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Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II.
Angewandte
A Journal of the Gesellschaft Deutscher Chemiker
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Chemie
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Accepted Article
Title: Necroptosis Induced by Ruthenium(II) Complexes as Dual
Catalytic Inhibitors of Topoisomerase I/II
Authors: Hui Chao, Kai Xiong, Chen Qian, Yixian Yuan, Lin Wei,
Xinxing Liao, Liting He, Thomas W. Rees, Yu Chen, Jian
Wan, and Liangnian Ji
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006089
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10.1002/anie.202006089
Angewandte Chemie International Edition
RESEARCH ARTICLE
Necroptosis Induced by Ruthenium(II) Complexes as Dual
Catalytic Inhibitors of Topoisomerase I/II
Abstract: Inducing necroptosis in cancer cells is an effective
approach to circumvent drug-resistance. Metal-based triggers have,
however, rarely been reported. Ruthenium(II) complexes containing
1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine were developed with a
series of different ancillary ligands (Ru1-7). The combination of the
main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with
superior nucleus-targeting properties. As a rare dual catalytic inhibitor,
Ru7 effectively inhibits the endogenous activities of topoisomerase
(topo) I and II and kills cancer cells by necroptosis. The cell signaling
pathway from topo inhibition to necroptosis was elucidated.
Furthermore, Ru7 displays significant antitumor activity against drugresistant cancer cells in vivo. To the best of our knowledge, Ru7 is the
first Ru-based necroptosis-inducing chemotherapeutic agent.
Introduction
Cancer drug resistance is a major problem in chemotherapy,
which can be caused by the regulation of transporters, upregulation of detoxification systems or modulation of cell death
pathways.[1] Conventional anticancer agents, such as cisplatin,
camptothecin, and paclitaxel, mostly induce apoptosis regardless
of target and mechanism.[2] It has been demonstrated that many
cancer cells exhibit drug resistance via dysregulation of apoptotic
machinery, including the overexpression of antiapoptotic proteins
and defects in apoptotic signalling.[3] As the apoptotic machinery
is composed of dozens of antiapoptotic and proapoptotic proteins,
which are also affected by many oncogenic signals, it is highly
difficult to treat cancers with apoptotic resistance. The apoptotic
resistance of cancer cells can, however, be circumvented by
chemotherapeutic agents which induce non-apoptotic cell
death.[4]
[a]
[b]
[c]
K. Xiong, Dr. C, Qian, Dr. Y. Yuan, X. Liao, L. He, Dr. T. W. Rees,
Dr. Y. Chen, Prof. L. Ji and Prof. Dr. H. Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry
Sun Yat-Sen University
Guangzhou, 510275, P. R. China
E-mail: chenyu63@mail.sysu.edu.cn, ceschh@mail.sysu.edu.cn
L. Wei, Prof. Dr. J. Wan,
College of Chemistry,
Central China Normal University
Wuhan, 430079, P. R. China
Email: jianwan@mail.ccnu.edu.cn
Prof. Dr. H. Chao
College of Chemistry and Environmental Engineering
Shenzhen University
Shenzhen, 518071, P. R. China
Supporting information for this article is given via a link at the end
of the document.
Necrosis was originally considered to be random and
unregulated, however, increasing evidence reveals that necrosis
can be induced and proceed in a regulated manner like apoptosis.
This is known as necroptosis. Necroptosis is intricately connected
with many physiological processes and crucial for the
maintenance of tissue homeostasis-throughout life.[5] Reports on
necroptosis-inducing chemotherapeutic agents are rare,
especially metal-based agents. Cisplatin can trigger necrosis in
many types of cancer cells. This occurs only in the presence of
the caspase inhibitor (Z-VAD-FMK) however, indicating that
necroptosis is optimally induced when the apoptotic machinery is
compromised.[6] Recently, two rhenium(V) oxo complexes were
developed as necroptosis triggers and demonstrated excellent
therapeutic efficiency.[7] Zn(II), Ni(II), Os(II), and Fe(III) complexes
were also utilized to induce necroptosis in cancer cells.[8] It was
noted that cisplatin-induced necroptosis is caspase inhibitor
dependent. In contrast, the afore mentioned complexes induce
necroptosis in the absence of caspase inhibitors. Although
interesting, these complexes need further evaluation of their
cellular targets and in vivo therapeutic effects. Aside from these
few studies, the area remains underexplored.
Topoisomerases (topo) are critical enzymes for the control of
DNA supercoiling and entanglement, and are therefore involved
in cell proliferation. Topo I and topo II are two types of
topoisomerases which are ordinarily over-expressed in tumors.[9]
Their respective inhibitors, camptothecin and etoposide, are
among the most commonly used anticancer drugs. As is the case
for many antitumor drugs, these inhibitors are susceptible to drug
resistance. The down-regulation of the targeting enzyme, and
Scheme 1. Schematic illustration of the dual catalytic inhibitor of topoisomerase
I and II inducing necroptosis.
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Accepted Manuscript
Kai Xiong,[a] Chen Qian,[a] Yixian Yuan,[a] Lin Wei,[b] Xinxing Liao,[a] Liting He,[a] Thomas W. Rees,[a] Yu
Chen,*[a] Jian Wan,*[b] Liangnian Ji,[a] and Hui Chao*[a][c]
10.1002/anie.202006089
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RESEARCH ARTICLE
apoptotic machinery dysregulation, are responsible for this.[10]
When one of these topoisomerases is suppressed, the other is
commensurably overexpressed. Furthermore, simultaneous
treatment with the respective inhibitor antagonizes cytotoxic
effects.[11] Therefore, simultaneous inhibitors of both topo I and II
(dual inhibitors) are expected to have a broader spectrum of
activity as expression levels of the two enzymes are variable
between different types of cancers and possess a significant
therapeutic advantage over agents targeting one type of
topoisomerase alone.[12]
Metal complexes have been developed as topo inhibitors and
although those reported have displayed remarkable antitumor
activity, most of the research so far has focused on either topo I
or II alone.[13] Some Ru(II) complexes, such as [Ru(bpy)2(pscl)]2+
and [Ru(bpy)2(psbr)]2+, have been found to efficiently inhibit both
topo I and II.[11b] However, they still have several shortcomings: 1)
These Ru(II) complexes are all classed as topoisomerase poisons
and induce apoptosis. 2) Their nuclear accumulation properties
are yet to be confirmed, therefore, the relationship between
apoptosis and topo inhibition is in doubt. 3) Their in vivo antitumor
activity remains uninvestigated. In this context, ruthenium(II)
complexes
containing
1,1-(pyrazin-2-yl)pyreno[4,5e][1,2,4]triazine were developed as dual catalytic inhibitors of topo
I and topo II (Ru1-7, Figure 1). The nucleus-targeting of Ru7 was
achieved by adjustment of the auxiliary ligands and thereby the
overall properties of the complex. Ru7 induces necroptosis and
the cell signaling pathway involved is herein demonstrated
(Scheme 1). The antitumor activity of Ru7 as the first metal-based
topo I/II dual catalytic inhibitor that triggers necroptosis was also
evaluated in vivo.
Results and Discussion
The main ligand 1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine
(pzpp) was obtained in a good yield by the reaction of pyrene-4,5dione with pyrazine-2-carboxamide hydrazine. Cellular uptake
and organelle distribution are a key factor affecting the efficacy of
metal-based antitumor agents. Tuning the lipophilicity or charge
of a complex is an effective approach to achieve this.[14] With this
in mind, auxiliary ligands with increasingly conjugated systems
were added to Ru1-6, while a cyclometalated ligand was
introduced to Ru7. Ru1-6 were synthesized in a similar manner
Figure 2. Inhibitory effects of Ru1-7 on the catalytic activity of topoisomerases
I (A) and topoisomerase II (B) were determined with relaxation assays.
by heating pzpp with corresponding precursor cis-Ru(L)2Cl2 [L =
2,2’-bipyridine (bpy, Ru1); di(pyridin-2-yl)amine (dpa, Ru2); 4,4'dimethyl-2,2'-bipyridine (dmb. Ru3); 1,10-phenanthroline (phen,
Ru4); 4,7-dimethyl-1,10-phenanthroline (dmp, Ru5) and 4,7diphenyl-1,10-phenanthroline (dip, Ru6)] in ethylene glycol at 120
ºC for 6 h. For cyclometalated Ru7, stoichiometric equivalents of
[(η6-C6H6)Ru(bpy)Cl] and 2-phenyl-pyridine (ppy) were combined
and refluxed in DMF for 4 h, before the addition of 1 equivalent of
pzpp and reflux for further 4 h. All the crude products were purified
by chromatography on Al2O3 with acetonitrile and toluene
mixtures as the eluents. These complexes were fully
characterized by elemental analysis, ESI-MS and 1H NMR
spectroscopy (Figure S1-S7). The crystal of Ru1, Ru2, Ru3, Ru5
and Ru6 were also obtained by solvent evaporation (acetonitrile
and toluene, 1:1, v/v) and the structures determined by single
crystal x-ray diffraction. ORTEP representations of the structures
including the atom numbering scheme are shown in Figure S8S12. The X-ray crystallographic data, including selected bond
lengths and angles are given in the Supporting Information (Table
S1-S6).
The results of Topo I and Topo II inhibition assays with
different concentrations of Ru1-7 are shown in Figure 2. Ru1-7
inhibit the ability of topo I and topo II to relax negatively
supercoiled plasmid DNA. These results are consistent with
known topo I inhibitor camptothecin (CPT) and topo II inhibitor
etoposide (VP-16). The inhibition efficacy is concentrationdependent. All the Ru(II) complexes completely inhibit topo I/II
activity at a concentration of 5 μM, indicating that their inhibition
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Figure 1. The chemical structures of the Ru(II) complexes Ru1-7.
10.1002/anie.202006089
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Figure 3. (A) IC50 values of cisplatin and Ru1-Ru7 after incubation with the lung cancer cell A549, lung drug-resistant cancer cell A549R and the lung normal cell
BEAS-2B for 48 h. (B) The octanol/water partition coefficients of Ru1-Ru7. Insert: the distributions of Ru1-Ru7 in octanol/water solutions. (C) Amounts of ruthenium
within the cytoplasm and nuclei of A549R cells after incubating with Ru1-Ru7 (5 μM) for 1, 3, 6, and 24 h.
abilities are independent of the auxiliary ligand. In contrast, no
significant inhibition was observed for 10 μM CPT and VP-16.
Topoisomerase inhibitors are split into two types: poison and
catalytic inhibitor.[15] Topoisomerase poisons selectively affect the
re-ligation of DNA and form stable enzyme-DNA-inhibitor ternary
complexes. Catalytic inhibitors prevent the binding of topo
enzymes to DNA. The majority of topo inhibitors are topo poisons
and reports on topo I/II dual catalysis inhibitors are rare.[16] This
may be due to the structural differences between the two
enzymes, resulting in design difficulties. [17] To investigate whether
Ru1-7 are topo poisons, the formation of topoisomerase-induced
DNA-strand breaks was measured. As shown in Figure S13A, the
poison CPT stabilizes the topoisomerase I cleavable complex,
resulting in the generation of open-circle plasmid DNA (lane 10,
marked with an arrow). In contrast, open-circle DNA bands were
not observed for the Ru(II) complexes (lane 3-9), implying the
existence of a different inhibition mechanism. Additionally, Ru1-7
antagonize the formation of open-circle DNA in the presence of
20 times higher concentrations of CPT (lane 11-17), suggesting
that the Ru(II) complexes act a step upstream of CPT. Similar
results were observed for topoisomerase II (Figure S13B). The
poison VP-16 selectively affects topo II-mediated DNA re-ligation,
which leads to the presence of a linear DNA band. None of the
Ru(II) complexes increase the level of DNA scission, but all of
them prevent cleavable complex formation in the presence of VP16, even when the concentration of VP-16 is 20 times that of the
complexes.
The ability of Ru1-7 to prevent topoisomerase-DNA binding
was studied by electrophoretic mobility shift assay (EMSA). [15b]
The results are presented in Figure S14. Topo I forms stable
complexes with plasmid DNA, resulting in a relatively immobile
band that is retained close to the application slot. Treatment of
these complexes with SDS and proteinase K released the DNA.
The topo poison CPT did not interfere with the binding and
scission steps of topo I. In contrast, topo I binding of DNA was
significantly affected by the complexes. A similar result was
obtained for topo II. Topo II binds to DNA in both the presence
and absence of VP-16, while Ru1-7 prevent this binding. These
observations demonstrate that the complexes act as topo I and II
dual catalytic inhibitors rather than poisons. This conclusion is
further demonstrated by the endogenous topoisomerase
inhibition and molecular docking experiments, which follow.
The remarkable topo I and II inhibition properties of Ru1-7
encouraged the investigation of their anticancer activity. Five
cancer cell lines (A549, SGC-7091, HeLa, HepG2, and BEL7402), two drug-resistant cell lines (A549R, and SGC-7901/DDP)
and two normal cell lines (LO2, and BEAS-2B) were tested.
Cisplatin, CPT, VP-16, and the ligand pzpp were used as controls.
The detailed IC50 data are shown in Table S7 and the results for
A549, A549R, and BEAS-2 are also presented in Figure 3A. The
ligand pzpp was inactive against all of the cell lines (IC50 > 200
μΜ). CPT and VP-16 exhibited moderate cytotoxicity towards all
the cancer cell lines but much less activity toward normal cells. In
contrast, cisplatin showed stronger activity against the cancer cell
lines than CPT and VP-16, with the exception of the drug-resistant
cell lines. The selectivity of cisplatin between cancer and normal
cells is, however, poor. Although Ru1-7 possess similar
topoisomerase inhibition abilities, their antitumor activities are
varied. Ru1 and Ru3 showed the lowest cytotoxicities with IC50 >
100 μM. Ru2, Ru4, and Ru5 displayed moderate antiproliferative
activity similar to CPT. Treatment with Ru6 inhibited the cancer
cells with IC50 values in the range of cisplatin. The cyclometalated
complex Ru7 exhibited the most potent anticancer activity.
Against A549, for example, the IC50 value of Ru7 is 3.0 ± 0.1 μM,
3.8, 26.3, and 17.5-fold lower than that of cisplatin, CPT, and VP16, respectively. Ru7 displayed similar activity towards both the
drug-resistant and corresponding cancer cell lines, demonstrating
its ability to overcome drug resistance. Moreover, Ru7 had a high
selectivity towards cancer cells over normal cells. The selectivity
ratio between normal lung cells BEAS-2B (IC50 = 18.0 ± 1.1 μM)
and lung cancer cells A549R (IC50 = 2.2 ± 0.2 μM) was found to
be 8.2. In contrast, the ratios for CPT, VP-16, and cisplatin are
1.4, 2.0, and 0.2, respectively. Although A549 cells are
characterized by multidrug resistance via P-glycoprotein
(transporter) expression rather than reducing apoptosis, A549R
cells are more resistant towards cisplatin-induced apoptosis than
SGC-7901/DDP. Therefore, the following experiments were
performed in A549R cells.
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increase in log PO/W value from -2.06 ± 0.23 to -0.62 ± 0.04.
Further addition of methyl groups (Ru5, -0.22 ± 0.02) and phenyl
groups (Ru6, 0.43 ± 0.07) also increased the lipophilicity. The
cyclometalated complex Ru7 with a charge of +1 compared with
the +2 charge of the other complexes has the highest lipophilicity
of the series, which is predictive of a higher cellular uptake (Figure
3B). To achieve topoisomerase inhibition, localization in the cell’s
nucleus is necessary. The nucleus-targeting properties of the
complexes were investigated by inductively coupled plasma mass
spectrometry (ICP-MS). The ICP-MS data match the trends
observed in lipophilicity and antitumor activity experiments
(Figure 3C). The cellular uptakes of Ru1-5 were poor. Containing
the lipophilic ligand dip, the cellular uptake of Ru6 was
significantly higher than those of Ru1-5. Although the nucleuslocalized amount of Ru6 was also increased, its cytosolic fraction
was still up to 52.3% (24 h). In contrast, the cyclometalated
complex Ru7 displayed the most potent nuclear-targeting activity.
After 24 h incubation, the nuclear fraction is 89.2%, confirming its
specific nuclear-targeting property. Due to its optimal antitumor
activity and selectivity, Ru7 was chosen for further studies in
A549R cells.
Figure 4. (A) Left: schematic illustration of the dual catalytic inhibition
mechanism. Right: endogenous topo inhibition assay. A549R cells were
pretreated with CPT, VP-16, or Ru7 for 24 h and then the nuclear lysates were
incubated with 100 ng of pBR322 plasmid at 37 °C for 30 min. (B) Molecular
docking experiments indicate that Ru7 can bind to the DNA-binding site of
topoisomerase I. (C-D) Molecular docking results for the interations between
Ru7 and the topoisomerase II DNA-binding site (C) and ATP-binding site (D).
The cellular uptake and organelle localization of the
complexes was investigated in order to determine the cause of
variation in cytotoxicity across the series. The ability of Ru(II)
complexes to pass through cell membranes is related to their
lipophilicity. The octanol/water partition coefficients (log PO/W) of
the complexes were therefore assessed.[18] As expected, the
lipophilicities of Ru1-6 increase with the degree of conjugation of
the auxiliary ligands. Ru1-5 are hydrophilic while Ru6 and Ru7
are lipophilic. Ru2, with the imino group, showed the most
negative log PO/W value (-2.15 ± 0.36). Replacing the auxiliary
bipyridyl ligand (Ru1) with phenanthroline (Ru4), gave an
To further confirm the nuclear localization of Ru7 and verify
whether Ru7 can inhibit topoisomerases in cells, a cell-based
DNA relaxation assay was performed. A549R cells were treated
with Ru7 for 24 h and the nucleus was extracted. The nuclear
extractions were lysis and used for the assay.[12b] If Ru7 cannot
enter the nucleus nor inhibit the topoisomerases, the result for
Ru7-treated and Ru7-free samples will be the same, that is, the
topoisomerases keep active. As shown in Figure 4A, in the
absence of Ru7, the endogenous topoisomerases in the nuclear
lysates made the supercoiled plasmids completely relaxed or
linearized (lane 2). Incubation with 100 μM CPT or VP-16 inhibited
the endogenous topoisomerases due to the formation of stable
enzyme-DNA-inhibitor complexes. Low concentration treatment
with Ru7 (5 μM) did not affect the topo-mediated relaxation. When
the concentration was increased to 20 μM, a strong inhibition was
observed (lane 6). Because the above cleavable complex assay
demonstrated that the Ru(II) complexes are topo I and II dual
catalytic inhibitors, this endogenous topo inhibition was assigned
to the binding of Ru7 to topo I and topo II. Molecular docking
experiments were performed to provide insight into the mode of
action of Ru7 as a topo catalytic inhibitor. With topo I, Ru7 is
predicted to bind to the DNA-binding pocket (Figure 4B). π-π
stacking between Ru7 and PHE723, ARG364, ARG488, and
ARG590, as well as the cation-π interaction with LYS532
accounts for the inhibition. Results with topo II show that there are
two potential binding modes for Ru7. The first one is similar to the
case of topo I. Ru7 could localize in the DNA-binding pocket of
topo II through hydrogen bonding with ARG820, and π-π stacking
with ARG503 and TYR821 (Figure 4C). As the activation of topo
II requires ATP, Ru7 could also bind to the ATP pocket of topo II
(Figure 4D), stabilized via π-π stacking (with PHE791, ARG945,
ARG488, and TRP947) and cation-π interactions (with LYS814
and LYS739).
The mechanism of cell death induced by Ru7 was evaluated
next. The cytotoxicity of Ru7 in the presence of inhibitors was
studied. A549R cells were pretreated for 0.5 h with Z-VAD-FMK
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Figure 5. (A) The cell signaling pathway from topo inhibition to necroptosis. (B)
The expression level of phospho-RIPK1 (p-RIPK1), phospho-RIPK3 (p-RIPK3),
and MLKL with or without the presence of inhibitors (DPQ, PARP-1 inhibitor;
Nec-1, RIPK1 inhibitor). (C) Nuclear DNA damage was examined by the comet
assay. (D) The expression level of PARP-1 with or without the presence of
inhibitor DPQ.
(apoptosis inhibitor), 3-methyladenine (autophagy inhibitor) and
necrostatin-1 (necrosis and necroptosis inhibitor) followed by Ru7
(2.5 μM) for a further 24 h.[19] As shown in Figure S15, with
addition of Z-VAD-FMK, no effect on the cell survival rate was
observed (cell viability = 70.1%) when compared with treatment
Ru7 alone (cell viability = 68.1%), indicating that apoptosis was
not involved in the cell death pathway induced by Ru7. this was
further demonstrated by caspase activation assay. Caspase is an
important protein family involved in apoptosis. As expected,
caspases were activated in response to cisplatin while treatment
with Ru7 antagonized activation (Figure S16). Similarly,
autophagy was not found to be involved in Ru7 induced cell death
as the presence of autophagy inhibitor 3-methyladenine did not
affect the cell survival rate (Figure S15) and no activation of LC3
was observed (Figure S17). In contrast, Ru7 driven cell death was
significantly reduced in the presence of necrostatin-1 (cell viability
= 90.5%, t test, p < 0.05). The cell death mechanism was further
investigated by Annexin V-FITC/propidium iodide (AV/PI) dual
staining assay (Figure S18). The results show that cisplatin, CPT,
and VP-16 treatment leads to increase in the number of AV+PI−
cells with a constant percentage of AV−PI+ cells. This results from
the externalization of phosphatidyl-serine on the cell surface, an
early indicator of apoptosis. Significantly different phenomena
were observed on treatment with Ru7 (1 μM). 19.5% of cells were
permeable to PI while that for control was 6.2%. Although the
percentage of AV+PI− cells increased slightly from 3.9% to 7.5%,
it did not increase further when the concentration of Ru7 was
increased to 2.5 μM. In contrast, 2.5 μM Ru7-treatment
remarkably increased the percentage of AV−PI+ cells, with
severely damaged plasma membranes. This result, together with
the above cytotoxicity results, demonstrate that Ru7 induces
necrosis or necroptosis
Figure 6. The in vivo antitumor activity of Ru7 towards A549R xenografted
tumor. (A) Representative photographs of mice after treatments with saline (0.2
mL, Control), Ru7 (high dose, 2.0 mg/kg), Ru7 (low dose, 0.5 mg/kg) and
cisplatin (4.0 mg/kg, Cis-Pt). Tumor volumes (B) and body weights (C) change
curves of various treatments as indicated. The error bars denote the standard
deviation of results from ten mice.
The main difference between necrosis and necroptosis is that
the latter is regulated. The core necroptotic pathway is receptorinteracting protein kinase 1 (RIPK1)–RIPK3–mixed lineage
kinase domain-like protein (MLKL) (Figure 5A).[20] The
immunoblotting results in Figure 5B show that with Ru7 treatment,
the expression of phospho-RIPK1 (p-RIPK1), phospho-RIPK3 (pRIPK3), tetramer-MLKL increased while the amount of monoMLKL was reduced in a concentration-dependent manner. This
activation could be blocked by the presence of RIPK1 inhibitor
necrostatin-1 (Nec-1),[13c] which is consistent with the above
cytotoxicity result (Figure S15). Upstream of RIPK1 and its
relationship with topo inhibition were also determined. Recent
reports have linked the activation of poly(ADP-ribose) polymerase
(PARP-1) to the expression of RIPK1.[21] As a DNA repair enzyme,
PARP-1 can be triggered by DNA damage.[13c,22] Nuclear DNA
damage was therefore examined by a comet assay (Figure 5C).
Treatment with Ru7 led to the appearance of a “broom-like” tail
indicating severe DNA damage. This result was consistent with
that of cisplatin, CPT, VP-16, and previously reported topo
catalytic inhibitors.[23] Subsequent activation of PARP-1 was
similar to that of p-RIPK1, p-RIPK3 and tetramer-MLKL (Figure
5D). Co-incubation with 3, 4-Dihydro-5[4-(1-piperindinyl)butoxy]1(2H)-isoquinoline (DPQ), a selective PARP-1 inhibitor, not only
inhibited the expression of p-RIPK1, p-RIPK3 and tetramer-MLKL
(Figure 5B) but also markedly decreased the cytotoxicity of Ru7
(Figure S15), confirming that PARP-1 acts as the upstream of the
necroptotic pathway and is involved in Ru7-induced cell death. In
addition, it has been reported that the RIP1-RIP3-MLKL complex
mediates downstream executing molecules and events such as
reactive oxygen species (ROS) burst, plasma membrane
permeabilization and cytosolic ATP reduction. ROS levels were
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As the in vivo antitumor activity of metal-based necroptosis
inducers remains an unexplored area of research, the in vivo
antitumor activity of Ru7 was evaluated. A549R xenografted
tumor-bearing mice were randomly divided into four groups,
named control (0.2 mL saline), high dose (Ru7, 2.0 mg/kg), low
dose (Ru7, 0.5 mg/kg) and Cis-Pt (cisplatin, 4.0 mg/kg).
Intraperitoneal injections were given every 4 days to all groups.
As shown in Figure 6, the cisplatin treated group showed similar
tumor development to the control due to the cisplatin resistance
of the cell line. In contrast, Ru7 significantly inhibited tumor
development. After 24 days of treatment, the tumor growth of the
high dose group was negligible, and the average volume was only
23.3% of that of the control group. Furthermore, the body weights
of all groups, except cisplatin, increased in a stable manner during
treatment (Figure 6) with no serious cellular structural changes,
pathological alterations or organ damage observed (Figure S20).
In contrast, the cisplatin treated group exhibited significant loss of
body weight and evident liver and kidney damage. These results
confirm the advantages of Ru7 as a necroptotic inducer for the
treatment of drug resistant cancer.
Experimental Section
Experimental details including synthetic details, characterization data,
experimental data and analysis are provided in the Supporting Information.
Single-crystal X-ray structure CCDC deposition codes: Ru1, 1941708;
Ru2, 1941709; Ru3, 1941706; Ru5, 1941707 and Ru6, 1941705
Acknowledgements
This work was supported by the National Science Foundation of
China (Nos. 21525105, 21778079, 21977126), the Ministry of
Education of China (No. IRT-17R111), the Fundamental
Research Funds for the Central Universities (No. 20lgjc01), and
the Pearl River S&T Nova Program of Guangzhou (No.
201806010136).
Keywords: Bioinorganic Chemistry • Medicinal Inorganic
Chemistry • Metals in Medicine • Ruthenium • Necroptosis
[1]
[2]
Conclusion
[3]
In this work, ruthenium(II) complexes, Ru1-7, were developed as
chemotherapeutic agents. Ru1-7 are highly novel catalytic
inhibitors of both topoisomerase I and II preventing the
topoisomerases from binding to DNA. The antitumor activities of
Ru1-7 vary with lipophilicity, resulting from differences in cellular
uptake. Introduction of the cyclometalated auxiliary ligand to Ru7
endows the complex with excellent nucleus-targeting properties.
Ru7 displays remarkable cytotoxicity against various cancer cell
lines including cisplatin-resistant cancer cells, demonstrating the
complexes ability to overcome drug resistance. Furthermore, the
cytotoxicity of the complex is highly selective towards cancer cells
over normal cell lines. Screening with inhibitors of different cell
death modes revealed Ru7 induces necroptosis. Characteristic
indicators of necroptosis were observed, including ROS burst,
plasma membrane permeabilization and cytosolic ATP reduction.
The cell signalling pathway from topo inhibition to necroptosis was
also demonstrated. Ru7 inhibits topo I and II, leading to DNA
damage and PARP-1 activation, subsequent RIPK1, RIPK3, and
MLKL activation results in necroptosis. Finally, the excellent in
vivo antitumor activity of the complexes was demonstrated
proving Ru7 is a necroptosis-inducing chemotherapeutic agent
with great clinical potential for circumventing drug resistance in
cancer. We hope that our results will provide new evidence that
the induction of necroptosis in cancer cells is an effective way to
circumvent drug resistance, and hopefully, in the future,
necroptosis inducers can be applied in the clinic treatment.
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Accepted Manuscript
measured by staining with DCFH-DA and ATP levels were
examined by luciferase assay. As shown in Figure S19, after
incubation with Ru7, enhancement of cellular ROS production
and decrease in ATP levels were observed. Plasma membrane
permeabilization was assessed by lactate dehydrogenase (LDH)
release. A 2.0 and 3.3-fold increase in the luminescence intensity
of released LDH was obtained after 1 and 2.5 μM Ru7 treatments
respectively (Figure S19).
10.1002/anie.202006089
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Ru(II) complexes were developed to
act as dual catalytic inhibitors of
topoisomerase I and II. Tuning of the
auxiliary ligands gave complex Ru7,
which targets the nucleus of cancer
cells effectively, inducing cell death
via necroptosis. The cell signaling
pathways were investigated and the in
vivo activity against drug resistant
cancer was found to be excellent.
Kai Xiong, Chen Qian, Yixian Yuan, Lin
Wei, Xinxing Liao, Liting He, Thomas W.
Rees, Yu Chen,* Jian Wan,* Liangnian
Ji and Hui Chao*
Page No. – Page No.
Necroptosis Induced by Ruthenium(II)
Complexes as Dual Catalytic
Inhibitors of Topoisomerase I/II
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