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Targeting cancer cell metabolism with mitochondria-immobilized phosphorescent cyclometalated iridium(iii) complexes.
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Cite this: Chem. Sci., 2017, 8, 631
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Targeting cancer cell metabolism with
mitochondria-immobilized phosphorescent
cyclometalated iridium(III) complexes†
Jian-Jun Cao,a Cai-Ping Tan,*a Mu-He Chen,a Na Wu,a De-Yang Yao,b Xing-Guo Liu,b
Liang-Nian Jia and Zong-Wan Mao*a
Cancer cell metabolism is reprogrammed to sustain the high metabolic demands of cell proliferation.
Recently, emerging studies have shown that mitochondrial metabolism is a potential target for cancer
therapy. Herein, four mitochondria-targeted phosphorescent cyclometalated iridium(III) complexes have
been designed and synthesized. Complexes 2 and 4, containing reactive chloromethyl groups for
mitochondrial fixation, show much higher cytotoxicity than complexes 1 and 3 without mitochondriaimmobilization properties against the cancer cells screened. Further studies show that complexes 2 and
4 induce caspase-dependent apoptosis through mitochondrial damage, cellular ATP depletion,
mitochondrial respiration inhibition and reactive oxygen species (ROS) elevation. The phosphorescence
of complexes 2 and 4 can be utilized to monitor the perinuclear clustering of mitochondria in real time,
which provides a reliable and convenient method for in situ monitoring of the therapeutic effect and
Received 1st July 2016
Accepted 22nd August 2016
gives hints for the investigation of anticancer mechanisms. Genome-wide transcriptional analysis shows
that complex 2 exerts its anticancer activity through metabolism repression and multiple cell death
signalling pathways. Our work provides a strategy for the construction of highly effective anticancer
DOI: 10.1039/c6sc02901a
agents targeting mitochondrial metabolism through rational modification of phosphorescent iridium
www.rsc.org/chemicalscience
complexes.
Introduction
The success of platinum-based drugs in the past few decades
has stimulated great interest in the search for other metal-based
anticancer agents.1 Non-platinum anticancer drugs act through
multiple mechanisms, different from those of platinum
drugs.2,3 These drugs are expected to have the capability to
overcome platinum resistance and reduce side effects.4,5 Very
recently, organometallic iridium complexes have emerged as
potential candidates for new metallo-anticancer drugs.6 As
demonstrated by Sadler and Meggers et al., organometallic
iridium complexes can exert their anticancer activity through
multiple mechanisms, which include catalyzing cellular redox
reactions7,8 and inhibiting enzyme activities.9,10 Additionally,
a
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry
and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, P. R. China.
E-mail: tancaip@mail.sysu.edu.cn; cesmzw@mail.sysu.edu.cn
b
Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of
Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology
and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, Guangzhou, People's Republic of China
† Electronic supplementary information (ESI) available: Experimental procedures,
gures and tables, references and X-ray crystallographic data. CCDC
1452036–1452038. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c6sc02901a
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cyclometalated iridium(III) complexes have rich photophysical
properties, e.g., high quantum yields, large Stokes shis, longlived luminescence, good photostability and cell permeability.
They have attracted increasing attention in bioimaging and
biosensing applications.11–15 Our group has endeavoured to
combine the unique photophysical and anticancer properties of
phosphorescent cyclometalated Ir(III) complexes to construct
novel multifunctional theranostic platforms that can induce
and monitor the therapeutic response simultaneously.16–19
The central metabolic pathways operating in malignant cells
are different from those in normal cells.20,21 The alteration in
cancer cell metabolism is important for oncogene revolution,
tumorigenesis and tumour cell proliferation.22–24 Compared
with their normal counterparts, tumour cells are characterized
by a metabolic phenotype with a shi from ATP generation
through oxidative phosphorylation to ATP generation through
glycolysis even under normal oxygen concentrations.20 As
mitochondria have well-recognized roles in the production of
ATP and the intermediates needed for macromolecule biosynthesis, targeting mitochondria metabolism has emerged as
a very effective strategy to kill cancer cells selectively.25
Mitochondria also play a vital role in a variety of cellular
processes, such as cell death regulation, calcium modulation
and redox signalling.26,27 Cancer cells exhibit various degrees of
alterations to mitochondrial function, e.g., a higher
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mitochondrial membrane potential (MMP) and increased
oxidative stress, which provides opportunities to target cancer
cell mitochondria for an optimal therapeutic outcome.28
Moreover, there has been growing interest in developing
emissive mitochondria-targeted multifunctional theranostic
agents that can monitor changes in the mitochondrial physiological status during the therapeutic process.17,29,30 However,
reports elucidating the consequence of targeting anticancer
metal complexes to mitochondria are limited.31,32
In this work, four cyclometalated iridium(III) complexes,
[Ir(N–C)2(N–N)](PF6) (N–N ¼ (2,20 -bipyridine)-4,40 -diyldimethanol (L1) or 4,40 -bis(chloromethyl)-2,20 -bipyridine (L2); N–C ¼ 2phenylpyridine (ppy) or 2-(2,4-diuorophenyl)pyridine (dfppy)),
were designed and synthesized (Scheme 1). Due to their positive
charge and lipophilicity, 1–4 were anticipated to accumulate in
mitochondria. The reactive chloromethyl subunits were expected to immobilize 2 and 4 within mitochondria as the result
of nucleophilic substitution with reactive thiols present in
various mitochondrial proteins.33,34 Complexes 1 and 3 incorporating non-reactive hydroxymethyl groups were used as
controls. The in vitro antiproliferative activities of 1–4 were
investigated against several cancer cell lines as well as a human
normal cell line. The anticancer properties of the mitochondriaimmobilized complexes 2 and 4, which included mitochondrial
damage, cellular ATP depletion, inhibition of mitochondrial
respiration, reactive oxygen species (ROS) elevation and induction of apoptosis, were explored using a variety of methods.
Time-dependent tracking of the mitochondrial morphology was
carried out for 2- and 4-treated cells. Additionally, the possible
anticancer mechanisms of complex 2 were elucidated by analysis of genome-wide gene expression proles.
Results and discussion
Synthesis, characterization and stability
Complexes 1–4 were obtained by reuxing two equivalents of
ligands and the corresponding cyclometalated Ir(III) dimers in
CH2Cl2/CH3OH (1 : 1, v/v) followed by anion exchange with
NH4PF6 and purication by column chromatography on silica
gel. Pure products of 1–4 obtained in high yields by recrystallization were characterized by 1H NMR spectroscopy (Fig. S1–
S4†), ESI-MS and elemental analysis (ESI†). Complexes 1, 3 and
4 were characterized by X-ray crystallography (Fig. 1, Tables S1
and S2†). The Ir atoms have a distorted octahedral coordination
Scheme 1
Chemical structures of complexes 1–4.
632 | Chem. Sci., 2017, 8, 631–640
X-ray crystal structures and atom-numbering schemes for
complexes 1, 3 and 4 at a 30% thermal ellipsoids probability level. The
hydrogen atoms, counter ions and solvents are omitted for clarity.
Fig. 1
geometry and the largest deviation is represented by the bite
angle (from 76.4 to 76.9 ) of the bipyridine ligand. The two Ir–C
bonds are in a mutual cis arrangement, and their high trans
inuence renders slightly shorter Ir–N bond lengths in the C–N
ligands than those in the cyclometalating N–N ligands. These
ndings are commonly observed for related cyclometalated
Ir(III) complexes.18,35
The absorption spectra of complexes 1–4 in phosphate buffer
saline (PBS), CH3CN and CH2Cl2 are characterized by multiple
bands (Fig. 2A and S5†). The high-energy bands (<350 nm) are
assigned to spin-allowed ligand-centered (1LC) p–p* transitions
for cyclometalated (C–N) and ancillary (N–N) ligands. The
relatively low-energy bands can be assigned to the mixed singlet
and triplet metal-to-ligand charge-transfer (1MLCT and 3MLCT)
and ligand-to-ligand charge-transfer (LLCT) transitions.11,18,36
Upon excitation at 405 nm, complexes 1–4 exhibit long-lived
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and cisplatin against all the human cancer cell lines tested
(Table 1). Complexes 2 and 4 are highly cytotoxic against
cisplatin-resistant A549R cells, indicating they are not crossresistant with cisplatin.
Notably, a relatively high selectivity for cancer cells is
observed for complex 2. For example, it shows approximately an
11 fold higher selectivity for cancerous A549 cells over
noncancerous LO2 cells. A co-culture model of LO2 and A549
cells was used to further demonstrate the capability of complex
2 to selectively kill cancer cells (Fig. S8†). The nuclei of A549
cells were prelabeled with Hoechst 33 342 (20 -(4-ethoxyphenyl)5-(4-methyl-1-piperazinyl)-2,50 -bi-1H-benzimidazole trihydrochloride). Aer the cocultured cells are treated with 2, most of
the A549 cells (blue nuclei) are stained positively by both
annexin V and PI, while the LO2 cells are still viable. The
hyperchromatic nuclei of the A549 cells indicate that 2 mainly
induces apoptotic cell death.
Lipophilicity, cellular uptake and localization
Fig. 2 (A) UV/Vis spectra of complexes 1–4 measured in CH3CN at
25 C. (B) Emission spectra of complexes 1–4 measured in CH3CN at
25 C. The excitation wavelength is 405 nm.
green to red phosphorescence (Fig. 2B, S6 and Table S3†). The
emission lifetimes of 1–4 in PBS, CH3CN and CH2Cl2 fall in the
range between 32 and 327 ns, indicating the phosphorescent
nature of the emissions. The emission lifetimes and quantum
yields of 1–4 are sensitive to solvent polarity. Generally, the
emission quantum yields and lifetimes of 1–4 increase upon
decreasing the solvent polarity, which is also observed for other
related phosphorescent Ir(III) complexes.37
As the reactive chloromethyl groups may undergo hydrolysis
in aqueous solutions, we chose complex 2 to evaluate its
stability in a DMSO-d6 and D2O mixture (v/v, 7/3) at 37 C. The
results show that about 98.5% of complex 2 is invariant aer
48 h incubation at 37 C, as veried by 1H NMR spectroscopy
(Fig. S7†). Aer 7 days, about 26.2% of complex 2 is transformed, which may be attributed to the hydrolysis of the
chloromethyl groups.
In vitro cytotoxicity and selective killing of cancer cells
The in vitro cytotoxicity of complexes 1–4 and cisplatin was
determined against human cervical carcinoma (HeLa), human
lung carcinoma (A549), cisplatin-resistant A549 (A549R), human
breast cancer (MDB-MA-231), human prostate carcinoma (PC3)
and human normal liver (LO2) cells by a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay aer 48 h
treatment. Complexes 2 and 4, with IC50 values ranging from 0.2
to 1.8 mM, show much higher cytotoxicity than complexes 1, 3
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It has been reported that the cellular uptake behaviour of metal
complexes is usually related to many factors, e.g., lipophilicity,
molecular size and substitute groups.18,38,39 The lipophilicity is
referred to as log Po/w (where Po/w ¼ the octanol/water partition
coefficient). The log Po/w values, determined by a classical
shake-ask method, for 1, 2, 3 and 4 are 0.23, 1.28, 1.09 and
2.12, respectively.
As iridium is an exogenous element, the cellular uptake
levels of Ir(III) can be quantitatively determined by inductively
coupled plasma-mass spectrometry (ICP-MS). The cellular
uptake efficacy is inuenced by the lipophilicity and the
substitution groups on the N–N ligand (Table S4†). It should be
noted that complexes 2 and 4 with the chloromethyl substituents show a much higher cellular uptake efficacy than 1 and 3
containing the hydroxymethyl groups.
The localization of 1–4 in A549 cells was investigated by laser
scanning confocal microscopy. All the complexes can be visualized in the A549 cells aer 1 h incubation (Fig. S9†). The
phosphorescence of 1–4 shows distinct lamentous and punctate patterns. Colocalization experiments of 1–4 with the mitochondrion-specic uorescent probe MitoTracker Deep Red
(MTDR) show that 1–4 can specically localize to mitochondria
(Fig. 3). The Pearson's colocalization coefficients obtained for 1–
4 with MTDR are 0.74, 0.84, 0.87 and 0.82, respectively. Similar
results are also observed for 2 and 4 by high resolution confocal
scanning laser microscopy (Fig. S10†). However, negligible
colocalization of 1–4 with LysoTracker Deep Red (LTDR) can be
detected (Fig. S11†). To further verify the distribution of 1–4 in
different cellular compartments, the mitochondrial, cytosolic
and nuclear fractions were isolated from A549 cells treated with
complexes 1–4 (Fig. S12†). As measured by ICP-MS, the content
of iridium in the mitochondria is much higher than that obtained in the cytosol and nuclei. These results collectively
indicate that complexes 1–4 can specically target mitochondria in A549 cells.
We further investigated the cellular uptake mechanisms of
1–4. Incubation of A549 cells with 1–4 at a lower temperature
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Table 1
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Cytotoxicity of the tested compounds against different cell lines
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IC50a (mM)
Compounds
HeLa
A549
A549R
MDB-MA-231
PC3
LO2
1
2
3
4
Cisplatin
10.0 0.9
0.52 0.04
2.7 0.2
1.8 0.1
18.8 1.4
24.0 2.0
0.40 0.02
16.8 1.2
0.21 0.02
22.4 2.0
42.2 2.7
0.64 0.04
17.8 1.4
0.74 0.05
120.2 6.5
24.5 1.9
0.33 0.02
12.3 1.1
0.66 0.05
27.5 2.5
>100
1.4 0.1
38.0 1.9
1.04 0.09
23.8 2.0
>100
4.5 0.3
8.1 0.5
1.5 0.1
26.9 1.9
a
Cells were incubated with the indicated compounds for 48 h. Data are presented as the means standard deviations (SD), and cell viability was
assessed aer 48 h of incubation.
mitochondrial dye, is easily washed out once the MMP is lost.41
As expected, the emission of Rhodamine 123, and complexes 1
and 3 is barely detectable in cells under the same conditions.
The higher cytotoxicity of 2 and 4 compared with 1 and 3 is
correlated with their higher uptake efficacy, which may be at
least partially attributed to their immobilization and prolonged
retention time in mitochondria.
The ability of 1–4 to undergo covalent conjugation to intracellular proteins was also conrmed by gel electrophoresis,
which separates the proteins puried under denaturing conditions from lysed A549 cells treated with Ir(III) (Fig. S17†).
Distinct emissive protein bands can be observed in the gel-
Fig. 3 Determination of colocalization of 1–4 with MTDR by confocal
microscopy. A549 cells were incubated with 1–4 (10 mM, 1 h), and then
stained with MTDR (100 nM, 30 min) at 37 C. 1 and 2: lex ¼ 405 nm;
lem ¼ 630 20 nm. 3 and 4: lex ¼ 405 nm; lem ¼ 560 20 nm. MTDR:
lex ¼ 633 nm, lem ¼ 655 20 nm. Scale bar: 10 mm.
(4 C) results in a reduced cellular uptake efficiency as revealed
by confocal microscopy (Fig. S13–16†). Pretreatment of the cells
with metabolic inhibitors, 2-deoxy-D-glucose and oligomycin,
can lower the cellular uptake levels of these complexes, while
the endocytosis modulator chloroquine shows no effect on the
ability of complexes to cross the plasma membrane. The results
suggest that complexes 1–4 penetrate the cell membrane mainly
through an energy-dependent mechanism and do not rely on
the endocytic pathways.40
Mitochondrial immobilization
As complexes 2 and 4 contain chloromethyl groups, they are
supposed to react with thiol groups of cysteine residues in
proteins and peptides in cells to form stable covalent bonds.34
MitoTracker™ probes, commercial thiol reactive mitochondrial
dyes containing chloromethyl groups, can be retained during
cell xation.33 Similar to that observed for MTDR, the emission
of complexes 2 and 4 is retained in cells aer the cells are xed
and washed (Fig. 4). Rhodamine 123, a conventional
634 | Chem. Sci., 2017, 8, 631–640
Sensitivity of emission intensity of Rhodamine 123 (Rh123),
MTDR and Ir(III) to fixation and washing. A549 cells were incubated
with 1–4 (10 mM) at 37 C for 1 h. The cells were fixed by paraformaldehyde and washed twice with PBS/DMSO (9/1, v/v). The cells
were imaged by confocal microscopy after each step. 1 and 2: lex ¼
405 nm; lem ¼ 630 20 nm. 3 and 4: lex ¼ 405 nm; lem ¼ 560 20
nm. Scale bar: 10 mm.
Fig. 4
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separated denatured protein mixtures isolated from 2- and 4treated cells. Meanwhile, no emissive bands are detected in
proteins isolated from 1- and 3-treated cells. Similar results are
observed in vitro using bovine serum albumin (BSA) as a model
protein that contains one free cysteine residue (Fig. S18†). The
interactions of complexes 1–4 with BSA have also been investigated using tryptophan uorescence quenching experiments
(Fig. S19†). The Stern–Volmer constants (KSV) determined for 1,
2, 3 and 4 are 7.0 104, 1.1 105, 7.2 104 and 8.5 104 M1,
respectively. Accordingly, an increase in the emission intensities is observed for 1–4 upon binding with BSA (Fig. S20†).
Complex 2 displays approximately an 11.6-fold emission
enhancement when the molar ratio of BSA and Ir(III) reaches
8 : 1. The enhancement in emission intensities can be attributed to the hydrophobic environment in the binding pockets of
proteins, which is favourable for their imaging applications.37
These results indicate that complexes 2 and 4 can be immobilized on mitochondria by covalent interactions with proteins.
Induction of mitochondrial dysfunction
As complexes 1–4 could be localized to mitochondria, their
impact on mitochondrial integrity was monitored by detecting
the changes in MMP (DJm). The red/green uorescence of
5,50 ,6,60 -tetrachloro-1,10 -3,30 -tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), a mitochondria-selective aggregate dye, was
detected by ow cytometry (Fig. 5A) and confocal microscopy
(Fig. S21†).42 At low membrane potentials, JC-1 exists in the
form of the “J-monomer” with green uorescence. At high
membrane potentials, JC-1 forms “J-aggregates” and displays
red uorescence. The control cells show red uorescence, which
indicates that the mitochondrial membranes retain a high
voltage. The capability of these complexes to depolarize mitochondria is correlated with their cytotoxicity. In cells treated
with 1 and 3, a small portion of cells lose their DJm. Complexes
2 and 4 cause a marked decrease in MMP, as evidenced by the
uorescence shi from red to green. Aer a 6 h treatment, the
percentage of cells with mitochondrial membrane depolarization increases from 6.6 0.7% to 85.7 1.7% and 82.4 7.6%
for 2 (10 mM, 6 h) and 4 (10 mM, 6 h), respectively.
To further investigate the effects of complexes 1–4 on the
mitochondrial metabolic status, we measured their impact on the
intracellular ATP level and mitochondrial respiration. The capability of 1–4 to reduce the ATP content in A549 cells is correlated
with their cytotoxicity (Fig. 5B). The impact of complexes 1 and 3
on intracellular ATP levels is not obvious. Meanwhile, complexes
2 and 4 cause a signicant dose-dependent decrease in ATP
production as compared with the control cells. At a concentration
of 10 mM, the ATP levels decrease from 86.3 1.0 to 30.2 2.4
and 20.2 3.3 nM for 2 and 4, respectively.
Complexes 2 and 4 were chosen as model compounds to
further investigate their impact on the mitochondrial bioenergetic status. Mitochondrial respiration was quantied by
measuring the oxygen consumption rate (OCR) directly using
a Seahorse XF24 Extracellular Flux Analyzer.43 Several key
parameters were measured to assess mitochondrial oxidative
phosphorylation (OXPHOS) by using modulators of respiration
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that target components of the electron transport chain (ETC)
(Fig. 5C and D). Cells treated with 2 and 4 display a decrease in
basal OCR (Fig. 5E). The mitochondrial respiration comprises
coupled respiration for ATP synthesis and uncoupled respiration
to drive the futile cycle of proton pumping and proton leak back
across the inner mitochondrial membrane.44 The coupled respiration and proton leak were determined using the ATP synthase
inhibitor oligomycin. Cells treated with 2 and 4 show a dosedependent decrease in ATP production as compared with the
control cells (Fig. 5F). Proton leak is increased at a lower
concentration (0.25 mM) with a decline observed at higher
concentrations (1 mM) (Fig. 5G). Aer injection of the carbonyl
cyanide 4-(triuoromethoxy)phenylhydrazone (FCCP), a potent
mitochondrial uncoupling agent that can dissipate the proton
gradient and eliminate the control of respiration by ATP synthase, a decrease in the OCR peak of Ir(III)-treated cells is observed
as compared with the resilient control cells. The observation
suggests that the Ir(III)-treated cells have lost their spare respiratory capacity.45 Then, a mixture of antimycin A (a mitochondrial
complex III inhibitor) and rotenone (a mitochondrial complex I
inhibitor), which can shut down the mitochondrial respiratory
chain thoroughly, was injected to determine the fraction of nonmitochondrial O2 consumption including substrate oxidation
and cell surface oxygen consumption. A decrease in non-mitochondrial respiration is detected in cells treated with 2 and 4
(Fig. 5H). These results collectively indicate that the inhibition of
both mitochondrial and non-mitochondrial respiration contributes to the cytotoxicity of 2 and 4.
Real-time tracking of mitochondrial morphology
Mitochondria are highly dynamic organelles that undergo
constant fusion and ssion. Mitochondria are actively transported in cells, which is essential for maintaining physiological
functions of cells.46 However, the molecular mechanisms regulating these behaviours have not been well understood yet. Thus,
tracking the changes in mitochondrial morphology may give
insight into the investigation of cell death mechanisms.47 Realtime tracking of the mitochondrial morphology was carried out
by monitoring the emission of 2 and 4 in A549 cells using
confocal microscopy (Fig. 6A and B). A549 cells can be effectively
labelled by 2 and 4 aer 0.5 h incubation, when mitochondria
show the normal tubular network and distribution. Prolonged
incubation causes a collapse of the normal tubular mitochondrial
network into mitochondrial aggregates and large perinuclear
clusters. Mitochondrial swelling, fragmentation and perinuclear
clustering in cells treated with 2 and 4 are also conrmed by
transmission electron microscopy (TEM, Fig. 6C). It can be seen
that in control cells, mitochondria are distributed evenly in the
cytoplasm. In Ir(III)-treated cells, the fragmented mitochondria
tend to localize around the nucleus.
Intracellular ROS detection
Apart from producing energy, mitochondria are also a major
source of ROS. Mitochondrial damage and intracellular ROS
production are closely related.27 Intracellular ROS elevation
induced by complexes 1–4 was detected by ow cytometry and
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Induction of mitochondrial dysfunction by complexes 1–4. (A) Effects of 1–4 on MMP analyzed by flow cytometry at the indicated
concentrations. A549 cells were treated for 6 h then stained with JC-1. lex ¼ 488 nm. lem ¼ 530 30 nm (green) and 585 30 nm (red). (B)
Intracellular ATP levels in A549 cells. The cells were treated with the vehicle and 1–4 at the indicated concentrations for 6 h. (C and D) Respiratory
profiles of control and 2- and 4-treated A549 cells under basal conditions, and after the addition of oligomycin (0.75 mM), FCCP (0.6 mM) and the
mixture of rotenone (0.5 mM) and antimycin A (0.5 mM) measured by a Seahorse XF24 Extracellular Flux Analyzer. The OCR values were
normalized to 1 mg protein determination by the BCA assay. (E) Basal respiration was calculated by subtracting OCR values after the addition of
the mixture of rotenone (0.5 mM) and antimycin A (0.5 mM) from basal OCR. (F) ATP production was calculated by subtracting OCR values after the
addition of oligomycin from basal OCR. (G) Proton leak was calculated by subtracting OCR values after the addition of the mixture of rotenone
(0.5 mM) and antimycin A (0.5 mM) from OCR values obtained after the addition of oligomycin. (H) Non-mitochondrial respiration was the OCR
value after the addition of the mixture of rotenone (0.5 mM) and antimycin A (0.5 mM). *p < 0.05, **p < 0.01.
Fig. 5
confocal microscopy with 20 ,70 -dichlorodihydrouorescein
diacetate (H2DCFDA) staining. H2DCFDA can be converted to
the highly uorescent 20 ,70 -dichlorouorescein (DCF) by cellular
ROS.48 Aer a 6 h treatment, a dramatic concentration-dependent ROS elevation is observed for 2- and 4-treated A549 cells. At
a concentration of 10 mM, treatment with 2 and 4 increases the
mean uorescent intensity (MFI) by approximately 5.5- and
13.7-fold, respectively (Fig. 7). Similar results are also obtained
by confocal microscopy, with markedly concentration-dependent increases in the intensity of the DCF uorescence observed
in 2- and 4-treated cells (Fig. S22†). Additionally, pre-treatment
of A549 cells with N-acetyl-L-cysteine (NAC), a ROS scavenger,
signicantly diminishes the antiproliferative potency of 2 and 4
(Fig. S23†). These results indicate that 2 and 4 induce ROSdependent cell death.
Cell cycle arrest and induction of apoptosis
In addition to their role as ATP generators, mitochondria
mediate essential cell functions such as cell-cycle control and
636 | Chem. Sci., 2017, 8, 631–640
apoptosis.49 Cell cycle arrest may occur in response to the
blockage of macromolecule biosynthesis caused by a lack of
ATP production and mitochondrial dysfunction.50 Mitochondria play a key role in apoptosis by regulating the release of
cytochrome c and other pro-apoptotic proteins from the space
between the inner and outer mitochondrial membranes to the
cytosol.26
Cell cycle arrest in A549 cells induced by 2 and 4 was
analyzed by ow cytometry using propidium iodide (PI) staining
(Fig. 8A). Complexes 2 and 4 cause a dose-dependent G0/G1 cell
cycle arrest. Aer treatment with Ir(III) for 24 h, the percentage
of cells in the G0/G1 phase increases from 57.4 3.1% (control)
to 85.3 4.4% and 88.8 5.2% for 2 (2.5 mM) and 4 (2.5 mM),
respectively.
The morphology of apoptotic cells is characterized by cell
shrinkage, nuclear fragmentation, chromatin condensation
and membrane blebbing.51 First, the ultrastructural changes in
the morphology of cells were examined by TEM (Fig. 8B). The
control cells show a normal morphology. In contrast, the cells
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Fig. 6 (A and B) Confocal microscopic tracking of the mitochondrial morphology in real-time using 2 (10 mM) and 4 (10 mM) over a period of 8 h.
2: lex ¼ 405 nm; lem ¼ 630 20 nm. 4: lex ¼ 405 nm; lem ¼ 560 20 nm. The cell borders are recognizable and outlined with the red curve.
Scale bar: 10 mm. (C) Representative TEM images showing the perinuclear clustering of abnormal swollen mitochondria of A549 cells treated with
2 (d–f) and 4 (g–i) at a concentration of 5 mM for 24 h. (b) and (c) are enlarged views of the red zones in (a). (e) and (f) are enlarged views of the red
zones in (d). (h) and (i) are enlarged views of the red zones in (g).
treated with 2 and 4 show obvious morphological evidence of
different stages of apoptosis including condensed chromatin,
nuclear fragmentation and apoptotic bodies. In addition,
similar phenomena are also observed in Ir(III)-treated A549 cells
stained with Hoechst 33 342 (Fig. S24†). The control cells
exhibit homogeneous nuclear staining, and the Ir(III)-treated
cells display typical apoptotic changes, e.g., bright staining,
condensed chromatin and fragmented nuclei.
The activation of caspases is one of the best recognized
biochemical hallmarks of apoptosis.52 The effect of Ir(III) treatment on caspase-3/7 activity was determined using the CaspaseGlo assay. Treatment of A549 cells with 2 and 4 stimulates the
activation of caspase-3/7 in a dose-dependent manner
(Fig. S25†). Aer a 12 h treatment at a concentration of 10 mM,
the activity of caspase-3/7 is increased by approximately 1.6- and
1.7-fold in 2- and 4-treated cells, respectively. Similar to that
observed for cisplatin, pre-treatment of the cells with the pancaspase inhibitor, z-VAD-fmk, inhibits the antiproliferative
activity of 2 and 4 (Fig. S26†).
During apoptosis, phosphatidylserine (PS) is exposed externally due to loss of the asymmetry of plasma membrane phospholipids, and externalized phosphatidylserine emits eat-me
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signals to neighboring cells.51 The percentage of apoptotic cells
was determined by annexin V and PI double labeling, measured
by ow cytometry (Fig. S27†) and confocal microscopy
(Fig. S28†). Aer treatment of cells with 2 and 4 for 24 h, the
percentages of cells in both the early apoptotic (annexin Vpositive and PI-negative) and the late/necrotic (annexin V-positive and PI-positive) stages increase dose-dependently. Aer
24 h of treatment, apoptotic cells increase from 1.4 0.02% to
17.5 0.8% and 61.5 1.1% for 2 (10 mM) and 4 (10 mM),
respectively. Moreover, pre-treatment of A549 cells with NAC
decreases the apoptosis-inducing capability of 2 and 4
(Fig. S27†). These results collectively indicate that 2 and 4
induce caspase-dependent and ROS-mediated apoptotic cell
death.
Microarray analysis
Complex 2 shows comparable cytotoxicity to complex 4 against
the cancer cells tested, while it is less cytotoxic towards normal
LO2 cells. We chose complex 2 as the model compound for
microarray analysis to further elucidate the underlying mechanisms. Genome-wide gene transcriptional proles of 2-treated
cells were determined by Affymetrix human U133 plus
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Fig. 7 Intracellular ROS production measured by DCF fluorescence
(lex ¼ 488 nm, lem ¼ 530 30 nm) with flow cytometry. A549 cells
were treated with 2 and 4 at the indicated concentrations for 6 h.
2.0 microarray analysis in triplicate runs (Table S5†). 507 gene
sets are identied as being up-regulated and 715 gene sets are
down-regulated by more than two-fold in 2-treated A549 cells
(Table S6†). The heat shock 70 kDa protein 6 (HSP70B0 ) gene, is
markedly increased (about 79-fold). HSPs are anti-apoptotic
proteins, their expression can be induced in response to anticancer chemotherapy, and HSP70 can also prevent both caspase-dependent and caspase-independent cell death.53 The
elevated expression of HSP70B0 conrms that 2 initiates
apoptotic machinery in A549 cells. The mitochondrion-encoded
critical subunits I, II and III of cytochrome c oxidase (COX1,
COX2 and COX3) decrease signicantly with up to a 15-fold
decrease observed for COX1. The decrease in transcription of
these genes in the respiratory chain conrms that treatment
with 2 causes dysfunction of aerobic respiration.
In order to identify the possible anticancer mechanisms,
a connectivity map (Cmap, http://www.broad.mit.edu/cmap/),
which contains genome-wide transcriptional expression data
from a panel of cell lines treated with a library of 1309 bioactive
small molecules, was used to analyze the list of transcripts
regulated by 2.55 The results of the Cmap analysis in the case of
2 are summarized in Table S7.† A high correlation is obtained
with 2 and pyrvinium, which can inhibit the mitochondrial
respiration.56
638 | Chem. Sci., 2017, 8, 631–640
Fig. 8 (A) Effects of 2 and 4 on cell cycle distribution analyzed by flow
cytometry. A549 cells were stained by PI after treatment for 24 h. (B)
Representative TEM images showing different stages of apoptosis in
A549 cells treated with 2 and 4 (10 mM) for 24 h.
To mine the core biological functions from the enormous
dataset of a microarray, the impacted genes have been divided
into three Gene Ontology (GO) database categories: biological
process, cellular component and molecular function. Using the
predened gene sets by gene ontology, there are 43 pathways
that are signicantly enriched with the differentially expressed
genes from the 2-treated group (Table S8†). Treatment with 2
inuences several important biological processes such as the
mitotic cell cycle, mitotic nuclear division and DNA replication.
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The regulated genes derived from microarray experiments
were analyzed by a web-based bioinformatics tool, DAVID
(database for annotation, visualization, and integrated
discovery).54 The modulation of cell signalling pathways by 2 is
summarized in Table S9.† The results suggest that 2 affects
several pathways known to regulate cell death, including the cell
cycle, DNA replication and the p53 signalling pathway. The
mitochondrion is a central metabolic organelle, and it executes
critical functions for the metabolism of fatty acids, amino acids
and nucleotides. Treatment with 2 inuences metabolic pathways related to mitochondrial functions, such as D-glutamine
and D-glutamate metabolism, alanine, aspartate and glutamate
metabolism and pyrimidine metabolism. These ndings are
consistent with the experimental results.
In order to further conrm that 2-induced cell death occurs
through mitochondrial dysfunction, cell cycle perturbation and
apoptosis, we validate the differential expression of 21 genes
involved in several key pathways (mitochondrial metabolism,
the p53 signalling pathway and the cell cycle) in response to 2treatment by quantitative real-time PCR (RT-PCR). The primer
sequences of the selected genes studied in RT-PCR are listed in
Table S10,† and the functions of these genes are listed in Table
S11.† The heat map of the 2-induced expression prole of A549
cells to the untreated control is shown in Fig. S30.† The fold
changes of expression as determined by RT-PCR for these genes
are concordant with those obtained by microarray analysis
(Fig. S31†). Based on the results of bioinformatic analyses, the
proposed anticancer mechanisms of action of 2 are depicted in
Fig. S32.† The cytotoxicity of 2 is suggested to be related to
mitochondrial damage, elevation of ROS, initiation of DNA
damage responses, cell cycle arrest and induction of apoptosis.
Conclusions
In summary, four cyclometalated Ir(III) complexes with 40 ,40 substituted 20 ,20 -bipyridyl ligands have been developed as
mitochondria-targeted anticancer agents. Among them,
complexes 2 and 4 containing a reactive chloromethyl group can
be xed on mitochondria through nucleophilic substitution
with reactive thiols present in mitochondrial proteins. The
immobilization of complexes 2 and 4 on mitochondria results
in a much higher cytotoxicity than the non-xable complexes 1
and 3, which may attributed to their higher cellular penetration
capability as well as a longer retention time on mitochondria.
Mechanism studies show that 2 and 4 mainly induce caspaseand ROS-mediated apoptotic cell death. Complexes 2 and 4 can
lower the intracellular ATP levels, attenuate the mitochondrial
bioenergetic function and induce mitochondrial depolarization. Interestingly, complexes 2 and 4 can be also utilized to
monitor mitochondrial morphological changes, which provides
the possibility for in situ monitoring of the therapeutic effect,
and gives insights into their anticancer mechanisms. Moreover,
complex 2 can selectively kill cancer cells over normal cells.
Overall, our work shows that targeting mitochondrial metabolism by immobilization is a very effective strategy for the
construction of multifunctional phosphorescent metal
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complexes with enhanced anticancer activity and selectivity
towards cancer cells.
Acknowledgements
This study was supported by the 973 program (no.
2014CB845604 and 2015CB856301), the National Science
Foundation of China (no. 21572282, 21231007 and 21571196),
the Ministry of Education of China (no. IRT1298), the Guangdong Natural Science Foundation (2015A030306023), the
Science and Technology Program of Guangzhou (2014J4100140)
and the Fundamental Research Funds for the Central
Universities.
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