7th February 2013
DOI: 10.1039/c3dt33077j
www.rsc.org/dalton
5-fluorouracil, cisplatin and a series of non-platinum metal
complexes have been prepared. These kinds of agents exhi-
Introduction
In the past decades, much attention has been paid to the inter- bited their therapeutic effects on cancer cells by induction
action between small molecules and DNA.1 DNA has been of DNA damage and activation of downstream signaling
identified as a primary target for anticancer drug design and pathways.3
remains one of the most promising biological receptors for the
With the successful application as anticancer drugs, metal
development of chemotherapeutic agents. The interaction complexes, especially cisplatin, have stimulated extensive
between small molecules and DNA could probably cause DNA investigations and the search for alternative transition metal
damage in cancer cells, which in turn blocked cell division complexes with anticancer activities.4 Recently, many metal-
and finally results in cell death.2 Therefore, the design of based compounds, such as Pt and Ru complexes, have been
effective and selective DNA binding agents is essential for found to exhibit desirable antitumor activities.5 However, the
development of new potential DNA-targeted antitumor drugs. accumulation of metal ions in the body can lead to deleterious
Till now, a large number of DNA-targeted drugs, such as effects and unavoidable toxicity. Thus biodistribution and
clearance of the metal complex as well as its pharmacological
specificity need to be considered.6
As a vital metal ion for cellular processes, zinc (Zn), the
second most important transition metal in the human body, is
essential for cell growth and cell division.7 Studies have
showed that several families of regulatory proteins containing
Zn were important for DNA synthesis, gene expression, and
induction of cell apoptosis.8 Recently, evidence also suggested
an intriguing link between zinc and cancer.9 For instance,
increase in the Zn level could decrease the proliferation of
aDepartment of Chemistry, Jinan University, Guangzhou, 510632, P. R. China.
E-mail: tchentf@jnu.edu.cn, tzhwj@jnu.edu.cn
bDepartment of Chemistry Science and Technology, Zhanjiang Normal University,
Zhanjiang, 524048, P. R. China
†Electronic supplementary information (ESI) available: Experimental details for
synthesis of ligand (bpbp) and crystallographic data for the complexes. CCDC
874326 (bpbp), 874327 (complex 1), 874328 (complex 2). For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c3dt33077j
‡Authors contributed equally to the work.
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prostate cancer cells.10 Zn has been shown to be responsible absorption bands resulting from the skeletal vibration of the
for the functional conformation of tumor suppressor p53, and aromatic rings in the 1392–1590 cm−1. The appearance of 1439
the addition of physiological Zn concentrations could mediate and 1455 cm−1 of ligand and complexes are assigned to the
the renaturation of wild-type p53.11 However, little attention vibration of CvN bond. The ligand and complexes gave satis-
has been paid to the biological roles of synthetic Zn-based factory 1H NMR spectra and element analysis. By comparing
compounds, especially those containing bioactive molecules with bpbp, the δ values of H atom in complexes 1 and 2
and exhibiting both effective antitumor and DNA-targeted shifted to the lower field.
activities.
The molecular structures of the ligand and complexes were
Because DNA has been identified as a key pharmacological established by X-ray crystallography as shown in Fig. 1. The
target of chemotherapeutic agents, many DNA-targeting crystal data are summarized in ESI Table S1.† Selected bond
groups such as intercalators have been conjugated to the lengths and angles of complexes 1 and 2 are listed in Tables
metal ions in the past years.6,12 The development of metal S2–S4,† respectively. As illustrated in Fig. 1A, two benzene
complexes containing bioactive molecules as ligands offers the rings of the ligand bpbp substituted the N–H bond of hydro-
possibility of discovering novel anticancer drugs with gen atoms of benzimidazole groups. The average N–C bond
enhanced and targeted biological activities.4a Studies have length between substituted benzene rings and nitrogen atoms
showed that the intercalating abilities of the complexes was 1.4364 Å. The substituted rings, benzimidazole rings and
depended on the types of metal ions, the ligand donor atoms, pyridine rings were not in the same plane. The dihedral angle
the planarity of ligands and the coordination geometry.13 Ben- between the substituted ring plane and the benzimidazole
zimidazole derivatives are important pharmacophores in drugs plane was 68.7°. As illustrated in Fig. 1B, the central Zn(II) ion
that display a diversity of pharmacological activities, such as was pentacoordinated and surrounded by N3Cl2 environment,
anti-inflammatory, antioxidant, gastroprotective and antipara- adopting distorted trigonal bipyramidal geometry.
sitic activities.14 Many DNA minor groove binders containing
The ligand (bpbp) afforded three N atoms to coordinate to
one or more benzimidazole heterocycles, endowed with prom- Zn(II) and two chloride ions located on each side of the planar
ising antitumor and antiparasitic activities, have been reported pyridine ring. The bond angles of N(1)–Zn(1)–N(4), N(3)–Zn(1)–
to date.15 In our previous study, a series of Ru complexes con- N(4) and N(3)–Zn(1)–N(1) were 144.52(10)°, 72.44(9)° and
taining bis-benzimidazole derivatives were synthesized and 72.10(9)°, respectively. The average bond length of Zn–N was
found to be able to target mitochondria and induce caspase- 2.170(3) Å. The Zn(1)–Cl(1) and Zn(1)–Cl(2) bond lengths were
dependent apoptosis in cancer cells through superoxide over- 2.2558(10) Å and 2.2731(10) Å, respectively. The two benzimi-
production.16 Because both Zn and benzimidazole derivatives dazole rings and the pyridine ring in the ligand were nearly in
had potential in anticancer design, the combination of them the same plane (r.m.s. deviation = 0.3196 Å). The two substi-
in a single molecule could probably achieve enhanced anti- tuted phenyl rings were inclined with the two benzimidazole
cancer effects. Interestingly, in the present study, two zinc(II) rings. The dihedral angles between the substituted phenyl
complexes containing bis-benzimidazole derivatives have been rings and the benzimidazole rings were 45.1° and 57.4°,
synthesized and evaluated for their in vitro anticancer activi- respectively. The ORTEP drawing for complex 2 with atom
ties. The underlying molecular mechanisms by which they numbering is shown in Fig. 1C, which demonstrates that
caused cancer cell death were also elucidated. The results complex 2 is a mononuclear structure and consists of a
−
revealed that the complexes were able to induce p53-depen- [Zn(bpbp)2]2+ dication, ClO4 counter anion, ethanol and
dent apoptosis in cancer cells by triggering DNA damage in an water solvent molecule. In the [Zn(bpbp)2]2+ cation, the central
intercalating mode. Therefore, the Zn(II) complexes containing Zn(II) ions adopted a distorted octahedral geometry with all six
bis-benzimidazole derivatives may be candidates for further coordination positions occupied by six nitrogen atoms from
evaluation as a chemotherapeutic agent for human cancers.
two bpbp ligands. The longest and shortest bond lengths for
Zn–N were 2.191 Å (Zn1–N9) and 2.117 Å (Zn1–N3) respectively,
while the average bond length for Zn–N was recorded at
2.150 Å. The dihedral angle between the plane formed by Zn1
N1 N3 N4 and the plane formed by Zn1 N6 N8 N9 (r.m.s. devi-
ation = 0.0197 Å) was 95.9°. The four substituted phenyl rings
were inclined with the benzimidazole rings being attached.
Results and discussion
Synthesis and molecular structural characterization of Zn
complexes
The ligand bpbp (bpbp = 2,6-bis(1-phenyl-1H-benzo[d]imida- The dihedral angles between the substituted phenyl rings and
zol-2-yl)pyridine) and two new Zn complexes were prepared fol- the benzimidazole rings were 67.3°, 74°, 57.4° and 59.1°,
lowing the method shown in Scheme S1.† The ligand bpbp respectively. Taken together, the clear characterization of the
was synthesized by displacement of the Br atom of bromoben- structure of the complexes facilitates the clarification of the
zene by the deprotonated 2,6-bis(benzimidazole)pyridine. The antitumor mechanisms and the structure–activity relationship.
reaction of bpbp with one equivalent of ZnCl2 in DMF resulted
In vitro cytotoxic effects of Zn complexes
in complex 1. The reaction of two equivalents of bpbp with
Zn(ClO4)2 in ethanol yielded complex 2. The IR spectra for Studies have shown that metal complexes may act by enhan-
the free ligand bpbp, complex 1 and complex 2 showed all cing the delivery of the active ligands to target sites inside the
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new metal-based drugs. The metal could probably act as a
carrier and stabilizer for the drug ligands until it reaches its
target. At the same time, the organic drug ligands could
protect the metal and prevent side reactions in its transit
toward a second target of biological action. Therefore, the
combined effects between the metal and the ligands could
result in enhancement of biological activities or activate new
action mechanisms. For instance, the coordination of a well-
known breast cancer drug tamoxifen to iron achieved signifi-
cant enhancement of anticancer effects.18 In the present study,
benzimidazole derivatives were used as active ligands to be
coordinated to Zn, because this kind of compound has been
identified as an important pharmacophore in drug design and
has been found to demonstrate promising anticancer activities
and DNA-binding properties. The antiproliferative activities of
the complexes were screened by MTT assay against a panel of
five human cancer cell lines, including human breast adeno-
carcinoma MCF-7 cells, human osteosarcoma MG-63 cells,
human cervix adenocarcinoma Hela cells, human melanoma
A375 cells and human liver cancer Hep G2 cells. As the
balance between therapeutic potential and toxic side effects of
a compound is important for evaluating its usefulness as a
pharmacological drug, experiments were also carried out to
examine the cytotoxicity of the synthetic complexes against
human normal cell lines. Therefore, HK-2 human kidney cells
were chosen to evaluate the cytotoxicity for these compounds.
As shown in Table 1, the ligand and the synthetic Zn com-
plexes exhibited broad-spectrum inhibition on the growth of
five human cancer cell lines with IC50 values ranging from
2.9 μM to 18.1 μM after a 72 h treatment, by using cisplatin as
a positive control. The results demonstrated that the tested
cancer cell lines were all susceptible to the ligand and com-
plexes. Especially, complex 2, containing two active ligands,
was more active than complex 1 and the ligand itself toward all
the tested cell lines. The IC50 value of MCF-7 cells was found
at 2.9 μM, which was significantly lower than that of cisplatin
and the ligand bpbp. These results suggest the contribution of
the ligands to the anticancer activity of complexes and confirm
the enhancement between the metal and the ligand in this
kind of complex. However, the exact metabolism pathways and
the active forms of the complexes inside the cellular remain
elusive, which will be the focus of our future study.
Despite this potency, the synthetic complexes and the
ligand were less toxic to HK-2 human normal cells, with IC50
values found at 28.4, 23.7 and 14.1 μM respectively, which
were slightly higher than that of cisplatin (10.3 μM). These
results demonstrated the lower toxicity of the complexes than
cisplatin. Taken together, our results suggest that the Zn com-
plexes possess better selectivity between cancer and normal
cells than cisplatin and have potential application in cancer
chemotherapy.
Fig. 1 Crystal structures of complexes studied in this work. (A) The molecule
structure of bpbp (ORTEP; 30% ellipsoids; hydrogen atoms removed for clarity).
(B) The molecule structure of complex 1 (ORTEP; 50% ellipsoids; hydrogen
atoms removed for clarity). (C) Molecular structure of complex 2. All unlabeled
atoms are carbon atoms (ORTEP; 30% ellipsoids; hydrogen atoms removed for
clarity).
Induction of apoptotic cell death by complex 2
Because of the highest sensitivity of MCF-7 cells towards
cells.17 Coordination of organic compounds with known thera- complex 2, this cell line was used for further investigation on
peutic values to metal ions is an approach to the discovery of the underlying mechanisms of complex 2-induced cell growth
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Table 1 Growth inhibition of the ligand and complexes on selected human cancer and normal cellsa
IC50 (μM)
Compounds
MCF-7
MG-63
HeLa
A375
Hep G2
HK-2b
bpbp
1
2
11.2 1.3
6.8 1.5
2.9 0.3
3.0 1.4
15.9 1.7
5.2 1.2
3.6 0.2
12.5 2.1
18.1 2.9
9.4 0.5
5.2 0.5
15.9 1.7
14.6 0.2
9.5 1.0
5.4 0.5
7.3 0.8
12.0 0.9
9.4 0.2
6.5 0.2
13.6 2.0
28.4 4.3
23.7 1.7
14.1 1.9
10.3 2.1
Cisplatin
a Cell viability after treatment for 72 h was determined by MTT assay as described in the Experimental section. Each IC50 value represents the
mean SD of three independent experiments. b Human normal cells.
Fig. 2 Growth inhibition effects of complex 2 on MCF-7 cells. (A) Cells were
treated with different concentrations of complex 2 for 24, 48, and 72 h and cell
viability was examined by MTT assay. (B) Changes in the morphology of MCF-7
cells under different treatments of complex 2 for 72 h as examined by phase-
contrast microscopy (magnification, 200×).
inhibition. Generally the inhibition of cancer cell proliferation
by anticancer drugs could be the result of induction of cell
Fig. 3 Effects of complex 2 on cell apoptosis and cell cycle distribution in
MCF-7 cells. (A) The cells treated with complex 2 for 72 h were collected and
cycle arrest or apoptosis, or a combination of these two modes.
Therefore in this study, firstly, the inhibitory effect of complex
2 on the growth of MCF-7 cells was also determined by MTT
assay. As shown in Fig. 2A, treatment of MCF-7 cells with
complex 2 resulted in a dose- and time-dependent inhibition
of cell growth, accounting for 7–40, 11–62, and 22–71% inhi-
bition after 24, 48, and 72 h of treatment with various concen-
trations of complex 2 (0.625–40 μM), respectively. In the phase-
contrast observation, as shown in Fig. 2B, MCF-7 cells treated
with different concentrations of complex 2 for 72 h showed
dose-dependent decrement of cell numbers and the change in
cell morphology, such as cell shrinkage, cell rounding, and the
appearance of apoptotic bodies. Moreover, we determined the
effect of complex 2 on cell cycle distribution using flow cyto-
metry. As shown in Fig. 3A, exposure of MCF-7 cells to indi-
cated concentrations of complex 2 resulted in marked dose-
dependent increase in the proportion of apoptotic cells as
reflected by the sub-diploid peaks. For instance, the sub-G1
population was noticeably enhanced from 6.3% to 74.9% after
72 h of treatment with 8 μM of complex 2. Meanwhile, a slight
increase in the G0/G1 phase population was also observed in
cells exposed to 4 μM and 8 μM of complex 2. The increase in
the G0/G1 phase cell population was accompanied by a
stained with PI after fixation. (B) Quantitative analysis of apoptotic cell death
induced by complex 2 by measuring the sub-G1 cell population. Cells treated
with 8 μM of complex 2 for various periods of time were fixed with 70%
ethanol, stained with PI, and analyzed by flow cytometry. Each value represents
the mean of three independent experiments.
cytometry. As shown in Fig. 3B, complex 2 induced a notice-
able time-dependent increase in the sub-G1 cell population,
which reached 38.9% after the treatment for 48 h. However, no
significant change in the cell cycle distribution was observed
in the results (data not shown). On the whole, apoptosis could
be a major mode of cell death induced by complex 2 in cancer
cells. In order to further prove this finding, enzymatic labeling
assay (TUNEL) and DAPI staining assay were carried out to
detect DNA fragmentation and nuclear condensation. TUNEL
can detect the early stage of DNA fragmentation in apoptotic
cells prior to changes in morphology. Results shown in Fig. 4
reveal a dose-dependent increase in DNA fragmentation and
nuclear condensation in MCF-7 cells. Taken together, these
results demonstrate that the complex 2-induced MCF-7 cell
death is mainly caused by induction of apoptosis.
Induction of DNA damage in MCF-7 cells by complex 2
decrease in the S and G2/M phase cell populations. To study Accumulating evidence indicates that chemotherapeutic
the time course for this increment of sub-G1 and G0/G1 phase
cell populations, we treated the MCF-7 cells with 8 μM of
agents induced tumor regression through the inhibition of
proliferation and/or the activation of apoptosis.19 A broad
complex 2 for various periods of time and analyzed by flow array of metal complexes, especially cisplatin, with strong
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Fig. 5 Induction of DNA strand breaks in MCF-7 cells by complex 2. Cells were
treated with indicated concentrations of complex 2 for 12 h and then analyzed
by Comet assay. The tail indicates cells with damaged DNA. The length of the
tail reflects the degree of DNA damage. The images shown here are representa-
tive of three independent experiments with similar results.
Fig. 4 Complex 2-induced apoptotic cell death as examined by TUNEL and
DAPI assay in MCF-7 cells. Cells were treated with different concentrations of
complex 2 for 72 h. Representative images of DNA fragmentation and nuclear
condensation in response to the treatment of complex 2 as described in Results
and discussion (magnification, 200×).
DNA-binding behavior induce efficient apoptosis in cancer
cells and many of them occurred with the involvement of DNA
damage and direct interaction with DNA.6,20 In our previous
study,16 Ru complexes containing bis-benzimidazole deriva-
tives were found to be able to induce dose-dependent increase
in DNA fragmentation and apoptosis in cancer cells. Based on
these results, we predicted that complex 2-induced MCF-7 cell
apoptosis could be triggered by DNA damage. Therefore, the
induction of DNA damage by complex 2 was investigated by
Comet assay, which provides a simple and effective way to
evaluate both single- and double-strand breaks in DNA at the
single-cell level. Under the influence of an electric field, the
denatured or cleaved DNA fragments can migrate out of the
cells and display the amount of DNA present in the tail of the
comet, which is representative of DNA damage caused by the
added agent.21 As shown in Fig. 5, a significant elevation of
DNA strand breaks (DSBs) as evidenced by the increase in tail
DNA was detected in MCF-7 cells. A dose-dependent increase
in DNA damage was also observed in MCF-7 cells exposed to 2,
4 and 8 μM of complex 2 for 72 h. These results imply that
DNA damage was triggered by complex 2 in cancer cells.
Fig. 6 DNA-binding behavior of complex 2 with DNA extracted from MCF-7
cells. (A) Absorption spectra of complex 2 in Tris-HCl buffer upon addition of
increasing concentrations of MCF-7 cell DNA. [Zn] = 20 μM. (B) Emission spectra
of complex 2 in 3 mM of Tris-HCl buffer in the absence and presence of MCF-7
cell DNA. [Zn] = 20 μM. (C) The CD spectra of MCF-7 cell DNA in absence or
addition of increasing concentrations of complex 2. [DNA] = 300 μM. (D) Effect
of complex 2 on the relative viscosity of MCF-7 cell DNA at 30.0 °C ( 0.1 °C).
[DNA] = 400 μM.
With the addition of MCF-7 cell DNA, hypochromism (35%)
and red shift (Δλ = 3 nm) of MLCT transition (metal-to-ligand
charge transition) of complex 2 in electronic spectra were
observed (Fig. 6A). The calculated intrinsic binding constant
(Kb) for complex 2 was 2.0 × 105 M−1, which is typical for metal
DNA-binding properties of complex 2
To further confirm the complex 2-induced DNA damage in complexes that bind to DNA via intercalation. This result was
MCF-7 cells, the DNA-binding properties of complex 2 were further confirmed by fluorescence measurement in the
also examined by spectroscopic and viscosity measurement. absence or presence of different concentrations of MCF-7 cell
Spectrophotometry is the most common method to investigate DNA. The fluorescence intensity decreased obviously (Fig. 6B),
the interaction of transition metal complexes with DNA. In which demonstrates the strong interaction between complex 2
general, transition metal complexes exhibit hypochromism and MCF-7 cell DNA. To gain further information, we also
and red shift in their electronic spectra when they are bound recorded the CD spectra of MCF-7 cell DNA exposed to
to DNA. The degree of hypochromism (decrease in absorption complex 2 (Fig. 6C). The CD spectra showed a slight but sig-
of the DNA band) depends on the binding mode and affinity. nificant decrease in the intensity of the positive CD signal
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around 267 nm. The viscosity of the double strand DNA medicine, we detected the stability of the synthesized ligand
increases when a complex binds to DNA in an intercalating (bpbp), complex 1 and complex 2 during the incubation in
mode.22 In order to further determine the binding mode, vis- DMSO at room temperature (25 °C) for 72 h by UV-Vis. As
cosity measurement was carried out by adding various concen- shown in Fig. S1,† no change in the UV-Vis spectra of the
trations of complex 2 to MCF-7 cell DNA. As shown in Fig. 6D, ligand and complexes was observed. The ligand and complexes
the relative viscosity of MCF-7 cell DNA increased significantly remained stable at least for 72 h. Based on these results, we
in the presence of complex 2. Taken together, these results confirmed that the solvolysis does not appear when dissolving
indicate that complex 2 may bind to DNA through an interca- the ligand and complexes in DMSO, which means it does not
lating mode.
influence the results of biological studies. This stability sup-
ports their future applications in the treatment of cancers.
Complex 2 induces activation of the p53 pathway
Considerable evidence has shown that DNA damage could
cause apoptotic cell death via various signaling pathways.23 In
cell models, DNA damage activates ATM (ataxia telangiectasia
mutated) and ATR (ataxia telangiectasia and Rad3 related) pro-
teins, which signal downstream to checkpoint kinases, such as
CHK1 and CHK2, and tumor suppressor gene p53 and finally
result in apoptosis.24 As a transcription factor, p53, after acti-
vation by DNA damage,25 could directly or indirectly induce
cell apoptosis through both the extrinsic and intrinsic apopto-
sis pathways.26 Based on the above evidences, it is possible
that the DNA damage triggered by complex 2 may induce p53
phosphorylation. In order to confirm this hypothesis, we deter-
mined the protein level of total and phosphorylated p53 in
MCF-7 cells after being treated with 2–8 μM of complex 2 for
72 h. As shown in Fig. 7, the treatments resulted in elevation
of phosphorylated p53 at Ser15 in a dose-dependent manner.
As another DNA damage maker, H2A.X was rapidly phosphoryl-
ated (within seconds) at serine 139 when DSBs are introduced
into mammalian cells.27 Therefore, we analyzed the phos-
phorylation of histone H2A.X on serine 139 by Western blot-
ting. As shown in Fig. 7, a significant increase in this protein
was observed in MCF-7 cells treated with indicated concen-
trations of complex 2. These results suggest that the DNA
damage-mediated p53 activation is involved in cancer cell
apoptosis induced by complex 2.
Conclusions
In the present study, two Zn(II) complexes containing bis-ben-
zimidazole derivatives have been designed, synthesized and
evaluated for their in vitro anticancer activities. The underlying
molecular mechanisms through which they caused cancer cell
death were also elucidated. The complexes were identified as
potent antiproliferative agents against a panel of five human
cancer cell lines. Especially, complex 2 demonstrated higher
growth inhibition on MCF-7 human breast carcinoma cells
than cisplatin. Despite this potency, the complexes possessed
high selectivity between human cancer and normal cells, in
comparison with cisplatin. Further investigation on intracellu-
lar mechanisms revealed that complex 2 was able to induce
p53-dependent apoptosis in cancer cells by triggering DNA
damage. On the basis of this evidence, we suggest that Zn(II)
complexes containing bis-benzimidazole derivatives may be
candidates for further evaluation as a chemotherapeutic agent
for human cancers. This study provides broader space for
improving the structure–activity relationships of Zn complexes
and opens up a new dimension in the biomedical area for
complexes in design of DNA-targeted anticancer agents.
Experimental section
Materials
Due to the stability of these Zn(II) complexes being an
important factor affecting their clinical applications in
Pyridine-2,6-dicarboxyl acid, o-phenylenediamine, bromoben-
zene, CuI, Cs2CO3, and 1,10-phenanthroline were purchased
from Shanghai Aladin Reagent Company. All the chemicals
and solvents were analytically pure and were used without
further purification. Thiazolyl blue tetrazolium bromide
(MTT), propidium iodide (PI), 4′,6-diamidino-2-phenylindole
(DAPI) and a bicinchoninic acid (BCA) kit for protein determi-
nation were purchased from Sigma-Aldrich. All antibodies
used in this study were purchased from Cell Signaling Technol-
ogy (Beverly, MA). The water used in cellular experiments was
ultrapure, supplied by a Milli-Q water purification system from
Millipore.
Fig. 7 Regulation of phosphorylated p53, total p53 and phosphorylated Synthesis and characterizations
histone H2A.X expression levels by complex 2 in MCF-7 cells. Cells were exposed
2,6-Bis(1-phenylbenzimidazol-2-yl)pyridine (bpbp). The
to complex 2 with indicated concentrations for 72 h. Changes in the levels of
protein expression were shown as ratios of selected groups. All experiments
were carried out at least in triplicates.
ligand was synthesized according to a previous method.31
Briefly, a mixture of bromobenzene (1.75 g, 11 mmol), 2,6-bis-
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(benzimidazolyl)pyridine (1.65 g, 5.0 mmol), CuI (0.40 g, penicillin (100 units ml−1), and streptomycin (50 units ml−1) at
2.1 mmol), 1,10-phenanthroline (0.8 g, 0.88 mmol), and 37 °C in a humidified incubator with 5% CO2 atmosphere.
Cs2CO3 (14.5 g, 45 mmol) was suspended in 50 ml of DMF.
MTT assay
The mixture was refluxed for 24 h and then cooled to room
temperature. The resulting sticky residue was purified by The effects of the complexes on cell proliferation were deter-
column chromatography (1 : 2 THF–hexane) and gave a color- mined by MTT assay. Briefly, cells were seeded in 96-well
less ligand (1.14 g, yield: 45%). The crystals were obtained culture plates at different densities. After 24 h, different con-
from the evaporation of the ethanol solution. Selected IR data centrations of compounds were added and incubated for the
(KBr, cm−1): 3422, 3058, 1597, 1503, 1439, 1392, 1336, 1250, indicated time. Then, 20 μl per well of MTT solution (5 mg
997, 752. 1H NMR: (CDCl3): 6.99 (d, 4H), 7.184–7.367 (m, 12H), ml−1 phosphate buffered saline) was added and incubated for
7.876 (t, 3H), 8.08 (d, 2H).
5 h. The medium was aspirated and replaced with 200 μl per
[Zn(bpbp)Cl2] (1). A solution of ZnCl2 (0.136 g, 1.0 mmol) in well of DMSO to dissolve the formazan salt formed. The color
10 ml of ethanol was added to a hot stirred solution of bpbp intensity of the formazan solution, which reflects the cell
(0.464 g, 1.0 mmol) in 50 ml of ethanol. The reaction mixture growth condition, was measured at 570 nm using a microplate
was stirred for 20 minutes at room temperature. After recrystal- spectrophotometer (VERSA max).
lization from ethanol, colourless crystals were obtained. Yield
0.240 g (80%). Anal. calcd for C31H21Cl2N5Zn ([Zn(bpbp)Cl2]):
Flow cytometric analysis
C, 62.07%; H, 3.53%; N, 11.68%. Found: C, 62.29%; H, 3.51%; Induction of apoptosis by complex 2 was quantified by flow
N, 11.73%. Selected IR data (KBr, cm−1): 3460, 3067, 1597, cytometric analysis according to our previous method.28
1494, 1455, 1408, 1336, 1005, 750, 697. 1H NMR: (d6-DMSO): Briefly, treated or untreated cells were trypsinized, washed
7.21 (d, 4H), 7.49 (q, 12H), 8.01 (m, 3H), 8.06 (d, 2H).
with PBS and fixed with 70% ethanol overnight at −20 °C. The
[Zn(bpbp)2](ClO4)2·CH3CH2OH·H2O (2). A solution of Zn- fixed cells were washed with PBS and incubated with a PI
(ClO4)2·6H2O (0.186 g, 0.5 mmol) in 10 ml of ethanol was working solution (1.21 mg ml−1 Tris, 700 U ml−1 RNase,
added to a hot solution of bpbp (0.464 g, 0.5 mmol) in a 50 ml 50.1 μg ml−1 PI, pH 8.0) for 4 h in darkness. The stained cells
ethanol solution. The reaction mixture was stirred for were analyzed with an Epics flow cytometer (Beckman Coulter,
20 minutes at room temperature. After recrystallization from Fullerton, CA). Cell cycle distribution was analyzed using Multi-
ethanol, colourless crystals were obtained. Yield 0.505 g Cycle software (Phoenix Flow Systems, San Diego, CA). The pro-
(80.67%). Anal. calcd for C64H50Cl2N10O10Zn ([Zn(bpbp)2]- portion of cells in G0/G1, S, and G2/M phases was represented
(ClO4)2·CH3CH2OH·H2O): C, 72.76%; H, 4.77%; N, 13.26%. as DNA histograms. Apoptotic cells with a hypodiploid DNA
Found: C, 72.29%; H, 4.51%; N, 12.93%. Selected IR data (KBr, content were measured by quantifying the sub-G1 peak in the
cm−1): 3437, 3058, 1590, 1502, 1439, 1329, 1084, 744, 682, 618. cell cycle pattern. For each experiment, 10 000 events per
1H NMR: (d6-DMSO): 8.08 (t, 2H), 7.59 (m, 4H), 7.42 (d, 28H), sample were recorded.
7.21 (d, 8H).
TUNEL–DAPI co-staining assay
DNA fragmentation was examined by the TUNEL apoptosis
detection kit (Roche) following the manufacturer’s instruction.
Briefly, cells cultured in chamber slides were fixed with 3.7%
formaldehyde for 1 h and permeabilized with 0.1% Triton
X-100 in PBS. Then, the cells were incubated with 50 μl of
TUNEL reaction mixtures for 1 h at 37 °C. For nuclear staining,
cells were incubated with 1 mg ml−1 of DAPI for 15 min at
37 °C. Stained cells were then washed with PBS and examined
on a fluorescence microscope (Nikon Eclipse 80i).
X-ray crystallography
Single crystal structure determination for the ligand and com-
plexes 1 and 2 was performed on a Siemens Smart-CCD diffr-
actometer equipped with a normal focus, 3 kW sealed tube
X-ray source and graphite monochromated Mo-Kα radiation
(λ = 0.71073 Å) at 173 K. The structures were solved by direct
methods by using the program SHELXTL. Absorption correc-
tion was carried out by semi-empirical method, Fourier differ-
ence techniques, and refined by full-matrix least-squares. All
non-hydrogen atoms in both structures were refined anisotro-
pic displacement parameters. All hydrogen atoms were theo-
retically added.
Comet assay
Comet assay for detection of DNA damage was performed
using a kit (Trevigen). According to our previous method,29
cells treated with complex 2 were harvested by trypsinization,
centrifuged and suspended in PBS. The cell suspensions
Cell culture
The cell lines used in this study, including human breast adeno- mixed with melted LM agarose were coated on the standard
carcinoma MCF-7 cells, human osteosarcoma MG-63 cells, slides and electrophoresis was performed for 30 min in a
human cervix adenocarcinoma Hela cells, human melanoma Savant ps 250 system set at 300 mA and 1 V cm−1 in darkness.
A375 cells, human liver cancer Hep G2 cells and the normal After electrophoresis, the slide was rinsed with distilled water,
human kidney HK-2 cells, were obtained from American Type fixed in 70% ethanol for 5 min and air-dried overnight. DNA
Culture Collection (ATCC, Manassas, VA) and maintained in was stained with SYBR Green I (Trevigen) and visualized under
DMEM medium supplemented with fetal bovine serum (10%), a fluorescence microscope (Nikon, Eclipse E-600).
5938 | Dalton Trans., 2013, 42, 5932–5940
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DNA binding experiments
holder. The cell path length was 1 cm. Spectra were recorded
in the range of 230–500 nm in 0.2 nm increments with an aver-
aging time of 1 s. CD spectral characteristics were compared
for MCF-7 cell DNA in the absence and presence of complex 2,
respectively.
Viscosity measurements were carried out using a Schott
Gerate AVS 310 automated viscometer maintained at 30.0
0.1 °C in a thermostatic bath. The increasing concentrations of
complex 2 were added into 400 μM of DNA solution. The flow
time was measured by a digital timer and each sample was
measured three times. The relative viscosity of DNA in the
absence and presence of the metal complex was calculated
using eqn (2).
DNA binding experiments were done in Tris-HCl/NaCl buffer
(5 mM Tris-HCl, 50 mM NaCl, pH 7.2) using DMSO solution of
the complexes. The DNA was extracted from MCF-7 cells using
a Mammalian genomic DNA extraction kit (Beyotime). Briefly,
A375 cells were harvested by centrifugation at 2000 rpm. The
cell pellets were suspended in lysis buffer containing protease
and vortexed with the presence of phenol. Then the DNA was
separated from other cell components. After addition of a
1 : 1 mixture of ethanol and ammonium acetate and shaking,
DNA was precipitated and then washed with 75% ethanol
twice. The obtained DNA was resuspended with nuclease-free
water for further use. Solutions of DNA in Tris-HCl/NaCl buffer
gave a ratio of UV absorbance (260/280 nm) of 1.8–1.9 : 1, indi-
cating that the DNA was sufficiently free of protein. The con-
centration of DNA diluted 100-folds was determined
spectrophotometrically by using the molar absorption coeffi-
cient of 6600 M−1 cm−1 (260 nm).
η ¼ ðt ꢀ t0Þ=t0
ð2Þ
where t is the flow time of the DNA-containing solution with
different concentrations of complex 2 and t0 is the flow time of
the DNA-containing solution alone. Data are presented as
(η/η0)1/3, where η0 is the viscosity of DNA in the absence of
complex 2 and η is the viscosity of DNA in the presence of
complex 2.
Absorption spectra of the synthetic complex
2 were
recorded on a Cary 5000 UV-2450. Spectroscopic titrations
were carried out by keeping a constant concentration of
complex 2 (20 μM) dissolved in 3.0 ml of Tris-HCl buffer (pH
7.2, [NaCl] = 50 mM) with increasing concentrations of MCF-7
cell DNA. Each spectrum was recorded after equilibration of
the sample for 10 min. The absorbance (A) of the most red-
shifted band of complex 2 was recorded after successive
additions of MCF-7 cell DNA. The intrinsic equilibrium
binding constant (Kb) values were calculated according to the
decay of the Soret absorption by eqn (1).30
Western blot analysis
The cells were harvested after centrifugation, washed with PBS
twice. Total cellular proteins were extracted by incubation of
cell pellets with cell lysis buffer (Cell Signaling Technology)
overnight at −20 °C. The protein concentrations of the extracts
were determined using a BCA protein assay kit according to
the manufacturer’s instructions. SDS-PAGE was done in 12%
tricine gels loading 40 μg of cell lysates per lane. After electro-
phoresis, separated proteins were transferred to nitrocellulose
membranes for 75 min at 110 V and blocked with 5% non-fat
milk in TBS buffer for 1 h. Then, the membranes were washed
with TBST buffer and incubated with primary antibodies at
1 : 1000 dilutions in 5% BSA overnight at 4 °C with continuous
agitation; secondary antibodies were conjugated with horse-
radish peroxidase at 1 : 5000 dilutions for 2 h at room tempera-
ture, followed by three washes in TBST buffer. The target
proteins were detected on X-ray film (Kodak) using an
enhanced chemiluminescence reagent. β-Actin was used to
confirm the equal loading and transfer of proteins.
1=2
ðεa ꢀ εfÞ=ðεb ꢀ εf Þ ¼ ðb ꢀ ðb2 ꢀ 2Kb2Ct½DNAꢁ=sÞ Þ=2KbCt
ð1Þ
where b = 1 + KbCt + Kb[DNA]/2s and εa is the extinction coeffi-
cient observed for the spectral band at a given DNA concen-
tration, εf is the extinction coefficient of the complex free in
solution, εb is the extinction coefficient of the complex when
fully bound to DNA, Kb is the equilibrium binding constant, Ct
is the total complex concentration, [DNA] is the DNA concen-
tration in nucleotides and s is the fitting parameter giving an
estimate of the binding site size in base pairs. The non-linear
least-squares analysis was done using OriginLab.
Statistical analysis
Emission spectra of the synthetic complex 2 were measured
on a 970CRT spectrofluorometer with excitation at 363 nm.
Spectroscopic titrations were carried out under room tempera-
ture to determine the binding affinity between DNA and
complex 2. Briefly, 3.0 ml of Tris-HCl buffer (pH 7.2, [NaCl] =
50 mM) was placed in the reference cuvette and complex 2
(20 μM), dissolved in 3.0 ml of Tris-HCl buffer (pH 7.2, [NaCl]
= 50 mM), was added into the sample cuvette. Then the MCF-7
cell DNA at 2.0 mM in base pairs was added into both the
reference and the sample cuvettes. Finally, the spectra were
recorded in the range of 373–700 nm.
Experiments were carried out at least in triplicate and results
were expressed as mean
formed using the SPSS statistical program version 13 (SPSS
Inc., Chicago, IL). The difference between two groups was ana-
lyzed by a two-tailed Student’s t test. A difference with P < 0.05
(*) or P < 0.01 (**) was considered statistically significant.
SD. Statistical analysis was per-
Acknowledgements
The circular dichroism (CD) spectra of MCF-7 cell DNA This work was supported by the Natural Science Foundation of
modified by complex 2 were recorded on a Jasco J-810 spectro- China and Guangdong Province, the Program for New Century
photometer equipped with a thermoelectrically controlled cell Excellent Talents in University, Key Project of Science and
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 5932–5940 | 5939

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Paper
Dalton Transactions
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5940 | Dalton Trans., 2013, 42, 5932–5940
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