A selenium-containing ruthenium complex as a cancer radiosensitizer, rational design and the important role of ROS-mediated signalling

Article abstract of DOI:10.1039/c4cc07926d

A novel selenium-containing ruthenium complex Ru(phtpy)(phenSe)Cl(ClO₄) (phtpy = 4-phenyl-2,2′:6′,2′′-terpyridine, phenSe = 2-selenicimidazole[4,5-f]1,10-phenanthroline) has been synthesized and found be able to enhance radiation-induced DNA damage through superoxide overproduction, which leads to G2/M arrest and apoptosis in cancer cells by activating ROS-mediated pathways. This journal is

Full text of DOI:10.1039/c4cc07926d

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A selenium-containing ruthenium complex as a
cancer radiosensitizer, rational design and the
important role of ROS-mediated signalling†
Cite this: Chem. Commun., 2015,
51, 2637
Received 7th October 2014,
Accepted 2nd January 2015
Zhiqin Deng,‡ Lianling Yu,‡ Wenqiang Cao, Wenjie Zheng* and Tianfeng Chen*
DOI: 10.1039/c4cc07926d
www.rsc.org/chemcomm
A novel selenium-containing ruthenium complex Ru(phtpy)(phenSe)- been regarded as appropriate substitutes of Pt complexes.10
Cl(ClO4) (phtpy = 4-phenyl-2,20:60,200-terpyridine, phenSe = 2-seleni- Our previous work has proved that Ru complexes as a novel
cimidazole[4,5-f]1,10-phenanthroline) has been synthesized and found class of anticancer agents could induce DNA damage of cancer
be able to enhance radiation-induced DNA damage through super- cells followed by triggering apoptosis or cell cycle arrest.11
oxide overproduction, which leads to G2/M arrest and apoptosis in Studies have demonstrated photo-activated properties of Ru
cancer cells by activating ROS-mediated pathways.
complexes,9,12 which could be used as potential photodynamic
therapy (PDT) agents. Inspired by these discoveries, we proposed
More than 50% of diagnosed cancer patients receive radiotherapy, that Ru complexes can probably be developed into radiosensitizers,
alone or in combination with other therapies worldwide.1 Ionizing since the X-ray possesses much higher energy than visible light,
radiation (IR) as one of the primary treatments for various cancers is might activate these Ru complexes as well. Selenium (Se) is a
prized because of its unique advantages of being noninvasive and necessary trace element with potential anticancer activities.13,14
low systemic toxicity.2 However, despite radiotherapy achieving Organic Se, especially selenoheterocyclic compounds, has attracted
varying degrees of success, many patients still suffer from recurrence more and more attention because of their unique pharmacological
and unexpected side effects.3 Because the effect of radiotherapy is activities.15 Our previous studies have indicated that seleno-
strongly limited by the radioresistance of cancer cells, the combi- heterocyclic compounds could effectively induce DNA damage
nation of radiotherapy with radiosensitizers as an experimental and and apoptosis of cancer cells.16 We also showed that seleno-
clinical strategy has been established to reduce radioresistance.4 compounds could effectively enhance the anticancer efficacy of
In the past few decades, radiosensitizers are widely used clinically X-ray through activation of diversified ROS-mediated signaling
and are considered to be able to improve the local-regional effects pathways.17 Based on the interesting physical and biochemical
of radiotherapy.5 Most radiosensitizers (such as cisplatin and characteristics and therapeutic advantages of Se, we attempt to
carboplatin) could target DNA and thus sensitize cancer cells to improve the anticancer activities and radiosensitization of Ru
radiation via enhancing DNA damage and inhibiting the DNA complexes by introducing seleno-ligands. Therefore, in this study, a
repair process.6 Therefore, based on this action mechanism to novel class of Ru complexes, Ru(phtpy)Cl3 (1), Ru(phtpy)(ip)Cl(ClO4)
design new metal complexes is becoming a novel strategy for (2a), Ru(phtpy)(pip)Cl(ClO4) (2b) and Ru(phtpy)(phenSe)Cl(ClO4) (2c)
discovery of new anticancer drugs.
(phtpy = 4-phenyl-2,20:60,200-terpyridine, ip = imidazole[4,5-f ]1,10-
In the past few decades, an increasing number of metal-based phenanthroline, pip = 2-phenylimidazole[4,5-f ]1,10-phenanthroline
complexes, especially platinum (Pt) complexes, were developed as and phenSe = 2-selenicimidazole[4,5-f ]1,10-phenanthroline)
radiosensitizers due to their DNA-binding properties.7 However, have been synthesized (Fig. 1A) and their anticancer activities
the application of Pt complexes was limited by serious toxic side and radiosensitization effects against human melanoma A375
effects, drug resistance and weak selectivity between tumour and cells were also examined as well. Among these complexes, the
normal tissues.8 Ruthenium (Ru) complexes, possessing favour- Se-containing one, 2c, possessed potent anticancer activity and
able properties suitable for flexible antitumor drug design,9 have radiosensitization effects. The studies on the action mechanisms
revealed that 2c sensitized A375 cells to radiation by enhancing
Department of Chemistry, Jinan University, Guangzhou 510632, China.
radiation-induced ROS-mediated DNA damage and down-
stream signalling pathways, eventually resulting in G2/M arrest
and apoptosis.
In this study, complex 1 was synthesized by mixing equal
quantities of RuCl3 and the phtpy ligand in ethanol to reflux at
E-mail: tchentf@jnu.edu.cn, tzhwj@jnu.edu.cn; Fax: +86 20 85221263
† Electronic supplementary information (ESI) available: Synthesis details, ESI-
MS, 1H NMR and UV spectrum analysis of synthesized compounds with/without
ionizing radiation. See DOI: 10.1039/c4cc07926d
‡ These authors contributed equally to the work.
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Table 1 Growth inhibition of 2c and 2c-radiation treatment on A375 and
HK-2 cellsa
IC50 (mM)
Complex A375
A375 + IRb SERc HK-2
HK-2 + IR SERd
2c
9.7 ꢀ 2.9 4.4 ꢀ 1.4 2.2
110.9 ꢀ 4.4 99.4 ꢀ 5.9 1.1
10.4 ꢀ 2.2 7.3 ꢀ 2.3 1.4
Cisplatin 7.5 ꢀ 1.3 4.9 ꢀ 2.2 1.5
a
Cell viability was determined by MTT assay after treatment for 72 h.
The dose of IR (X-ray) is 8 Gy. SER (sensitivity enhancement ratio) =
b
c
d
IC50 (A375)/IC50 (A375 + IR). SER = IC50 (HK-2)/IC50 (HK-2 + IR).
The in vitro radiosensitization of 2c against A375 and HK-2
cells was examined by MTT assay using cisplatin as a positive
control. The cells were incubated with different concentrations
of complex 2c or cisplatin for 6 h, followed by radiation at a
dose of 8 Gy, then cells were cultured for another 66 h before
examining their cell viability. As shown in Table 1, 2c effectively
sensitized A375 cells to radiation with a sensitivity enhancement
ratio (SER) of 2.2, which was much higher than that of cisplatin
(SER = 1.5). Moreover, for the human normal cell line (HK-2 human
kidney cells), complex 2c demonstrated low cytotoxicity toward HK-2
cells (IC50 = 110.9 mM), which was about 10 times lower than
that of cisplatin (IC50 = 10.4 mM). The SER value of 2c (1.1) was
also lower than that of cisplatin (1.4), which demonstrates the
higher selectivity of the synthetic complexes.
Studies were also carried out to examine the reason account-
ing for the different selectivity and radiosensitization effects
of 2c between cancer and normal cells. As shown in Fig. 2A, the
combined treatment of 2c and radiation was more cytotoxic
to A375 cells than 2c or X-ray alone. A remarkable decrease in
cell numbers and the change in cell morphology (such as cell
shrinkage and cell rounding) were observed in the cells that
received the combined treatment (Fig. S6, ESI†). In contrast, 2c
Fig. 1 (A) Structure of Ru complexes. (B) IC50 values and lipophilicity of
the Ru complexes. A375 cells were incubated with complexes for 72 h, and
the IC50 was determined by MTT assay. (C) Cellular uptake of complexes
1–2c (10 mM) in A375 cells as determined by ICP-MS analysis.
85 1C for 4 h. Complexes 2a–2c were synthesized by refluxing
the same quantity of 1 and the corresponding ligand in ethanol
for 6 h under a N2 atmosphere, followed by purifying by neutral
alumina column chromatography with methylbenzene and
acetonitrile as an eluent. The chemical structure and the purity
of the synthetic complexes were characterized and confirmed
by ESI mass spectrometry, 1H NMR spectroscopy and elemental
analysis (Fig. S1–S5, ESI†).
To examine the effects of Se on the biological application of Ru
complexes, firstly, MTT assay was applied to assess the anticancer
activities of the synthetic complexes. As shown in Fig. 1B, complex
1 exhibited slight growth inhibition on A375 cells after a 72 h
treatment, with the IC50 value of 59.6 mM. Meanwhile, 2a demon-
strated higher anticancer activity after coordination with the ip
ligand (IC50 = 52.4 mM), suggesting that the introduction of ip
analogs could enhance the anticancer activities of the Ru–phtpy
system. Though complexes 2a–2c shared similar chemical struc-
ture, their anticancer efficacy was totally different. Complex 2b
with the pip ligand did not exhibit effective suppression on the
growth of A375 cells, which may be due to its poor solubility as
a result of the introduction of the hydrophobic phenyl group.
Moreover, complex 2c with Se on the ip ligand shows a great
enhancement of antiproliferative activities towards A375 cells
(IC50 = 9.7 mM), indicating that the introduction of Se into Ru
complexes could improve their antitumor activities. Previously,
Barton and co-workers have shown that the cellular uptake and
anticancer activity of complexes were affected by their lipo-
philicity.18 Therefore, we measured the partition coefficient
(log P) and cellular uptake of 1–2c to determine their relation-
ship with the anticancer efficacy. As shown in Fig. 1B and C, the
cellular uptake of complexes 1, 2a and 2c was well correlated
with their partition coefficients. However, complex 2b with
high log P showed lowest anticancer activity and low cellular
uptake, which may due to its poor solubility in the aqueous cell
culture. Among these complexes, 2c displayed the highest log P,
highest cellular uptake and anticancer activity. These results suggest
that, the introduction of Se into Ru complexes could effectively
increase their lipophilicity, thus enhancing the cellular uptake and
anticancer efficacy.
Fig. 2 Relationship between radiosensitization effects and cellular uptake
of complex 2c. (A) Growth inhibition of different treatments on A375 cells.
Cells were exposed to different treatments for 72 h, and the cell growth
inhibition was determined by MTT assay. (B) Growth inhibition of different
treatments on HK-2 cells (72 h). (C) Cellular uptake of complex 2c (10 mM)
in A375 and HK-2 cells as determined by ICP-MS. (D) Flow cytometry
analysis of A375 cells treated for 24 h.
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alone showed slight growth inhibition on HK-2 cells, and it didn’t cell arrest and apoptosis eventually.20 Importantly, the X-ray-
enhance the cytotoxicity of X-ray toward the cells (Fig. 2B). The induced ROS generation has been identified as the major cause
different effects of 2c on cancer and normal cells could be due to of DNA damage.4b Therefore, we measured the ROS level in
the difference in cellular uptake. Consistent with this hypothesis, A375 and HK-2 cells by examination of dihydroethidium (DHE)
we found that, the uptake of 2c in A375 cells was much higher than fluorescence intensity. As shown in Fig. 3B, the co-treatment
that in HK-2 cells (Fig. 2C), which contributes to the higher growth remarkably increased the intercellular ROS generation in A375
inhibition and radiosensitization.
cells to over 200% of control, but no significant change was
Flow cytometric analysis was performed to examine the observed in cells exposed to 2c or X-ray alone. However, in HK-2
action modes of radiosensitization induced by Ru complexes. human normal cells, X-ray alone activated the intracellular ROS
As shown in Fig. 2D, 2c and radiation co-treatment induced generation to about 130% of control group (Fig. 3C). However,
G2/M arrest in A375 cells, as reflected by the increase of the the co-treatment of the cells with complex 2c reduced the ROS
percentage of cells at the G2/M phase (co-treatment at 31.6% vs. generation to the control level, which was much lower than that in
control at 17.0%). In addition, 2c enhanced the radiation-induced A375 cells. Furthermore, we found that, in A375 cells, the phos-
cell apoptosis, as evidenced by the increase in the Sub-G1 phase phorylation of ATM, ATR and histone (Fig. 3D), three important
from 7.4% to 14.9% (co-treatment). These results suggested that biochemical hallmarks of DNA damage,21 was more obvious than
2c-radiation co-treatment could induce disruption of cell-cycle those in HK-2 cells after being treated with 2c and X-ray. These results
progression and apoptotic cell death.
DNA has been regarded as the main target of X-ray and most effects of X-ray by triggering ROS-mediated DNA damage.
metal-based anticancer drugs. In order to examine the role of The chemical interaction between the complexes and X-ray
suggest that Se-containing Ru complexes enhance the anticancer
DNA in the anticancer action of 2c, firstly, we used the cell was also examined by ESI-MS and 1H NMR. As shown in Fig. S7
model to examine the cellular distribution of the complex by (ESI†), no significant change in the mass spectra and chemical
monitoring its autofluorescence. As shown in Fig. 3A, 2c mainly shift was observed in the complexes after radiation. The con-
located in the cytoplasm, suggesting that 2c doesn’t interact sistency of the UV-Vis spectra of the complexes before and after
with DNA directly. In the cytoplasm, cellular proteins have been radiation further confirmed the stability of the synthetic com-
proposed to be favourable targets for cytotoxic metal complexes.19 plexes (Fig. S8, ESI†). We also found that, the UV-Vis spectra of
Che and co-workers have discovered that metal complexes could complex 2c remained stable during incubation in aqueous solutions
inhibit some cellular proteins (such as TrxR) to cause the accumula- for 24 h (Fig. S9, ESI†). Even in the presence of 50–500 mM
tion of reactive oxygen species (ROS) and DNA damage, resulting in H2O2, the UV-Vis spectra of complex 2c didn’t show a change
after 30 min (Fig. S10, ESI†). The stability of this kind of
synthetic complex supports their future application in the
chemo- and radio-therapy of cancers.
To further elucidate the signalling mechanisms contributing
to the radiosensitization effects of 2c, we measured the level of
proteins related to the regulation of G2/M arrest and apoptosis.
As shown in Fig. 4A, the co-treatment up-regulated the level of
cyclin-B, a crucial cell cycle regulator necessary for the progression
Fig. 3 ROS-mediated DNA damage induced by complex 2c and X-ray.
(A) Cellular location of complex 2c in A375 cells. (B) Changes in the intra-
cellular ROS level induced by different treatments in A375 cells. (C) Changes
in the intracellular ROS level induced by different treatments in HK-2 cells.
(D) Western blot analysis for the expression of p-ATM, p-ATR and p-histone.
b-Actin was used as loading control. The concentration of 2c was 10 mM,
and the dose of radiation was 8 Gy.
Fig. 4 Signalling pathways triggered by complex 2c and X-ray. (A) Western
blot analysis of the expression of related proteins. b-Actin was used as loading
control. The concentration of 2c was 10 mM, and the dose of radiation was
8 Gy. (B) The main signalling pathways accounting for the radiosensitization
effects of complex 2c.
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Meanwhile, the combined treatment also induced the proteo-
lytic cleavage of PARP and obvious decrease in the expression
levels of total caspase-3, -8 and -9, indicating the proteolytic
cleavage of these proteins, which confirmed the involvement of
the extrinsic and mitochondria-mediated intrinsic apoptosis
pathways in the co-treatment-induced apoptosis. As expected,
the combined treatment also increased the expression of FADD
and suppressed the expression of Bcl-xl, a pro-survival member
of Bcl-2 family protein. The observation of ROS accumulation
and activation of mitochondria-mediated apoptosis proves the
induction of mitochondrial dysfunction by 2c. Considerable
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and phosphorylation of p53 at the ser 15 site and histone. Taken
together, these results indicate that 2c sensitizes cancer cells to
X-ray by triggering ROS-mediated DNA damage and activation
of the p53 pathway (Fig. 4B).
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