Rational design of ruthenium complexes containing 2,6-bis(benzimidazolyl)pyridine derivatives with radiosensitization activity by enhancing p53 activation

Article abstract of DOI:10.1002/cmdc.201500127

Abstract The rational design of metal-based complexes is an effective strategy for the discovery of potent sensitizers for use in cancer radiotherapy. In this study, we synthesized three ruthenium complexes containing bis-benzimidazole derivatives: Ru(bbp)Cl₃ (1), [Ru(bbp)₂]Cl₂ (2 a) (in which bbp=2,6-bis(benzimidazol-1-yl)pyridine), and [Ru(bnbp)₂]Cl₂ (2 b) (where bnbp=2,6-bis-(6-nitrobenzimidazol-2-yl)pyridine). We evaluated their radiosensitization capacities in vitro and mechanisms of action. Complex 2 b was found to be particularly effective in sensitizing human melanoma A375 cells toward radiation, with a sensitivity enhancement ratio of 2.4. Along with this potency, complex 2 b exhibited a high degree of selectivity between human cancer and normal cells. Mechanistic studies revealed that 2 b promotes radiation-induced accumulation of intracellular reactive oxygen species (ROS) by reacting with cellular glutathione (GSH) and then causing DNA stand breaks. The subsequent DNA damage induces phosphorylation of p53 (p-p53) and upregulates the expression levels of p21, which inhibits the expression of cyclin-B, leading to G2M arrest. Moreover, p-p53 activates caspases-3 and -8, triggers cleavage of poly(ADP-ribose) polymerase (PARP), finally resulting in apoptosis. Taken together, the results of this study provide a strategy for the design of ruthenium-based radiosensitizers for use in cancer therapy.

Full text of DOI:10.1002/cmdc.201500127

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DOI: 10.1002/cmdc.201500127
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Rational Design of Ruthenium Complexes Containing
2,6-Bis(benzimidazolyl)pyridine Derivatives with
Radiosensitization Activity by Enhancing p53 Activation
Zhiqin Deng, Lianling Yu, Wenqiang Cao, Wenjie Zheng, and Tianfeng Chen*[a]
The rational design of metal-based complexes is an effective
strategy for the discovery of potent sensitizers for use in
cancer radiotherapy. In this study, we synthesized three ruthe-
nium complexes containing bis-benzimidazole derivatives:
Ru(bbp)Cl3 (1), [Ru(bbp)2]Cl2 (2a) (in which bbp=2,6-bis(benzi-
midazol-1-yl)pyridine), and [Ru(bnbp)2]Cl2 (2b) (where bnbp=
2,6-bis-(6-nitrobenzimidazol-2-yl)pyridine). We evaluated their
radiosensitization capacities in vitro and mechanisms of action.
Complex 2b was found to be particularly effective in sensitiz-
ing human melanoma A375 cells toward radiation, with a sensi-
tivity enhancement ratio of 2.4. Along with this potency, com-
plex 2b exhibited a high degree of selectivity between human
cancer and normal cells. Mechanistic studies revealed that 2b
promotes radiation-induced accumulation of intracellular reac-
tive oxygen species (ROS) by reacting with cellular glutathione
(GSH) and then causing DNA stand breaks. The subsequent
DNA damage induces phosphorylation of p53 (p-p53) and up-
regulates the expression levels of p21, which inhibits the ex-
pression of cyclin-B, leading to G2M arrest. Moreover, p-p53 ac-
tivates caspases-3 and -8, triggers cleavage of poly(ADP-ribose)
polymerase (PARP), finally resulting in apoptosis. Taken togeth-
er, the results of this study provide a strategy for the design of
ruthenium-based radiosensitizers for use in cancer therapy.
Introduction
Radiation therapy has been used widely as a clinical treatment
for controlling or killing malignant cells for more than 100
years with varying degrees of success.[1] More than 50% of
cancer patients will undergo radiation therapy of some form,
alone or in combination with other treatments.[2] Although ra-
diation therapy has improved significantly over the last few de-
cades, many patients still suffer from localized side effects fol-
lowing treatment, such as fatigue, skin irritation, intestinal
damage, and oral mucositis.[3] Therefore, any improvement in
radiation therapy will benefit numerous people.
cisplatin and its analogues has been limited by serious side ef-
fects, a restricted spectrum of activity, and poor tumor selectiv-
ity.[6] Ruthenium-based complexes are potential alternatives to
cisplatin because of their suitability for the design of antitumor
agents; this includes a favorable ligand exchange rate, a good
range of accessible oxidation states, and, most importantly,
their photoactivated biological applications.[7] James and co-
workers demonstrated that Ru complexes of 4-nitroimidazoles
have good radiosensitizing properties and relatively low toxici-
ty.[8] Recently, Sgambellone et al.[9] discovered that the complex
[Ru(tpy)(5CNU)3]2+ (tpy=2,2:6,2-terpyridine and 5CNU=5-cya-
nouracil) shows much greater cytotoxicity after irradiation with
visible light, showing its potential for use in photodynamic
therapy (PDT). A similar potential can be applied to the Ru–
polypyridyl complexes discovered by Howerton et al.[10] Benzi-
midazoles possess the characteristics of favorable bioactivity
and fluorescence, are important pharmacophores for many
drugs, and exhibit a variety of pharmacological activities, such
as antiparasitic, antioxidant, and anti-inflammatory activities.[11]
Our previous work has shown that Ru complexes containing
bis-benzimidazole derivatives are able to effectively induce
DNA damage in cancer cells, and these complexes show great
selectivity between human cancer and normal cells.[12] More-
over, Gupta et al. and Wright et al. discovered that nitrobenzi-
midazole can effectively sensitize tumor cells to radiation.[13] In-
spired by these results, we hypothesized that Ru complexes
containing bis-benzimidazole derivatives could be activated by
X-ray radiation, as it possesses higher energy than visible light,
thereby sensitizing cancer cells to radiation. For this purpose,
three Ru–bis-benzimidazole complexes were synthesized
In the past decades, radiosensitizers have been widely used
as an effective strategy to improve the local–regional effects of
radiotherapy. The low absorption of radiation across biological
tissues limits the targeting of tumor cells, thus decreasing the
therapeutic efficacy. Metals exhibit higher radiation absorption
across tissue sections, leading to potentiation of the effective-
ness of radiation.[4] Therefore, the rational design of metal-
based complexes is a good strategy toward the discovery of
potent radiosensitizers. Moreover, considerable evidence has
shown that enhancing DNA damage and inhibiting DNA repair
processes can sensitize tumor cells to radiation.[5] For this
reason, many metal-based complexes, especially platinum
complexes, have been developed as radiosensitizers, owning
to their DNA-targeting properties. However, the application of
[a] Z. Deng, L. Yu, Dr. W. Cao, Prof. W. Zheng, Prof. T. Chen
Department of Chemistry, Jinan University
Guangzhou 510632, (P.R. China)
E-mail: tchentf@jnu.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500127.
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Results and Discussion
Synthesis and characterization
The ligands 2,6-bis(benzimidazo-1-yl)pyridine (bbp) and com-
plex 1 and 2a were synthesized as previously described, and
ESI-MS spectra were collected to demonstrate their puri-
ty.[12a,c,14] Briefly, bbp (or bmbp) was prepared by mixing ortho-
phenylenediamine (or methoxy-ortho-phenylenediamine), pyri-
dine-2,6-dicarboxylate, and polyphosphoric acid, then irradiat-
ing by microwave, followed by filtration and recrystallization.
The reaction of bmbp with concentrated sulfuric acid and
nitric acid results in bnbp (Scheme S1, Supporting Information
[SI]). Complexes 1, 2a, and 2b (Scheme S2, SI) were synthe-
sized by holding RuCl3 at reflux with the corresponding ligand
in ethanol for 24 h, followed by evaporation of the solvent.
The crude products were then purified by alumina column
chromatography with acetonitrile as eluent. All complexes dis-
played satisfactory characterization, with mass spectra
(Figures 2A, S2 and S3), elemental analyses, and UV/Vis spectra
(Figures 2B, S4 and S5) demonstrating their purity. 1H NMR
spectra showed all d values of hydrogen atoms in complex 2b
(Figure 2C). In the UV/Vis spectra, the absorption band located
at 300–400 nm originates from intra-ligand p!p* transitions
of bbp/bnbp. Poorly resolved absorption in the visible region
arises from metal-to-ligand charge transfers (MLCT) to bbp/
Figure 1. Structures of the ruthenium complexes in this study.
(Figure 1), and their radiosensitization toward A375 cells was
evaluated. The results showed that these synthetic complexes,
especially 2b, could effectively sensitize A375 cells to radiation
by reacting with cellular glutathione (GSH) and increasing the
levels of intracellular reactive oxygen species (ROS), thus en-
hancing radiation-induced G2M cell-cycle arrest and apoptosis
by activating the DNA-damage-mediated p53 signaling path-
way. Our study demonstrates that Ru–bis-benzimidazole com-
plexes could be developed as potential sensitizers for radio-
therapy, having illustrated their mechanism of action.
Figure 2. Stability of 2b: A) ESI-MS analysis, B) 1H NMR ([D6]DMSO) spectra, and C) UV/Vis (DMSO) spectra of 2b having received radiation at 0 and 8 Gy, as
indicated. D) UV/Vis spectra of 2b (5 mm) after treatment (30 min, room temperature) with various concentrations of H2O2, as indicated.
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bnbp ligands.[14] For complex 2b (Figure 2B), the absorption
band at l 295 nm belongs to the NO2 group.
complex, thus enhancing the electronegativity of the ligands,
further decreasing the energy of MLCT state.[15]
Importantly, the stability of these Ru complexes in DMSO
before and after radiation was characterized by ESI-MS,
1H NMR, and UV/Vis spectroscopy. No significant structural
change was observed after radiation, suggesting these Ru
complexes are stable in DMSO and under radiation conditions.
We also monitored the reactivity of bovine serum albumin
(BSA; Figure S6, SI) and H2O2 (Figure 2D) with 2b at various
concentrations by UV/Vis. As expected, no change in the spec-
tral structure was observed. These results demonstrate that
these Ru complexes are stable; therefore, their biological activ-
ities and further applications can be ascribed to the structures
as characterized.
Barton and co-workers demonstrated that the cellular
uptake and cytotoxicity of metal complexes are influenced by
their lipophilicity.[16] Therefore, the relationship between lipo-
philicity, cellular uptake, and anticancer activities of the com-
plexes of this study were determined (Figure 3B). As we ex-
pected, the anticancer activities of these complexes are pro-
portional to their lipophilicity and degree of cellular uptake.
The capacities of these Ru complexes as radiosensitizers
against A375 human melanoma cells were examined by MTT
and clonogenic assays, with cisplatin used as positive control
(Table 1). Relative to cisplatin, complex 2b was found to be
Table 1. HK-2 and A375 cell growth inhibition by treatment with Ru com-
plexes alone and in radiation co-treatment.
IC50 [mm][a]
SER[b] logP
Structure–activity relationships and radiosensitization
Complex
HK-2
A375
A375+X-ray
The anticancer activities of these Ru complexes against human
melanoma A375 cells were examined by MTT assay. As shown
in Figure 3A, complex 1 had very little inhibitory effect toward
A375 cells. After combining with another bbp ligand, the re-
sulting complex 2a showed greater growth suppression
toward A375 cells, indicating that the introduction of bbp li-
gands into Ru complexes could improve their antitumor activi-
ties. Among these complexes, 2b was found to have the high-
est anticancer efficacy. This is possibly due to the presence of
NO2 groups, which decrease the overall electron density of the
cisplatin
1
2a
2b
10.4Æ2.2
7.5Æ1.3
>100
4.9Æ2.2
>100
1.5
–
1.6
2.4
–
>100
0.86
1.55
1.82
74.3Æ5.9
67.3Æ4.1
15.1Æ2.5
14.3Æ2.7
9.4Æ1.9
5.9Æ1.7
[a] Cell viability was determined by MTT assay after treatment for 72 h;
data are the meanÆSD of n=3 independent experiments performed in
triplicate. [b] Sensitivity enhancement ratio (SER): [IC50 (A375)]/
[IC50 (A375+X-ray)].
more effective in sensitizing A375 cells to radiation, with a sen-
sitivity enhancement ratio (SER) of 2.4. Consistent with this
result, corresponding microscopy images (Figure 4A) support
the ability of 2b to effectively sensitize A375 cells to X-ray radi-
ation. As shown in Figure 4B, treatment by X-ray irradiation
and 2b caused 8.4 and 51.5% inhibition of cell growth, respec-
tively. Combined treatment with 2b and X-ray irradiation re-
sulted in no living cells remaining. Similar results were ob-
served by phase-contrast microscopy (Figure S7, SI); cells re-
ceiving a combination treatment showed remarkable depletion
of cell numbers along with changes in cell morphology, such
as cell shrinkage, cell rounding, and an increased density of
apoptotic bodies.
To determine whether these Ru complexes are tumor selec-
tive, HK-2 human kidney cells were chosen as normal cells to
evaluate the cytotoxicity of these complexes (Table 1). Relative
to cisplatin, 2b exhibited much milder cytotoxicity toward HK-
2 cells; it has 10-fold lower potency in inhibiting the growth of
HK-2 cells than A375 cells, demonstrating great selectivity be-
tween cancer and normal cells.
Cellular uptake of 2b by A375 and HK-2 cells was examined
by inductively coupled plasma mass spectrometry (ICP-MS). As
shown in Figure 5, a higher quantity of 2b was found in A375
cells (28.8 mmol per 107 cells) than in HK-2 cells (15.6 mmol per
107 cells), suggesting that the uneven cellular uptake of 2b in
cancer versus normal cells may be a primary reason for its
cancer selectivity. Taken together, these results show that 2b
could effectively sensitize cancer cells to radiation with great
selectivity for cancer over normal cells.
Figure 3. A) Activities of Ru complexes against A375 cells after treatment for
72 h. B) Relationship between cellular uptake and lipophilicity: cells were
incubated with Ru complexes for 12 h; error bars represent SD of n=3 data
sets.
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cantly increased radiation-induced G2M arrest in A375 cells, as
reflected by the enhancement in G2M peaks (X-ray at 38.5%
vs. X-ray+1 at 72.9%). Meanwhile 2a slightly increased the
cell population at S phase from 40.8% in response to radiation
alone, to 55.3% in response to co-treatment (Figure S9, SI). In-
terestingly, complex 2b enhanced the percentage of radiation-
induced apoptotic cells (Figure 6A), as reflected by the in-
Figure 4. Complex 2b can effectively sensitize A375 cells to X-ray radiation.
A) A375 cell colony formation under various treatment conditions as indicat-
ed; [2b]=5 mm, radiation dose: 8 Gy. B) Quantitative analysis of A375 cell
colony formation following the indicated treatments; statistical significance:
*p<0.05.
Figure 6. Complex 2b sensitizes radiation-induced apoptosis and G2M arrest
of A375 cells. Cells were treated with 2b for 24 h, alone or in combination
with X-ray radiation (8 Gy), and then subjected to flow cytometric analysis.
crease in the sub-G1 population (X-ray at 7.4% vs. X-ray plus
2b at 24.5%). On the other hand, co-treatment with 2b and X-
ray irradiation also increased the cell population at G2M phase
from 17.0% to 33.6% (Figure 6B). This result indicates that ra-
diation-induced apoptosis can be further sensitized by 2b,
while radiation-induced G2M arrest is maintained.
Complex 2b sensitizes radiation-induced apoptosis though
death receptor apoptotic pathway
Figure 5. Difference in cellular uptake of 2b (20 mm) in A375 and HK-2 cells.
Error bars represent SD of n=3 data sets.
Caspases, a family of cysteine aspartic proteases, are important
known mediators in the initiation and execution of apopto-
sis.[17] Because 2b can enhance radiation-induced apoptosis,
the expression levels of caspases and other related proteins
were examined by western blot analysis. As shown in Figure 7,
exposure of A375 cells to co-treatment with radiation and 2b
resulted in the activation of caspases-3 and -8, as well as the
cleavage of poly(ADP-ribose) polymerase (PARP), a DNA repair
enzyme that acts as a biochemical signature of cells undergo-
ing apoptosis.[18] However, no significant change was observed
Ru complexes promote radiation-induced cell-cycle
disruption and apoptosis
To determine the mechanisms behind the radiosensitization ef-
fects of these Ru complexes, flow cytometric analyses were
carried out to measure the effect of the complexes on cell-
cycle distribution. As shown in Figure S8 (SI), complex 1 signifi-
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