Rhodium(III) complexes with isoquinoline derivatives as potential anticancer agents:: In vitro and in vivo activity studies

Article abstract of DOI:10.1039/c9dt01951k

Two rhodium complexes Rh1 and Rh2 with isoquinoline derivatives were synthesized and characterized. Both complexes displayed strong anticancer activity against various cancer cells and low cytotoxicity against non-cancer cells. These complexes triggered a

Full text of DOI:10.1039/c9dt01951k

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14 March 2017
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Rhodium(III) complexes with isoquinoline derivatives as potential
anticancer agents: in vitro and in vivo activity studies
Taj-Malook Khan1, Noor Shad Gul1, Xing Lu1, Rajesh Kumar1,2, Muhammad Iqbal Choudhary2, Hong
Liang1,*, and Zhen-Feng Chen1,*
Two rhodium complexes Rh1 and Rh2 with isoquinoline derivatives were synthesized and characterized. Both
complexes displayed strong anticancer activity against various cancer cells and low cytotoxicity against non-cancer cells.
These complexes triggered apoptosis via mitochondrial dysfunction that increased the levels of ROS and Ca2+ and
released cytochrome C which ultimately activated caspases and the apoptosis pathway. The different biological
activities of Rh1 and Rh2 could be associated with the presence of methoxy substituents on the ligands. In vivo studies
showed that Rh1 effectively inhibited tumor growth in a T-24 xenograft mouse model with less adverse effect than
cisplatin. Overall, Rh1 and Rh2 induced apoptosis via mitochondrial pathways and could be developed as effective
anticancer agents.
based complexes possessing different mechanisms of action and
improved anticancer activity and safety profile than cisplatin.6
Introduction
The serendipitous discovery and successful development of cisplatin
in cancer therapy has opened an avenue for researchers to discover
more potent metal complexes with improved therapeutic efficacy. A
plethora of platinum complexes has been synthesized and studied as
potential therapeutic agents for different kinds of cancer.1 Among
these platinum complexes, carboplatin and oxaliplatin have been
used worldwide for the treatment of different types of cancers.2 The
primary mechanism of action of platinum complexes is to disrupt the
replication and transcription of DNA.3 However, the use of
platinum-based metallodrugs in cancer therapy is limited by their
lack of specificity in targeting cancerous cells over healthy cells,
which leads to severe side effects.4,5 To overcome these drawbacks,
tremendous efforts have been focused on discovering non-platinum
It is well recognized that the metabolic behavior of cancer cells
is different from that of normal cells and that mitochondria are the
organelle at the center of these metabolic pathways.7,8 The
mitochondria of cancerous cell undergo major changes, including
depolarization of the mitochondrial membrane potential and
increased generation of reactive oxygen species (ROS).9 Thus,
targeting mitochondrial metabolic pathways by metal complexes has
become a main focus in ongoing metallodrug research.10 In fact, a
handful of metal complexes that directly target the mitochondria are
in various phases of in vitro and in vivo investigation. These
complexes disrupt the mitochondrial membrane potential by
inducing apoptosis and cell death.11 Transition metal complexes
other than platinum offer an excellent opportunity to develop
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anticancer drugs with interesting coordination chemistry, variable Recently
rhodium
complexes
of
camphor-derived
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DOI: 10.1039/C9DT01951K
oxidation states, novel structural features, and useful chemical and bis(pyrazolylpyridine) and terpyridine ligand were synthesized,
physical characteristics.12 Among these non-platinum complexes, which showed a high affinity towards ct-DNA and BSA (bovine
rhodium(ІІІ) and iridium(ІІІ) complexes have captured the attention serum albumin). The molecular docking studies showed that all these
of the researchers, due to their unique chemical and biological complexes showed potential for DNA intercalation and affinity for
properties.13 They were found to have different modes of action and the minor groove of DNA. In vitro cytotoxicity showed that Rh(ІІІ)
cellular targets against cancer cells as compared to the traditional complex with a camphor-derived ligand having four methyl group is
platinum-based metallodrugs.14,15 However, there are still too few more effective and cytotoxic than having three methyl group and
Rh(ІІІ) complexes being studied.
terpyridine against HCT-116 cancer cell line23. Cyclometalated
The biological activity of metal complexes can be increased by iridium(ІІІ) complexes with diimine ligand exhibited high anticancer
combination with cytotoxic ligands and engagement with multiple activity against a panel of cancer cells including HepG2, A549,
cellular targets.16 Additionally, ligand substitution can further A549R, HeLa, PC3, HeLaρ0 and HLF with IC50 values higher than
improve the cytotoxicity of rhodium complexes. For example, the cisplatin. These complexes were accumulated in mitochondria. As a
trichlorido complex of Rh(ІІІ) bonded with
a 5,6-dimethyl result, it is affecting the function of mitochondria in term of ATP
substituted ligand of phenanthroline demonstrated 18-times higher depletion, metabolic disturbance and respiration inhibition which
anticancer activity than the non-substituted phenanthroline. This ultimately induced apoptotic cell death and mitophagy. Further
molecule induced toxicity by targeting the mitochondria and mechanistic studies revealed that these complexes are mainly
triggering apoptosis in Jurkat leukemia cells, along with an increase targeting the mtDNA and mitochondrial genome which may be the
in reactive oxygen species (ROS).17 Similarly, rhodium complexes target of metallodrugs to circumvent the resistance of cisplatin24.
that possess novel benzimidazole chelating ligands show excellent Half sandwich iridium(ІІІ)-NHC complexes exhibited better
anti-cancer activities against A2780, HT29, T47D, and A2780 CisR anticancer activity than cisplatin against A549 and HeLa cancer cell
cancer cell lines.18 Some Rh(ІІІ) complexes target plasmid DNA as line. Cytotoxic activity was correlated with the number of phenyl
an intercalator by binding to and disrupting regions close to the base groups attached to the central metal ion. The mechanistic studies of
pair mismatches.19 Rhodium butyrate complexes inhibit the these complexes showed that these complexes converted NADH to
synthesis of DNA by arresting the cell cycle at S phase.20 NHC- NAD+ by capturing the hydrogen from the molecule that producing
rhodium complexes trigger apoptosis via dysfunction of ROS which led to the decrease of mitochondrial membrane potential
mitochondrial membrane potential accompanied by oxidative and lysosomal damage and ultimately induced apoptotic cell death25.
damage of DNA and cell cycle arrest. In addition, NHC-rhodium Similarly, rhodium metalloinsertors complexes bind to DNA base
complexes show selective cellular targeting ability, thioredoxin pair mismatches and kill the cells which are deficient mismatch
reductase inhibition, and antimetastatic properties in mammalian repair than mismatch repair proficient counterpart at low
cells and E. coli.21 Similarly, a cyclometaleted rhodium complex concentrations.26 Iridium complexes with aroylthiourea ligands
exhibited cytotoxicity by inhibiting angiogenesis and essential signal proved to be not less than cisplatin in chemo-sensitivity against
transduction pathways.22
HCT-116 and ARPE-19 cancer cell lines.27
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Coordination of a biologically active ligand with the transition
metal core may form complexes with unique biological and physical
properties.28 Therefore, the selection of appropriate ligands to
modulate the cytotoxic properties of the complexes is crucial. In this
respect, isoquinoline is an excellent choice because of its various
biological and pharmacological properties, including anti-
inflammatory and anticancer activity.29 Many isoquinoline metal
Scheme 1 Synthesis of rhodium complexes. ChemicaVlierweaAgrteicnletOsn(lain)e.
DOI: 10.1039/C9DT01951K
SOCl2, benzene (24 h at room temperature), yield: 70-90% (b).
POCl3, CH3CN (12 h at 80°C) yield: 60-80% (c). SnCl2.2H2O, EtOAc
(12-24 h at 90°C) yield: 50-60% (d). La, Rh(DMSO)Cl3, MeOH,
acetonitrile(1:3), (72h at 80°C) yield: 7080% (e) Lb, Rh(DMSO)Cl3,
MeOH, acetonitrile(1:3), (72h at 80°C) yield: 8090%.
complexes demonstrate different cytotoxic mechanisms of action Synthesis and crystal structure of Rh1 and Rh2
and cellular targets compared to that of cisplatin.30,31
Two new rhodium(III) complexes were synthesized by mixing La or
In this paper, we report the synthesis and characterization of two Lb with Rh(DMSO)Cl3 in methanol and acetonitrile (1:3) in a sealed
new rhodium complexes with isoquinoline as a ligand. We also glass tube. The structures of these complexes were characterized by
1H, 13C-NMR, ESI-MS (Figures S1S6, supporting information),
studied their in vitro and in vivo anticancer activities, and propose a
possible anticancer mechanism.
Elemental analysis, and was further confirmed by single-crystal x-
ray diffraction analysis. Summary of the crystallographic data and
refinement details are available in Table S1. Selected bond lengths
and angles are listed in Table S2 (supporting information).
Results and discussion
Synthesis of Ligands
To prepare the ligands, 6,7-dimethoxy-1-(2-aminophenyl)-3,4-
dihydroisoquinoline (La) and 6-methoxy-1-(-2-aminophenyl)-3,4-
dihydroisoquinoline (Lb), a Bischler-Napieralski synthesis procedure
was followed as reported in the literature.32 The only difference in
the structure of the ligands is the number of methoxy groups
attached to the benzene ring of isoquinoline where La has methoxy
groups at C6 and C7, whereas Lb has only one at C6.
Structural features
In Rh1 and Rh2, one aminophenyl dihydroisoquinoline, one
dimethyl sulfoxide, and three chloride atoms are coordinated with
Rh(III) to give a six-coordinated distorted octahedral geometry
(Figures 1, 2). The coordination of N1 and N2 atoms of bidentate
aminophenyl dihydroisoquinoline ligand with Rh atom is responsible
for the formation of a six-membered ring (N1-N2/C1/C6/C7) in a
half-chair conformation. The RhN2 [Rh1N2 = 2.100 Å, Rh2N2
= 2.085 Å] and RhS1 [Rh1S1 = 2.28 Å, Rh2S1 = 2.265 Å] are
assembled perpendicular to each other, and three chloride atoms
[Rh1Cl1 = 2.33 Å, Rh1Cl2 = 2.35 Å, Rh1Cl3 = 2.34 Å,
Rh2Cl1 = 2.37 Å, Rh2Cl2 = 2.35 Å, Rh2Cl3 = 2.34 Å] and N1
[Rh1N1 = 2.066 Å, Rh2N1 = 2.089 Å] of the bidentate
aminophenyl dihydroisoquinoline ligands are located on the basal
positions of the square. Rings C1C6 and C8C13 of aminophenyl
dihydroisoquinoline are both planar, having the dihedral angles of
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57.4(3),
and
60.11(15)
between
them.
Ring
bonds in complexes Rh1 and Rh2. In Rh1, an N(1)H(1B)O (3)
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DOI: 10.1039/C9DT01951K
(N2/C7/C8/C13/C14/C15) exists in a boat conformation [(Q) =
0.490(6) Å, = 114.1(7) °,  = 82.9(7) °, (Q) = 0.505(3) Å,  =
66.4(3) °,  = 264.9(3) ° for Rh1, and Rh2, respectively]. In Rh1,
disorder at C16 was resolved using the 50% site occupancy factor
intra-molecular interaction was found to be the strongest one, having
a bond length of 2.33Å. Other interactions are summarized in Table
S3 (supporting information). Similar to Rh1, among all hydrogen
bonds in Rh2 involved in unit cell packing, N(1)H(1A)O(2) was
and application of the EADP constrain. Torsion angles of found to be the strongest one having a bond length of 2.36Å. Other
C15/N2/Rh/Cl2, Cl1/Rh/S/O, Cl2/Rh/S/O, Cl3/Rh/S/O in Rh1, and interactions are summarized in Table S4 (supporting information).
Rh2 were found to be 36.7°, 90.7°, 179.4°, 89.1°, 52.2°, 88.7°, C(18)H(18C)Cl(1) in Rh1, and C(16)H(16B)Cl(2) in Rh2 are
179.6°, and 89.5°, respectively. The larger dihedral angles are responsible for the chain elongation in a zig-zag manner along the b-
attributed to the steric hindrance around Rh(III).33
axis. NHCl intermolecular interactions are responsible for the S8
ring motif in both complexes. (Figures S7, S8, supporting
information).
Hirshfeld surface analysis
A two-dimensional Hirshfeld surface36 was generated for both
complexes, showing bright red spots that represent the location of
atoms with the potential to form the hydrogen bonds (Figure S9).
The brightness of these red spots indicates the potential strength of
the hydrogen bonds. Figure 3 shows the Hirshfeld surface interacting
Fig. 1 ORTEP view of the complex Rh1
with neighboring molecules. 2D fingerprint plots revealed the
contributions of HH at its maximum because the overall molecular
surface has its hydrogen atoms more exposed towards the
neighboring molecules. The ClH contact was found to be the
strongest one in both complexes as it was confirmed by the spikes
extending towards the bottom having 19.3%, and 20.6% in
complexes Rh1, and Rh2 respectively. Other important contacts are
CH, and OH having 14.2%, 13.5% for CH, 12.5%, and 11.6%,
Fig. 2 ORTEP view of the complex Rh2
respectively37 (Figures S10, S11, supporting information).
Supramolecular features
Intra- and intermolecular interactions exert significant influence on
the obtained structures, their physical properties34, and their
biological activities.35 Topological analysis of hydrogen bonding
reveals the presence of conventional and non-conventional hydrogen
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methoxy groups and their electron donating effects. TVhieiswhArytipcloetOhnelsinies
DOI: 10.1039/C9DT01951K
is supported by the analysis of the electrostatic potential surface38
mapped over the Hirshfeld surface, which reveals more potential
sites available in Rh1 than in Rh2 (Figure: S11, Supporting
information). It was evident from the IC50 values in Table 1 that Rh1
and Rh2 showed a significantly increased cytotoxicity as compared
with the corresponding ligand and rhodium(III) salt towards the T-24
cells. Against T-24, Rh1 and Rh2 exhibited higher cytoxicity than
cisplatin did. In the case of normal human liver cells (HL-7702), the
cytotoxicity of Rh1 are higher than Rh2, corresponding ligands and
rhodium(III) salts. Against BEL-7704, Rh1 was more toxic than
Rh2, corresponding ligands, rhodium(III) salt and cisplatin. Also, the
cytotoxic effect of Rh1 was better than Rh2, corresponding ligands
and rhodium(III) salt against HeLa cells, but it was worse than
cisplatin. In comparison with corresponding ligands and rhodium(III)
salt, rhodium complexes did not show significant cytotoxicity
towards MGC80-3. Against A-549 cell lines, Rh2 exhibited better
cytotoxicity than Rh1, corresponding ligands and rhodium(III) salt.
Although the rhodium(III) complexes displayed an improved
potency compared to ligands and rhodium(III) salt to most of the
tested cells, but to SK-OV-3 cell lines, it was exceptional. In case of
SK-OV-3, the rhodium(III) salt exhibited better cytotoxicity than
rhodium complexess and corresponding ligands. In a word, viewing
from the Table 1, it was found that T-24 was the best cell lines
exhibited anticancer activity in response to the rhodium complexes.
Fig. 3 Interaction with Hirshfeld surface for complexes Rh1 and Rh2
Stability of complexes in PBS solution
Under physiological conditions, the stability of Rh1 and Rh2 were
investigated in PBS (phosphate buffer saline) containing 1% DMSO,
pH 7.4 at room temperature for 0, 4, 12 and 24 h by UV-Vis
spectroscopy. Further stability and purity studies were carried out
and confirmed by HPLC. The spectral data showed that both Rh1
and Rh2 are stable at the given conditions in a DMSO stock solution
which was 2000 µM (Figures S12S15, supporting information).
Assessment of in vitro cytotoxicity by MTT
The in vitro cytotoxicity of rhodium(III) complexes and the
corresponding ligands were assessed against different tumor cell
lines including BEL-7704, MGC80-3, HeLa, A-549, T-24, SK-OV-
3, and HL-7702 (human normal liver cells) by the MTT assay.
Cisplatin and the corresponding rhodium(III) salt were taken as the
control. Except SK-OV-3, to the tested cell lines, synergistic effects
were observed upon coordination of the isoquinoline ligand to the
rhodium core. Except to A-549, the cytotoxic effect of Rh1 is higher
in comparison with Rh2, which may be due to the number of
Table 1. IC50 (μM) values of Rh1 and Rh2, on selected tumor cells for 48 h
Compound
La
HL-7702
T-24
BEL-7704
61.3±0.4
HeLa
MGC80-3
﹥100
A-549
SK-OV-3
46.3±0.5
20.04±0.7
95.03±0.3
﹥100
﹥100
Lb
95.0±0.3
56.02±0.5
75.02±0.5
95.04±0.4
86.04±0.5
74.01±0.5
41.4±0.3
Rh(DMSO)Cl3
Rh1
85.0±0.5
35.0±0.9
48.06±0.6
4.08±0.3
74.05±0.4
18.04±0.3
61.02±0.2
22.02±0.6
67.9±0.5
92.02±0.2
75.1±0.9
25.4±0.2
62.05±0.6
44.02±0.2
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Rh2
53.02±0.3
20.6±0.9
10.02±0.8
27.02±0.3
70.03±0.5
24.03±0.6
59.03±0.1
9.5±0.8
65.7±0.4
8.02±0.8
30.4±0.5
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DOI: 10.1039/C9DT01951K
cisplatin
19.9±0.5
22.05±0.3
Fig. 4 Cellular uptake and distribution of Rh1, Rh2 and cisplatin
contents in different parts of T-24 cell line measured by ICP-MS.
Control was provided with 1% DMSO. Rh1, Rh2 (10 μM) of
complexes were treated for 24 h at 37°C, respectively. Cisplatin (2
µM) was taken as a positive control. *P < 0.05, **p < 0.01 and ***P
< 0.001 determined by ANOVA.
Cellular uptake and distribution of rhodium in cancer cells
The cellular uptake can affect the biological activity of a drug.39 The
uptake and distribution of rhodium complexes in various parts of the
cell, including cytoskeletal, cytosolic, membrane/particulate, and
nuclear fractions were examined in T-24 cells treated with Rh1, Rh2
and cisplatin (treated control) for 24 h using the inductively coupled
plasma mass spectroscopy (Figure 4). The accumulation of Rh1 and
Rh2 was higher than untreated and treated control in the
membrane/particulate fraction (4377, 2745 ng), cytoskeletal fraction
(2084, 602 ng), and cytosolic fraction (1941, 1553 ng) respectively.
In addition, the concentration of Rh1 is almost double of the Rh2 in
the membrane fraction. In case of cisplatin (treated control) there has
been no significant accumulation of platinum in any part of cell. The
distribution of different concentrations of rhodium in different parts
of cells could be associated with the cytotoxicity and various cellular
and apoptotic pathways involving various mechanisms of action.40
Due to the uptake and accumulation of rhodium in T-24 cells, a
detailed investigation of Rh1 and Rh2 was conducted to explore the
possible mechanism of action of the complexes.
Cell cycle arrest
The cell cycle plays an important role in cell division and consists of
a sequential series of events within the cell. Cell cycle arrest is often
observed in treatments against cancer and other diseases and is
closely related to the induction of cell apoptosis and cell death.41,42
To explore the arresting effect of Rh1 (2.0 4.0, 8.0 μM), Rh2 (5.0,
10.0, 20.0 μM) and cisplatin (2 µM) on cell cycle progression, T-24
cells were exposed for 24 h and analyzed by flow cytometry (Figure
S16, supporting information). A dose-dependent increase in cell
population in S phase was observed in Rh1 treated cells (48.6%,
56.9% and 71.3%) versus the control (G1: 51.6%, G2: 10.1%, S:
38.3%). Although there is no significant effect of cisplatin (treated
control) on cell cycle arrest was observed. Whereas in the case of
Rh2 treated cells (22.7%, 32.5%, and 39.4%), an increase of cells in
G2 phase occurred as compared with the control (G1: 51.6%, G2:
10.1%, S: 38.3%). From the above result, it was concluded that Rh1
cause cell cycle arrest in S phase whereas Rh2 in G2 phase (Figure
5).
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cisplatin (treated control) had no significant effect on the induction
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DOI: 10.1039/C9DT01951K
of apoptosis. Therefore, both complexes can induce apoptosis in the
T-24 cell line significantly (Figure 6).
Fig. 5 Effect of rhodium complexes and cisplatin (treated control),
on the arrest of the cell cycle of T-24 cells. Mean population (%) of
T-24 cells in different phases of the cell cycle by 24 h treatment
compared with control. Presented data are the mean ± SD obtained
from three independent experiments. *P < 0.05, **p < 0.01 and
***P < 0.001 determined by ANOVA.
Fig. 6 Effect of rhodium complexes and cisplatin (treated control),
on the induction of apoptosis in T-24 cell. Mean population (%) of
cells in early and late apoptosis after the 24 h treatment with Rh1,
Rh2 and cisplatin compared with untreated control. Data were
presented as the mean ± SD acquired from three independent
experiments. *P < 0.05, **p < 0.01 and ***P < 0.001 determined by
ANOVA.
Induction of apoptosis
Apoptosis is a conserved process that regulates the growth of a
multicellular organism by eliminating unwanted and abnormal cells
through a controlled sequence of molecular events. In cancer cells,
the process of apoptosis is dysregulated so that it could be exploited
as an anti-cancer target.43-45 In order to determine the ability of Rh1
(2.0, 4.0, 8.0 μM), Rh2 (5.0, 10.0, 20.0 μM) and cisplatin (2 µM) to
induce apoptotic cell death, T-24 cells were treated with various
concentrations of rhodium complexes and treated control for 24 h,
stained with annexin V and propidium iodide, and analyzed by flow
cytometry (Figure S17, supporting information). The result showed a
dose-dependent increase in the population of early apoptotic cells
from 0.8% in control to 70.2% at the maximum concentration of
Rh1. Late apoptosis was observed in the case of Rh2 from 0.5% in
control to 41.4% at the highest concentration. In comparison,
Measurement of reactive oxygen species (ROS) generation
Reactive oxygen species cause oxidative damage to the cell and play
a crucial role in cellular biological function and signaling.46 The
mitochondrion is the primary source of production of ROS, and the
unnecessary production of ROS is considered a warning feature
leading to mitochondrial damage, genetic instability, and ultimately
apoptotic cell death.47 Therefore, ROS targeting is accepted as a
viable strategy for the treatment of cancer.48 ROS production upon
24 h treatment with Rh1 (4 μM), Rh2 (10 μM) and cisplatin (2 µM)
in T-24 was determined using flow cytometry. Cisplatin was taken
as positive control. To confirm whether the apoptosis induced by the
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complexes and cisplatin (treated control) related to the level of ROS, and analyzed by flow cytometry. As shown in FigureV8ie,wthAerticlelevOenllionef
DOI: 10.1039/C9DT01951K
a fluorescent marker was used (Figure 7). It was shown that a high intracellular calcium in the negative control was lower than in
level of ROS was generated by Rh1, whereas Rh2 considerably complex-treated cells at maximum concentrations of Rh1(4 μM) and
elevated the level of ROS and cisplatin have an insignificant effect. Rh2 (10 μM) and cisplatin (2 µM). It was noteworthy that the level
It is well known that the induction of cytotoxicity depends on the of calcium in Rh1 treated cells was higher as compared to those with
level of ROS generation.49,50 Therefore, it is suggested that ROS- Rh2
induced apoptosis was activated by exposure to the rhodium
complexes and thereby damaging the function of the mitochondria.
and
cisplatin
(treated
control).
Fig. 8 Flow cytometric investigation of Ca2+ in T-24 cells treated with
Rh1, Rh2 and cisplatin (treated control) as compared with
untreated control.
Fig. 7 Flow cytometric exploration of reactive oxygen species
produced by the treatment of Rh1, Rh2 and cisplatin (treated
control) compared with untreated control.
Assessment of changes in mitochondrial membrane potential
Mitochondria play a major role in the induction of apoptosis and are
regarded as a new target for antitumor drugs.53 The membrane
potential of the mitochondria is tightly-controlled and well-regulated
in the normal cells.54,55 Any damage to the membrane potential can
activate apoptotic pathways that ultimately lead to apoptotic death
by the release of pro-apoptotic factors.56 To evaluate apoptosis
caused by rhodium complexes and cisplatin, T-24 cells were treated
for 24 h and analyzed for depolarization of membrane potential by
flow cytometry using a fluorescent dye JC-1. As depicted from the
Figure S18 (supporting information), in both complexes, a dose-
dependent increase took place where the number of cells with
disrupted mitochondrial membrane increased from 1.4% in control
Fluctuation of intracellular Ca2+
For regular cellular activity, a tightly controlled regulation of
intracellular levels of calcium is essential and associated with cell
proliferation and apoptosis.51 It is considered that cancer cells
possess a remarkably high level of calcium either due to their
malfunctioning mitochondria, which can preserve high levels of
calcium or due to their intemperate influx of extracellular calcium.
This may also be linked to cell apoptosis due to mitochondrial
damage and utilized for determining the efficacy of anticancer
drugs.52 To examine the effect of Rh1, Rh2 and cisplatin (treated
control) on calcium homeostasis in T-24 cell, the cells were treated
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to 51.2% in Rh1 treated cells, and 36.1 % in Rh2 treated cells. compared with the control. Similarly, the expression levels of
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DOI: 10.1039/C9DT01951K
Whereas cisplatin has no significant effect compared to control. The activated caspase-8 were increased from 1.3% to 30.2% and 17.4%
results demonstrated that apoptosis induced by rhodium complexes respectively, and to 56.4% and 29.4% respectively for caspase-9,
was through a mitochondrial-mediated pathway, although other compared with the controls whereas the treatment of cisplatin
pathways may also be involved (Figure 9).
(treated control) has minimal effect but not significant, in the
activation of caspases. The activation of
caspases gave evidence that rhodium complexes especially Rh1 was
an efficient activator of caspases and induced cell apoptosis by
triggering caspase-3/8/9 in T-24 cells.
Fig. 9 Graph bar represented depolarization (%) of T-24 cells at a
different concentration of Rh1, Rh2 and cisplatin (treated control)
for 24h. Data are displayed as a mean ± SD of three independent
experiments. *P < 0.05, **p < 0.01 and ***P < 0.001 determined by
ANOVA.
Fig. 10 Determination of activated caspases; -3, -8, -9 in T-24 cells
treated with Rh1, Rh2 and treated control (cisplatin) as compared
with untreated control. The determination was performed in
triplicate, and one representative experiment was displayed.
Caspase 3/8/9 activation assay.
Caspases are the most critical apoptosis-related proteins, and they
play an essential role in the mitochondrial-mediated pathway. It is
well-considered that upon the disruption of mitochondrial
membrane, cytochrome C is released which consequently causes a
cascade of caspase activation, which includes caspase 3 and caspase
9.57,58 Flow cytometry was performed to measure the activation level
of caspase-3, -8, and -9 and to better understand the anticancer
mechanisms of Rh1 and Rh2 in the induction of apoptosis via the
mitochondrial-mediated pathway in T-24 cells. Figure 10 shows that
the levels of caspase-3 upon exposure to Rh1 (4 μM) and Rh2 (10
μM) increased from 1.4% to 61.3% and 32.3%, respectively,
Western blotting of apoptosis-related proteins
A direct relationship exists between Bcl-2 family proteins and
apoptosis. There are two classes: anti-apoptotic Bcl-2, Bcl-xl, and
proapoptotic Bax, Bak and Bim. In the generation of a death signal,
the expression of proteins from both classes is of high
importance.59,60 By Western blot, the expression level of these
proteins could be measured upon the treatment of T-24 cells with
Rh1 and Rh2. It is shown in Figure 11 that Bax, and Bak were
upregulated, and Bcl-2 and Bcl-xl were downregulated.
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cyclin A, cyclin B, and cyclin D decreased, and significantly
View Article Online
DOI: 10.1039/C9DT01951K
increased the level of chk1 and chk2. The increase in the level of
these proteins may be a result of cell cycle arrest at S-phase and G2
phase. Further examination of Cdks binding protein confirmed the
upregulation of proteins including p21, p27, and p53 upon treatment
with rhodium complexes. These proteins inhibit the activity of cdk2
and consequently cause cell cycle arrest at S and G2 phases. All of
these results are consistent with the flow cytometry analysis
regarding
the
cell
cycle
arrest.
Fig. 11 (A) Expression levels of apoptosis-related proteins of T-24
cells for 24 h treatment. (B) (C) Western blotting bands were
quantified with Image J (three independent experiments). *P < 0.05
and **P < 0.01 determined by ANOVA.
Western blotting of cell cycle related proteins
Cell cycle proteins, such as cdc25 A, cyclin A, cyclin D, cyclin B,
chk1, chk2, and cdks, are involved in controlling the cell cycle.61,62
Therefore, we evaluated the expression of these proteins upon the
treatment of T-24 cells with Rh1 and Rh2 for 24 h by Western
blotting. As shown in Figure 12, the expression level of cdc25 A,
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blotting band with Image J (three independent trials).V*iePw<Ar0tic.l0e5Onalninde
DOI: 10.1039/C9DT01951K
**P < 0.01 determined by ANOVA.
In vivo investigation of rhodium complexes in a tumor xenograft
model of T-24
To further investigate the in vivo anticancer activity of rhodium
complexes, Rh1 was used in a mouse model bearing T-24
xenografts. The mice were randomly divided into the negative
control group, Rh1 treated, and positive control groups. Two groups
of mice were treated intraperitoneally (IP) with Rh1 at a high dose of
(15.0 mg/kg) and a lower dose of (7.5 mg/kg) daily for 15 days after
the volume of tumor was 90110 mm3. The control group was given
5% DMSO in saline (V/V), whereas the cisplatin group was given
2mg/kg of cisplatin and was used as the reference. A dose-dependent
tumor growth inhibition was observed as shown in Figure 13. After
15 days of treatment, the mice were sacrificed, and the rate of tumor
growth inhibition rate (IR%) was determined as 54.5% for Rh1 (15.0
mg/kg) at a high dose treatment, and 37.6% for Rh1 at a low dose.
The tumor weight inhibition rate (IR%) of cisplatin was 54.5%,
which was comparable with that of the high dose treatment with Rh1
(Figure 13 A, B). No apparent adverse effects were observed during
the days of treatment in the Rh1-treated group, which exhibited
satisfactory results without substantial losses in body weight,
whereas the cisplatin-treated group demonstrated some weight loss.
These findings suggested that the safety profile of Rh1 is higher than
that of cisplatin, and Rh1 could inhibit the growth of T-24 tumor in
vivo comparably to cisplatin.
Fig. 12 (A) Expression levels of cell cycle-related protein in T-24 cell
treated with Rh1 and Rh2. (B) Quantification of the Western
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(1.25200µM) to carried out different biological assays and
View Article Online
DOI: 10.1039/C9DT01951K
spectroscopic experiments. The concentration of DMSO in stock
solution was 100%, and the concentration of DMSO was 0.5% in the
working solution. In the pharmacological studies, all compounds of
high purity were used, which were more than 99%. All the assays
kits including MTT, cell cycle evaluation (RNAse, PI), apoptosis
detection (annexin V, PI), mitochondrial membrane potential
detection (JC-1), activated caspases determination, ROS detection,
and Ca2+ were obtained from Bio-Vision and BD Biosciences. All
antibodies were purchased from Abcam (USA). All cell lines were
provided by the Shanghai Institute of Biological Sciences. The
xenograft mouse model of T-24 was purchased from Beijing
Bioscience Co. Ltd. (Beijing, China)
Fig. 13 The in vivo antitumor activity of Rh1 in mice having T-24
tumor xenograft. (A) Effect on the growth of tumor by Rh1(7.5, 15.0
mg/kg), cisplatin (2 mg/kg) or control (5% DMSO in saline v/v) in
tumor xenograft model. The tumor growth was measured by the
mean tumor volume (mm3) ±SD and calculated as the relative
tumor increment rate (TC%). (B) The weight of the tumor was
determined comparatively with control after the mice were
sacrificed. *P < 0.05, *p < 0.01 and ***P < 0.001, p vs vehicle
control, determined by ANOVA. (C) Changes in body weight were
recorded and exhibited in % values from the initial weight. (D)
Photograph of the tumor-containing control group, rhodium-
treated and cisplatin-treated.
Instruments
A Bruker HCT mass spectrometer was used for acquiring the ESI-
MS spectra. Bruker AVANCE NEO-500 NMR spectrometer was
used for recording of NMR spectra. Bruker Smart APEX II was used
to collect the single-crystal X-ray data. TU-1901 ultraviolet
spectrometer was used to obtain the absorption spectra. HPLC
analysis was performed on an Elite P230ІІ (Dalian, China). MTT
assays were carried out on microplate reader M1000 of Tecan
Trading Co. Ltd., Shanghai, China. Analysis of cell cycle was
achieved by using a FACS Aria ІІ flow cytometer (BD Biosciences,
San Jose, USA). For confirmation and detection of protein
expression, Western blot assays were run on an ECL Western
blotting system.
Experimental
Synthesis and characterization of La, Lb, Rh1, and Rh2
Materials
Synthesis of Ligands
La and Lb were synthesized according to procedures reported by
Nussbaum and co-workers.63
All the reagents were purchased from Xilong Chemical Co., Ltd,
Alfa Aesar, and Sigma Aldrich and used without further purification.
DMSO was used for the dissolution of Rh1 and Rh2, to prepare a
stock solution of 2000µM. Furthermore, PBS (phosphate buffer
saline) was used to prepare different working solution in the range of
Synthesis of Rh1 and Rh2
A mixture of La, Lb (0.025 mmol) and Rh (DMSO)Cl3 (0.05 mmol,
0.010 g) in acetonitrile (0.15 mL) and methanol (0.45 mL) was
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placed in a thick Pyrex glass tube of 25 cm length and frozen in ORTEP366 was used to draw the 3D view of the molecule, and
View Article Online
DOI: 10.1039/C9DT01951K
liquid nitrogen, exposed to vacuum, and sealed by fire assembly. MERCURY67 was used for visualizing the interactions and crystal
Dark orange block crystals were harvested after three days of heating packing. Table S1 shows the crystallographic data of Rh1 and Rh2
at a constant temperature of 80 °C, appropriate for X-ray analysis.
and S2 show selected bond length and angles.
Hirshfeld surface analysis
H, 13C-NMR data of Rh1
Hirshfeld surface analysis and its properties were determined using
standard parameters. A 2D Hirshfeld surface dnorm was generated on
a surface scale of 0.3271.475 Å for Rh1, and 0.2651.295 Å for
Rh2 by utilizing the Crystal Explorer Software package.68 2D
fingerprint plot has been generated to visualize the percentage
contribution of contacts towards the overall crystal packing.
Electrostatic potential surface was generated by the HF method
utilizing the TONTO package incorporated in Crystal explorer.
Cell culture and other experimental methods
1H NMR (600 MHz, (CD3)2SO) δ 7.41(td, J = 1.5, 8.0 Hz, 1H), 7.29
(d, J = 7.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 2H), 7.14 (s, 1H), 6.74 (s,
1H), 3.92 (s, 3H), 3.63 (s, 3H), 3.42(s, 6H), 2.88 (t, J = 7.0 Hz, 2
H), 2.53 (m, 2 H). 13C NMR (150 MHz, (CD3)2SO) δ 169.8, 152.8,
146.6, 142.1, 134.3, 133.3, 131.7, 131.5, 125.2, 123.6, 121.6, 114.2,
110.6, 56.3, 56.1, 51.8, 41.1, 40.8, 26.6. H-RMS(EI): Calcd for
C19H24Cl3N2O3RhS, m/z 567.9628, found m/z 568.5673 for [M+H]+.
Elemental analysis: calcd (%) for C19H24Cl3N2O3RhS: C, 40.05; H,
4.25; N, 4.92; S, 5.63; found: C, 40.21; H, 3.57; N, 4.80; S, 5.62.
1H, 13C-NMR data of Rh2
T-24, HeLa, MGC80-3, A-549, HL-7702, SK-OV-3, and BEL-7704
cells were purchased from Shanghai Cell Bank of Chinese Academy
of Sciences. Cell culture was carried out in Dulbecco’s Modified
Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). All
assays, including MTT, cell cycle analysis, detection of apoptosis, in
vivo xenograft animal model, Western blotting was performed
according to procedures reported by Wei et al.69 Flow cytometric
1H NMR (600 MHz, (CD3)2SO) δ 7.42 (m, 1H), 7.25-7.21
(overlapped, 4H), 7.07 (m, 1H), 7.00 (dd, J = 3.0, 11.0 Hz, 1H), 3.89
(s, 3H), 3.42 (s, 6H), 2.94 (t, J = 8.5 Hz, 2 H), 2.55 (m, 2 H). 13C
NMR (150 MHz, (CD3)2SO) δ 169.9, 162.8, 142.4, 142.1, 133.2,
133.0, 131.8, 131.4, 125.2, 123.6, 122.6, 112.7, 112.5, 56.1, 51.6,
41.1, 40.8, 27.4. H-RMS(EI): Calcd for C18H22Cl3N2O2RhS, m/z
537.9523, found m/z 538.9420 for [M+H]+. Elemental analysis:
calcd (%) for C18H22Cl3N2O2RhS: C, 40.06; H, 4.11; N, 5.19; S,
5.94; found: C, 40.5; H, 3.01; N, 5.22; S, 5.99.
analysis of
activated caspases, intracellular calcium ion and
measurement of reactive oxygen species were performed according
to methods reported by Qin et al.70 The membrane potential of
mitochondria was detected according to methods reported by Hu et
al.71 The in vivo experimental procedure for mice was approved by
the 181st Chinese People’s Liberation Army Hospital (Guilin, China)
and was conducted according to the guidelines provided by NIH for
care and use of laboratory animals in research.72 All experimental
X-ray crystallography
Crystals structures of Rh1 and Rh2 were collected on a Bruker
SMART Apex ІІ CCD diffractometer equipped with a graphite
monochromated Mo-Kα (λ=0.710 73 Å) at room temperature.
Structures were solved using the direct method, Fourier
transformations, and SHELXL97 program.64 All non-hydrogen
atoms were refined using anisotropic thermal parameters.
PLATON65 was utilized for various crystal parameters calculations,
details are shown in supplementary materials.
Conclusions
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Supporting Information: Vendor codes for Rh1 and Rh2 crysVtaielwdaAtrati,clHe POLnlCine
Two rhodium(ΙΙΙ) complexes Rh1 and Rh2 with isoquinoline
derivatives as ligands were synthesized and characterized by various
physical methods. Rh1 and Rh2 demonstrated considerable in vitro
anticancer activity against a variety of tumor cells and promising
selectivity towards T-24 cell lines relative to cisplatin. To most of
the tested cells, both complexes presented the synergistic effect in
cytotoxicity as compared with the rhodium(III) salt and free ligands.
Rh1 and Rh2 generated their anticancer activity by arresting the cell
cycle at S-phase and inducing apoptosis via mitochondrial
dysfunction. Rh1 exhibited a more favourable in vivo safety profile
than cisplatin, and the tumor inhibition ratios were comparable. Rh1
is a potential leading compound for further development as an
DOI: 10.1039/C9DT01951K
data, 1H-NMR, 13C-NMR data, ESI-MS data. The supplementary
crystallographic data of this paper can be seen under the CCDC No.
1851232,1851233.
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