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Oncosis-inducing cyclometalated iridium(iii) complexes.
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Oncosis-inducing cyclometalated iridium(III)
complexes†
Ruilin Guan,a Yu Chen,a Leli Zeng, ab Thomas W. Rees,a Chengzhi Jin,a
Juanjuan Huang,a Zhe-Sheng Chen, b Liangnian Jia and Hui Chao *ac
Oncosis is a non-apoptotic form of programmed cell death (PCD), which differs from apoptosis in both
morphological changes and inner pathways, and might hold the key to defeating a major obstacle in
cancer therapy – drug-resistance, which is often a result of the intrinsic apoptosis resistance of tumours.
However, despite the fact that the term “oncosis” was coined and used much earlier than apoptosis, little
effort has been made to discover new drugs which can initiate this form of cell death, in comparison to
drugs inducing apoptosis or any other type of PCD. So herein, we present the synthesis of a series of
Received 11th March 2018
Accepted 2nd May 2018
mitochondria-targeting cyclometalated Ir(III) complexes, which activated the oncosis-specific protein
DOI: 10.1039/c8sc01142g
porimin and calpain in cisplatin-resistant cell line A549R, and determined their cytotoxicity against a wide
range of drug-resistant cancer types. To the best of our knowledge, these complexes are the very first
rsc.li/chemical-science
metallo-components to induce oncosis in drug-resistant cancer cells.
Introduction
Ever since cancer was identied as one of the leading causes of
human death, scientists and researchers worldwide have been
motivated by the urgency to nd a cure for this disease. As the
rst generation of anticancer drugs, platinum complexes such
as cisplatin, oxaliplatin, and carboplatin are unfortunately
limited by their severe side effects and, most importantly,
inherent or acquired resistance by their target cells.1–3 To overcome these limitations, a great deal of research has been done.
One strategy has been to change the metal core of the
complex. Different coordination modes or valence states of the
metal core have been introduced, including Pt(IV) prodrugs, and
half-sandwich and cyclometalated complexes.4–9 Lippard et al.
developed the idea of Pt(IV) complexes as anticancer agent
candidates, and have recently published a review of new platinum drugs and their design.7 Better selectivity towards cancer
cells was also achieved by Guo et al., with the introduction of
biotin or superparamagnetic iron oxide nanoparticles (MRI
a
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry,
Sun Yat-Sen University, Guangzhou, 510275, P. R. China. E-mail: ceschh@mail.
sysu.edu.cn
b
College of Pharmacy and Health Sciences, St. John's University, New York, NY 11439,
USA
c
MOE Key Laboratory of Theoretical Organic Chemistry and Functional Molecule,
School of Chemistry and Chemical Engineering, Hunan University of Science and
Technology, Xiangtan, 400201, P. R. China
† Electronic supplementary information (ESI) available: Experimental section,
NMR, ES-MS, photophysical properties, crystal structure and data, confocal
images, cell viability and IC50 values. CCDC 1578910. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c8sc01142g
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agents), in which a boost in cytotoxicity by glutathione was
observed.8,9
A variety of heavy metal cores including iridium, ruthenium,
osmium, titanium, copper, iron, gold, and other metal
compounds have also been developed in this eld, including
some which have made it as far as clinical studies.10–19 Sadler
et al. developed a series of Ru(II) complexes with a novel halfsandwich structure and achieved considerable anticancer
activity.16 Using a different approach, Ma et al. designed Ir(III)
complexes as inhibitors of bioactivity and bio-targets, such as
H-Ras/Raf-1 protein–protein interactions and tumor necrosis
factor-a.17–19
Some of the aforementioned groups adopted other
approaches, and selective activation of drugs by light is
a particularly appealing one.20–24 Sadler et al. reported precisely
designed Pt(IV) prodrugs, which can sustain the bioactive
reducing agent GSH and be photoactivated to Pt(II) by visible
light.20,21 Using a similar strategy, Gasser et al. developed a Ru(II)
complex, the irradiation of which caused the activation of
a protective cage moiety to release the drug.22 In another
example involving photoactivation, photodynamic therapy
(PDT) was employed by Gasser et al. to design Ru(II) polypyridyl
complexes23 and ruthenium–porphyrin conjugates24 as effective
photosensitizers and anticancer agents. Although many clinical
agents induce apoptosis in order to cause the death of cancer
cells, other modes of cell death have also been employed,
including necroptosis, autophagy and paraptosis.25–34 Using
NADH as a cofactor, Sadler et al. developed a new strategy for
anticancer drug design based on the catalytic properties of
Ru(II) compounds, and found the new reductive stress mechanism of cell death.28,29 Lippard et al. synthesized two Re(V) oxo
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Fig. 1 (a) Chemical structures of Ir(III) complexes. (b) Cyt. C content in the cytosol (Cyt. C, C) and mitochondria (Cyt. C, M). A549R cells were
incubated with OnIr1 (2 mM), OnIr2 (2 mM), OnIr3 (3 mM), OnIr4 (3 mM), and cisplatin (150 mM) for 24 h and then separated into two cellular
portions. (c) Caspase 3/7 activity assay. (d) Confocal images of co-localization assay and (e) ICP-MS results of Ir distribution in the cytosol (blue
bar), nucleus (red bar) and mitochondria (green bar). A549R cells were incubated with Ir(III) complexes (2 mM, 8 h) respectively, and then incubated
with MTG for 0.5 h after being washed with PBS. Scale bar in (d): 10 mm.
complexes, which induced programmed necrosis, also known
as necroptosis, and a Pt(II) complex reported by Guo et al. was
proven to induce autophagic cell death in human ovarian
carcinoma cells.30,31 Gasser et al. discovered that a Ru(II) polypyridyl complex, a PDT agent they synthesized, can induce
either apoptosis or paraptosis depending on the cell cycle phase
the cells were in.32 New subcellular locations have also been
introduced as new targets for therapeutics. Rhee et al. and Ang
et al. presented Ir(III) PDT agents and Ru(II) complexes which
were selectively localized in the endoplasmic reticulum (ER),
and caused severe damage to cells.33,34
Apoptosis has been thought to be the main death pathway of
PCD and it was not until recently that more pathways have been
discovered and studied,35 and these modes of cell death include
the perhaps underestimated oncosis. Since its discovery over
a century ago, this potential strategy for the treatment of drugresistant cancers has lain relatively forgotten.36,37 Derived from
the Greek word “swelling”, oncosis is a unique mode of cell
death with its characteristic whole cell swelling, accompanied
by severe mitochondrial damage, cytoplasmic vacuolization,
plasma membrane blebbing and cytoskeletal collapse.37,38 A
surface receptor, porimin, and Ca ion-related protein calpain
were reported as key elements in mediating oncotic cell
death.39–41
Based on the abundant experience of our group in the design
of metal complexes with specic subcellular localization,
especially in lysosomes and mitochondria,42–46 and because of
the fact that mitochondrial dysfunction plays a critical role at
a very early stage of cell death, we decided to synthesize a series
of mitochondria-targeting cyclometalated Ir(III) complexes.
Benzothiazole has been reported to possess considerable anticancer properties, and much research has been carried out
aimed at developing benzothiazole containing chemodrugs
with high anticancer activity.47–52 Therefore, we report in this
5184 | Chem. Sci., 2018, 9, 5183–5190
study a series of iridium(III) complexes with benzothiazole
substituted ligands: [Ir(ppy)2(bbtb)]+ (OnIr1), [Ir(DFppy)2
(bbtb)]+ (OnIr2), [Ir(2pq)2(bbtb)]+ (OnIr3), and [Ir(pbt)2(bbtb)]+
(OnIr4). The crystalline structure of OnIr3 was also obtained
(Fig. 1a and S1–S9†). We tested these complexes with more than
ten cancer cell lines, ten drug-resistant cell lines and two
normal cell lines, in which considerable cytotoxicity was
exhibited as well as good selectivity towards cancer cells and
normal cells. These complexes displayed mitochondria-targeting properties and further investigation suggested a relatively
unusual death mode as oncosis was activated by OnIr1–OnIr4 in
drug-resistant cell line A549R.
Results and discussion
Subcellular localization
The subcellular localization of drugs determines the initial
interactions between cells and drugs, the study of which,
fortunately, is facilitated by the good photophysical properties
of the Ir(III) complexes (Fig. S10†). We used A549R, a cisplatinresistant cell line, as a model, to study the behavior of these
complexes. The specic cellular target of the complexes was
conrmed by a co-localization assay and an ICP-MS assay. As
shown in Fig. 1d, the signal of the commercial mitochondrial
dye MitoTracker® Green (MTG) correlated well with those of the
complexes, giving correlation coefficients between 0.83 and
0.92. In the ICP-MS assay, a majority of the Ir(III) complexes were
shown to be localized in the mitochondria (Fig. 1e). Both of the
assays conrm that the cellular targets of the Ir(III) complexes
are mitochondria. We also conrmed that the mechanism of
cellular uptake of OnIr1–OnIr4 was endocytosis, as shown in
Fig. S11.† The stability of OnIr1–OnIr4 in FBS and culture media
is also shown in Fig. S12 and S13.†
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Mitochondrial morphological alteration
Subsequently, we focused on the cellular target of our
complexes, mitochondria, the abnormal changes of which
would cause severe failure in normal physiological activities.
Residing in the mitochondrial inner membrane (MIM), ChChd3
is an abundant protein and essential for maintaining the
mitochondrial cristae structure. The loss and reduction of
ChChd3 reects an injury in the MIM, which might result in
a breakdown in mitochondrial function and cristae architecture.53 A marked decrease in ChChd3 content in a time-dependent manner in the treated cells suggested the occurrence of
a traumatic alteration in mitochondria, and all four Ir(III)
complexes displayed similar results (Fig. 2c). Transmission
electron microscopy (TEM) experiments were than carried out
for the observation of the mitochondrial structure. In the TEM
images of untreated A549R cells, the double-membrane structure of the mitochondrion was observed and the mitochondrial
cristae were obvious and complex. Aer treatment with OnIr1,
the mitochondrial cristae became smoother, and the entire
mitochondrion was dilated and swollen (Fig. 2a), indicating
more severe damage and a major loss of mitochondrial
membrane potential.
Mitochondrial membrane potential (MMP) and overload of
reactive oxygen species (ROS)
Since one of the main pathways in mitochondria to initiate cell
death is the loss of mitochondrial membrane potential (MMP)
induced by the overload of reactive oxygen species (ROS),54 ow
cytometry with JC-1 and dichlorodihydrouorescein diacetate
(DCFH) was performed. The MMP loss of A549R cells would
prevent the aggregation of the MMP-dependent mitochondrial
dye JC-1, indicated by a uorescence change from red to green,
which is shown in Fig. 3a in a time-dependent manner, especially the rise in FITC signals. The cause of MMP loss was tested
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using DCFH, a ROS reactive reagent. In Fig. 3b, a signicant
increase in the DCFH signal indicated the over-generation of
ROS, also in a time-dependent manner, consistent with the JC-1
results.
ATP depletion and increase in the bcl-2/bax ratio
Mitochondria are the power plants of the cell, the dysfunction
of which can lead to an issue called ATP depletion.55 As shown
in Fig. 2b, the ATP level of cells aer treatment with the Ir(III)
complexes suffered a rapid decrease to less than 35% of that in
the control cells.56 As a pair of death-related proteins, the sites
of action of which are in mitochondria, bcl-2 and bax are also
inuenced by the Ir(III) complexes. Western blotting was applied
to detect the expression level of these proteins. The content of
bcl-2 increased in a time-dependent manner, while that of bax
decreased, as shown in Fig. 2a. The ratio of bcl-2/bax increased;
note that this pattern is the exact opposite of typical apoptosis,
where the expression of bcl-2 would decrease while that of bax
would increase, but consistent with a special death mode called
oncosis.57–59 All four Ir(III) complexes exhibited the same
tendency (Fig. 2c). This abnormal phenomenon implied
a different death mode from apoptosis.
Release of cytochrome C and caspase 3/7 activation
Aer ROS overload and MMP loss we would expect to observe
the release of cytochrome C (Cyt. C) from the mitochondria to
the cytosol and then the activation of the caspase family,
especially caspase 3/7, which would eventually lead to cell
death.60 In this study, the cytosol and mitochondria of A549R
cells were separated to test their Cyt. C content by western
blotting. To our surprise, there was no evidence of Cyt. C release
to be observed in the A549R cells (Fig. 1b). In spite of the severe
loss of MMP, the content of Cyt. C in the cytosol did not increase
and that of the mitochondria did not decrease. The results of
Fig. 2 (a) TEM image of swollen mitochondria. The cells were treated with OnIr1 (1 mM, 24 h). (b) ATP deletion assay for A549R cells incubated
with OnIr1–OnIr4 for 48 h. Incubation with OnIr1 and OnIr2 was performed at a concentration of (A) 0.5 mM, (B) 1 mM, and (C) 2 mM; incubation
with OnIr3 and OnIr4 was performed at a concentration of (A) 1 mM, (B) 2 mM, and (C) 3 mM. (c) Cellular content of bcl-2, bax and ChChd3 in
A549R cells.
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Fig. 4 (a) Confocal images and (b) TEM images of cytoplasmic
vacuolization. A549R cells were incubated with OnIr1 (1 mM), OnIr2
(1 mM), OnIr3 (2 mM) and OnIr4 (2 mM) for 24 h. (c) Confocal images of
plasma membrane blebbing. A549R cells were incubated with OnIr1
(2 mM), OnIr2 (2 mM), OnIr3 (3 mM) and OnIr4 (3 mM) for 24 h. Scale bar
in confocal images: 10 mm.
(a) Flow cytometry results of JC-1 assay and (b) ROS generation
assay; A549R cells were incubated with OnIr1 and OnIr2 at
a concentration of 1 mM, while incubation with OnIr3 and OnIr4 was
performed at a concentration of 2 mM.
Fig. 3
the whole-cell caspase 3/7 expression level assay agreed with
these results, where no obvious increase in the caspase 3/7 level
was observed even aer incubation with the four Ir(III)
complexes at a concentration of nearly 4 times their IC50 values
for one day, while incubation with cisplatin (CDDP) caused an
increased signal (Fig. 1c and S14a†). Western blotting was also
performed and similar results were obtained (Fig. S16†). Caspase inhibitors Ac-DEVD-CHO and Z-VAD-fmk were co-incubated with the Ir(III) complexes, and no signicant changes in
cell viabilities were observed (Fig. S14b†). Therefore, combined
with the results of the bcl-2/bax ratio, a different death mode
from classic caspase-dependent apoptosis was suggested.
Morphological alterations of the cells
To clarify which death mode occurred in the process, confocal
laser scanning microscopy (CLSM) and TEM were applied to
observe the alterations in the A549R cells. The most signicant
morphological alteration was the vacuolization in cytoplasm,
which none of the known forms of apoptosis would cause.
During the process, vacuoles generated from lysosomes lled
up the entire cytoplasm (Fig. S15†),37 taking up most of the
space (Fig. 4a), and showing the absence of organelles, as shown
in Fig. 4b. As the incubation time increased, the whole cells
were swollen and rounded, sometimes detaching from the
substrate, and started to bleb (Fig. 4c). This helped us to rule out
paraptosis as the mode of cell death,25 as neither swelling nor
5186 | Chem. Sci., 2018, 9, 5183–5190
blebbing occurs in paraptosis. Notably, the bubbles around the
cells were absolutely clear on the inside, suggesting that there
was no process of budding, a typical action of apoptosis, taking
place. Another thing worth mentioning is that there was also no
sign of interruptions in plasmalemmal continuity in both CLSM
and TEM micrographs, a typical feature of necrosis and necroptosis.61 The results of co-incubation of autophagy inhibitor
3-methyladenine, paraptosis inhibitor cycloheximide, lysosomal protease-mediated cell death inhibitor leupeptin and
necrosis inhibitor necrostatin-1, and the Ir(III) complexes
showed no increase in cell viability (Fig. S14c†).62,63 To have
a deeper look into it, western blotting was introduced to check
the level of LC3, activated during the process of autophagy,64,65
RIP3, activated during necrosis and necroptosis,66,67 and ERK/
p-ERK, a marker of paraptosis.68 Again, no signicant changes
aer treatment with OnIr1–OnIr4 were observed (Fig. S16†). All
of the evidence thus far pointed toward oncosis, and so we
decided to investigate oncosis more comprehensively.
Cytoskeleton and calpain 1 activation
The change of the shape and volume of the whole cell also
implied a profound impact on the cytoskeleton. Both actin and
tubulin were altered during this phase.61,69–71 By the use of
western blotting, a marked decrease of the basic elements of the
cytoskeleton, b-actin and a-tubulin, appeared in a time-dependent manner,72 suggesting a possible breakdown and collapse
of the cytoskeleton (Fig. 5c). Further research by CLSM and
staining with commercial dyes Actin-Tracker Green and
Tubulin-Tracker Red allowed the actual visualization of the
alterations of actin and tubulin. As shown in Fig. 5a and S17,†
control cells have prominent thick actin cables across the whole
cells.69 Aer incubation with 0.5 mM OnIr1, a punctate pattern of
actin appeared, and fewer actin cables were observed in many of
the cells. Upon increasing the concentration of OnIr1 to 1 mM,
a lower density of actin bers was observed as well as fragmentation of the actin lament. We could also see the
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(a) Actin-Tracker Green and (b) Tubulin-Tracker Red staining of fixed A549R cells pre-treated with OnIr1 for 24 h in a dose-dependent manner.
Confocal images of A549R cells treated with OnIr1 for 24 h. (b) Flow cytometry results of cell volume changes. A549R cells were incubated with OnIr1
(1 mM), OnIr2 (1 mM), OnIr3 (2 mM) and OnIr4 (2 mM). Cellular content of (c) a-tubulin and b-actin and (d) porimin and calpain 1. (e) LDH leakage assay for
A549R cells incubated with OnIr1–OnIr4 for 24 h. Incubation with OnIr1 and OnIr2 was performed at a concentration of (A) 0.5 mM, (B) 1 mM, and (C) 2
mM; incubation with OnIr3 and OnIr4 was performed at a concentration of (A) 1 mM, (B) 2 mM, and (C) 3 mM. Scale bar in (a): 10 mM.
Fig. 5
interruption of actin bers by the vacuoles (red arrowhead).
Aer incubation with up to 2 mM OnIr1, the pattern of actin
cables effectively disappeared, leaving behind the punctate
actin, sometimes only some fragments (Fig. S18†), and
somehow those fragments managed to slip out of the cell
membrane, probably through the bursting of the bubbles,70
around which a band of uorescent stain was observed.69 The
stain of Actin-Tracker Green faded along with the increase of
incubation concentration. Meanwhile, a network of microtubules radiated from the perinuclear centers in control cells,71
and the cells underwent mitosis metaphase as shown in Fig. 5a
and S19† with Tubulin-Tracker Red staining. Aer incubation
with 0.5 mM OnIr1, the pattern of the microtubules was less
rigid. Upon increasing the concentration of OnIr1 to 1 mM, the
loss in length and number of microtubules began and the
pattern was vague. A signicant interruption of microtubules by
the vacuoles (yellow arrowhead) appeared. Aer further
increasing the incubation concentration of OnIr1 up to 2 mM,
the shape of the microtubules became completely loose, and
only the carcasses remained both in the cells and the big blebs.
Similar to that of Actin-Tracker Green, the stain of TubulinTracker Red decreased with an increase in the incubation
concentration of OnIr1, and was completely lost in some of the
cells (Fig. S20†). These images showed the collapse of the
cytoskeleton vividly, and that the breakdown of it could be
induced by these Ir(III) complexes in a dose-dependent manner.
Growing evidence indicates that calpains, a calcium-activated protease family, are involved in processes from mitochondrial dysfunction to cytoskeletal collapse. These neutral
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cysteine proteases were considered to be among the iconic
proteins in the procedure of oncotic cell death, and their
substrates include cytoskeletal proteins, the activation of which
would cause cytoskeletal breakdown.41,72 Although the members
of the calpain family were considered as cytoplasmic proteins,
one of the calpains, calpain 1, was found to be not only rich in,
but also navigated by its own structure towards, mitochondria.73
The results of western blotting in calpain 1 displayed its activation by the appearance of a new band below the other one
aer treatment with Ir(III) complexes, and specically the
treatment with OnIr1 gave rise to the new band in a timedependent manner (Fig. 5d). The impact of calpain activation is
profound in oncosis. There are numerous pieces of evidence
suggesting that the activation of calpain leads to mitochondrial
dysfunction and the breakdown of not only the cytoskeleton but
also the plasma membrane.41
Plasma membrane permeability and porimin expression
Since the symbolic phenomenon of oncosis is cell swelling, ow
cytometry was employed to measure the enlargement of cell
volumes. Apparently, limited by the plasma membrane, a cell
cannot swell innitely, so the peak should not shi entirely to
a new volume section. As shown in Fig. 5b, instead of a shi, the
shape of the peak representing cell volumes of A549R was
changed and tended to a certain extremum, which is the
maximum of the control cells, showing an increase in average
cell volume. To elucidate how the cells were swollen, we
measured the membrane permeability by lactate dehydrogenase
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(LDH) leakage assay. A dramatic increase of signals was observed
(Fig. 5d), indicating a large leakage of LDH, and therefore an
increase in membrane permeability in a majority of cells. The
increase of cell membrane permeability is ascribed to the
increase of cell volume.
A surface receptor, porimin (pro-oncosis receptor inducing
membrane injury), is supposed to be responsible for the
abnormal membrane permeability, which directly causes the
iconic whole cell swelling in the process of oncosis, and the
activation of porimin is also a marker of oncotic cell death.39,40,74
In Fig. 5d, the increase in expression of porimin upon incubation with OnIr2–OnIr4, as well as OnIr1, is demonstrated in
a time-dependent manner.
Cytotoxicity against drug-resistant cancer cells
Now that we had conrmed that the Ir(III) complexes induced
a unique death mode, oncosis, which is quite the opposite of
apoptosis in many ways, we tried to apply these complexes to
ght against cancer cells, especially drug-resistant cancer cells.
11 cancer cell lines from various body parts including lungs,
colon, kidneys and liver were tested, along with 10 drug-resistant cell lines and 2 normal cell lines (HL-7702, normal liver
cells; HEK 293, normal kidney cells). All four Ir(III) complexes
exhibited considerable cytotoxicity in normal cancer cell lines,
drug-resistant cell lines and their parental cell lines (IC50 values
down to 500 nM, Table S3†). The drugs causing resistance
included cisplatin, another metal complex like our complexes;
mitoxantrone, a type II topoisomerase inhibitor; doxorubicin,
a multi-target drug and As2O3, a classic inorganic nonmetal
drug. None of the resistances of these different types of drugs
were sufficient for the cancer cells to prevent their death on
exposure to the Ir(III) complexes. They also exhibited moderate
cytotoxicity towards normal cell lines, but their selectivity
towards cancer cells and normal cells can be as signicant as
30 times the IC50 value (OnIr1, HL-7702/A549R).
Discussion
From subcellular localization to cell membrane injury, we look
into some major alterations the treatment with these Ir(III)
complexes led to in the cells. The verication of the death mode
caused by certain compounds is oen delicate, especially for
PCD modes like oncosis, because like any other death modes
and pathways, certain phenomena or syndromes of oncosis
might be shared with other modes and PCD modes are in
a much less acknowledged situation compared with apoptosis.
For example, blebbing in cellular membranes could occur both
in necrosis and in oncosis; cytoplasmic vacuolization is also
a signicant feature in paraptosis, etc. Moreover, there are
crosstalks between two death modes and that leads to concepts
like necroptosis, oncotic apoptosis and even oncotic necrosis, to
name a few. In other words, since the word “oncosis” originally
means “swelling”, every cell death accompanied by whole cell
swelling ought to be oncosis, perhaps a crosstalk such as
“apoptotic oncosis”. When it comes to drug-resistant cancer
cells, the situation gets even more complicated. As a matter of
5188 | Chem. Sci., 2018, 9, 5183–5190
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fact, aer treatment with cisplatin, a classic apoptosis-inducing
agent, A549R cells exhibited a sign of autophagy and necrosis
with a slight increase in LC3II and RIP3, as shown in Fig. S16.†
This situation was somewhat expected, since it was reported
that high-dose cisplatin can induce autophagy and necrosis.75,76
However, when the pieces of evidence were put together, the fog
began to clear, and some of the evidence has been mentioned in
previous paragraphs, e.g. the morphological changes of the cells
and the bcl-2/bax ratio. Although there is caspase-independent
apoptosis which makes the whole caspase 3/7 detection and
caspase inhibitor experiments seem less convincing for ruling
out this common death mode,77–79 apoptosis per se oen
exhibits other features that are in no way related to what we
observed in this paper. While cell shrinkage is a well-known
consequence of apoptosis, the exact opposite cell swelling was
observed, and cytoplasmic vacuolization is rare in the process of
apoptosis. We also carefully checked other PCD modes either by
their specic inhibitors or the detection of their characteristic
protein expression and activation. Moreover, oncosis matched
every single phenomenon perfectly, and most importantly, we
demonstrated the activation of calpain 1, which is highly related
to oncotic cell death in many aspects, and the expression of
porimin, which is supposed to be the marker of oncosis.
The process of oncosis induced by these complexes can be
briey outlined as follows: (1) the complexes entered the cells
and localized in the mitochondria; (2) this caused the overgeneration of ROS and (3) the loss of MMP, (4) followed by ATP
depletion and an increased ratio of bcl-2/bax, leading to (5) the
failure of the ionic pumps resulting in a high cellular
membrane permeability and vacuolization from the lysosomes,37 (6) rounding and swelling of the cells and blebbing in
the plasma membrane, during which (7) a collapse of the
cytoskeleton occurred, and nally (8) the bubbles burst and the
remainder would experience phagocytosis or inammation, the
very end of an oncotic cell.70
Conclusion
To conclude, we synthesized a series of Ir(III) complexes, OnIr1,
OnIr2, OnIr3, and OnIr4, and found that they can target mitochondria and eventually proved that they can induce oncosis in
drug-resistant cancer cell line A549R. Finally, we screened the
anticancer activity of these oncosis-inducing complexes. They
showed IC50 values indicating considerable cytotoxicity against
various cancer cell lines and drug-resistant cell lines, with
selectivity towards normal cells lines. These results suggest that
the unique oncosis-inducing cyclometalated Ir(III) complexes
can be potent candidates to ght against drug-resistant cancers.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 21525105, 21471164, 21501201 and
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21778079), the 973 Program (No. 2015CB856301), and the
Fundamental Research Funds for the Central Universities.
Notes and references
1 V. Brabec and J. Kasparkova, Drug Resist. Updates, 2005, 8,
131–146.
2 Z. H. Siddik, Oncogene, 2003, 22, 7265–7279.
3 L. Kelland, Nat. Rev. Cancer, 2007, 7, 573–584.
4 T. Zou, J. Liu, C. T. Lum, C. Ma, R. C. Chan, C. N. Lok,
W. M. Kwok and C. M. Che, Angew. Chem., Int. Ed., 2014,
53, 10119–10123.
5 N. P. E. Barry and P. J. Sadler, Chem. Soc. Rev., 2012, 41, 3264–
3279.
6 Z. T. Cao, Z. Y. Chen, C. Y. Sun, H. J. Li, H. X. Wang,
Q. Q. Cheng, Z. Q. Zuo, J. L. Wang, Y. Z. Liu, Y. C. Wang
and J. Wang, Biomaterials, 2016, 94, 9–19.
7 T. C. Johnstone, K. Suntharalingam and S. J. Lippard, Chem.
Rev., 2016, 116, 3436–3486.
8 Z. Z. Zhu, Z. H. Wang, Y. G. Hao, C. C. Zhu, Y. Jiao,
H. C. Chen, Y. M. Wang, J. Yan, Z. J. Guo and X. Y. Wang,
Chem. Sci., 2016, 7, 2864–2869.
9 N. Muhammad, N. Sadia, C. Zhu, C. Luo, Z. Guo and
X. Wang, Chem. Commun., 2017, 53, 9971–9974.
10 D. L. Ma, H. Z. He, K. H. Leung, D. S. Chan and C. H. Leung,
Angew. Chem., Int. Ed., 2013, 52, 7666–7682.
11 L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao and
Z. S. Chen, Chem. Soc. Rev., 2017, 46, 5771–5804.
12 R. J. Needham, C. Sanchez-Cano, X. Zhang, I. RomeroCanelon, A. Habtemariam, M. S. Cooper, L. Meszaros,
G. J. Clarkson, P. J. Blower and P. J. Sadler, Angew. Chem.,
Int. Ed., 2017, 56, 1017–1020.
13 Z. G. Yue, W. Wei, Z. X. You, Q. Z. Yang, H. Yue, Z. G. Su and
G. H. Ma, Adv. Funct. Mater., 2011, 21, 3446–3453.
14 R. W. Y. Sun and C. M. Che, Coord. Chem. Rev., 2009, 253,
1682–1691.
15 Z. J. Guo and P. J. Sadler, Angew. Chem., Int. Ed., 1999, 38,
1512–1531.
16 T. Sriskandakumar, H. Petzold, P. C. Bruijnincx,
A. Habtemariam, P. J. Sadler and P. Kennepohl, J. Am.
Chem. Soc., 2009, 131, 13355–13361.
17 L. J. Liu, W. H. Wang, S. Y. Huang, Y. J. Hong, G. D. Li, S. Lin,
J. L. Tian, Z. W. Cai, H. M. D. Wang, D. L. Ma and
C. H. Leung, Chem. Sci., 2017, 8, 4756–4763.
18 C. H. Leung, H. J. Zhong, H. Yang, Z. Cheng, D. S. Chan,
V. P. Ma, R. Abagyan, C. Y. Wong and D. L. Ma, Angew.
Chem., Int. Ed., 2012, 51, 9010–9014.
19 T. S. Kang, Z. Mao, C. T. Ng, M. Wang, W. Wang, C. Wang,
S. M. Lee, Y. Wang, C. H. Leung and D. L. Ma, J. Med.
Chem., 2016, 59, 4026–4031.
20 N. J. Farrer, J. A. Woods, L. Salassa, Y. Zhao, K. S. Robinson,
G. Clarkson, F. S. Mackay and P. J. Sadler, Angew. Chem., Int.
Ed., 2010, 49, 8905–8908.
21 Y. Zhao, J. A. Woods, N. J. Farrer, K. S. Robinson,
J. Pracharova, J. Kasparkova, O. Novakova, H. Li,
L. Salassa, A. M. Pizarro, G. J. Clarkson, L. Song, V. Brabec
and P. J. Sadler, Chem.–Eur. J., 2013, 19, 9578–9591.
This journal is © The Royal Society of Chemistry 2018
Chemical Science
22 T. Joshi, V. Pierroz, C. Mari, L. Gemperle, S. Ferrari and
G. Gasser, Angew. Chem., Int. Ed., 2014, 53, 2960–2963.
23 A. Frei, R. Rubbiani, S. Tubafard, O. Blacque, P. Anstaett,
A. Felgentrager, T. Maisch, L. Spiccia and G. Gasser,
J. Med. Chem., 2014, 57, 7280–7292.
24 C. Mari, V. Pierroz, R. Rubbiani, M. Patra, J. Hess,
B. Spingler, L. Oehninger, J. Schur, I. Ott, L. Salassa,
S. Ferrari and G. Gasser, Chem.–Eur. J., 2014, 20, 14421–
14436.
25 S. Sperandio, I. de Belle and D. E. Bredesen, Proc. Natl. Acad.
Sci. U. S. A., 2000, 97, 14376–14381.
26 R. R. Ye, C. P. Tan, Y. N. Lin, L. N. Ji and Z. W. Mao, Chem.
Commun., 2015, 51, 8353–8356.
27 K. N. Dalby, I. Tekedereli, G. Lopez-Berestein and B. Ozpolat,
Autophagy, 2010, 6, 322–329.
28 S. Betanzos-Lara, Z. Liu, A. Habtemariam, A. M. Pizarro,
B. Qamar and P. J. Sadler, Angew. Chem., Int. Ed., 2012, 51,
3897–3900.
29 J. J. Soldevila-Barreda, I. Romero-Canelon, A. Habtemariam
and P. J. Sadler, Nat. Commun., 2015, 6, 6582.
30 W. J. Guo, Y. M. Zhang, L. Zhang, B. Huang, F. F. Tao,
W. Chen, Z. J. Guo, Q. Xu and Y. Sun, Autophagy, 2013, 9,
996–1008.
31 K. Suntharalingam, S. G. Awuah, P. M. Bruno,
T. C. Johnstone, F. Wang, W. Lin, Y. R. Zheng, J. E. Page,
M. T. Hemann and S. J. Lippard, J. Am. Chem. Soc., 2015,
137, 2967–2974.
32 V. Pierroz, R. Rubbiani, C. Gentili, M. Patra, C. Mari,
G. Gasser and S. Ferrari, Chem. Sci., 2016, 7, 6115–6124.
33 J. S. Nam, M. G. Kang, J. Kang, S. Y. Park, S. J. Lee, H. T. Kim,
J. K. Seo, O. H. Kwon, M. H. Lim, H. W. Rhee and T. H. Kwon,
J. Am. Chem. Soc., 2016, 138, 10968–10977.
34 M. J. Chow, C. Licona, G. Pastorin, G. Mellitzer, W. H. Ang
and C. Gaiddon, Chem. Sci., 2016, 7, 4117–4124.
35 L. E. Broker, F. A. Kruyt and G. Giaccone, Clin. Cancer Res.,
2005, 11, 3155–3162.
36 P. Weerasinghe and L. M. Buja, Exp. Mol. Pathol., 2012, 93,
302–308.
37 P. Weerasinghe, S. Hallock, R. E. Brown, D. S. Loose and
L. M. Buja, Exp. Mol. Pathol., 2013, 94, 289–300.
38 A. A. Peters, S. Y. N. Jamaludin, K. Yapa, S. Chalmers,
A. P. Wiegmans, H. F. Lim, M. J. G. Milevskiy, I. Azimi,
F. M. Davis, K. S. Northwood, E. Pera, D. L. Marcial,
E. Dray, N. J. Waterhouse, P. J. Cabot, T. J. Gonda,
P. A. Kenny, M. A. Brown, K. K. Khanna, S. J. RobertsThomson and G. R. Monteith, Oncogene, 2017, 36, 6490–
6500.
39 C. Zhang, Y. Xu, J. Gu and S. F. Schlossman, Proc. Natl. Acad.
Sci. U. S. A., 1998, 95, 6290–6295.
40 F. R. Ma, C. H. Zhang, K. V. S. Prasad, G. J. Freeman and
S. F. Schlossman, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
9778–9783.
41 X. Liu, T. Van Vleet and R. G. Schnellmann, Annu. Rev.
Pharmacol. Toxicol., 2004, 44, 349–370.
42 K. Q. Qiu, H. Y. Huang, B. Y. Liu, Y. K. Liu, Z. Y. Huang,
Y. Chen, L. N. A. Ji and H. Chao, ACS Appl. Mater.
Interfaces, 2016, 8, 12702–12710.
Chem. Sci., 2018, 9, 5183–5190 | 5189
View Article Online
Open Access Article. Published on 03 May 2018. Downloaded on 5/2/2026 2:50:27 AM.
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Chemical Science
43 H. Huang, B. Yu, P. Zhang, J. Huang, Y. Chen, G. Gasser, L. Ji
and H. Chao, Angew. Chem., Int. Ed., 2015, 54, 14049–14052.
44 G. Y. Li, Y. Chen, J. Q. Wang, Q. Lin, J. Zhao, L. N. Ji and
H. Chao, Chem. Sci., 2013, 4, 4426–4433.
45 J. Liu, Y. Chen, G. Li, P. Zhang, C. Jin, L. Zeng, L. Ji and
H. Chao, Biomaterials, 2015, 56, 140–153.
46 J. Liu, C. Jin, B. Yuan, Y. Chen, X. Liu, L. Ji and H. Chao,
Chem. Commun., 2017, 53, 9878–9881.
47 E. Brantley, V. Trapani, M. C. Alley, C. D. Hose,
T. D. Bradshaw, M. F. Stevens, E. A. Sausville and
S. F. Stinson, Drug Metab. Dispos., 2004, 32, 1392–1401.
48 T. D. Bradshaw and A. D. Westwell, Curr. Med. Chem., 2004,
11, 1009–1021.
49 H. K. Kim, M. K. Kang, K. H. Jung, S. H. Kang, Y. H. Kim,
J. C. Jung, G. H. Lee, Y. Chang and T. J. Kim, J. Med.
Chem., 2013, 56, 8104–8111.
50 E. Kashiyama, I. Hutchinson, M. S. Chua, S. F. Stinson,
L. R. Phillips, G. Kaur, E. A. Sausville, T. D. Bradshaw,
A. D. Westwell and M. F. Stevens, J. Med. Chem., 1999, 42,
4172–4184.
51 M. S. Chua, E. Kashiyama, T. D. Bradshaw, S. F. Stinson,
E. Brantley, E. A. Sausville and M. F. Stevens, Cancer Res.,
2000, 60, 5196–5203.
52 S. Yu, X. Gong and W. Chan, Macromolecules, 1998, 31, 5639–
5646.
53 M. Darshi, V. L. Mendiola, M. R. Mackey, A. N. Murphy,
A. Koller, G. A. Perkins, M. H. Ellisman and S. S. Taylor, J.
Biol. Chem., 2011, 286, 2918–2932.
54 N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin,
A. Macho, T. Hirsch, S. A. Susin, P. X. Petit, B. Mignotte
and G. Kroemer, J. Exp. Med., 1995, 182, 367–377.
55 E. M. Mills, D. Xu, M. M. Fergusson, C. A. Combs, Y. Xu and
T. Finkel, J. Biol. Chem., 2002, 277, 27385–27392.
56 Q. Huang, R. Zhang, L. y. Zou, X. Cao and X. Chu, PLoS One,
2013, 8, e61345.
57 E. H. Y. Cheng, T. V. Sheiko, J. K. Fisher, W. J. Craigen and
S. J. Korsmeyer, Science, 2003, 301, 513–517.
58 S. Cory and J. M. Adams, Nat. Rev. Cancer, 2002, 2, 647–656.
59 P. Weerasinghe, S. Hallock and A. Liepins, Exp. Mol. Pathol.,
2001, 71, 89–98.
60 L. M. Dejean, S. Martinez-Caballero and K. W. Kinnally, Cell
Death Differ., 2006, 13, 1387–1395.
61 B. F. Trump, I. K. Berezesky, S. H. Chang and P. C. Phelps,
Toxicol. Pathol., 1997, 25, 82–88.
62 B. Li, J. Zhao, C. Z. Wang, J. Searle, T. C. He, C. S. Yuan and
W. Du, Cancer Lett., 2011, 301, 185–192.
5190 | Chem. Sci., 2018, 9, 5183–5190
Edge Article
63 M. E. Guicciardi, M. Leist and G. J. Gores, Oncogene, 2004, 23,
2881–2890.
64 N. Mizushima, T. Yoshimori and B. Levine, Cell, 2010, 140,
313–326.
65 D. J. Klionsky, F. C. Abdalla, H. Abeliovich, R. T. Abraham,
A. Acevedo-Arozena, K. Adeli, L. Agholme, M. Agnello,
P. Agostinis, J. Zschocke and B. Zuckerbraun, et. al.,
Autophagy, 2012, 8, 445–544.
66 D. W. Zhang, J. Shao, J. Lin, N. Zhang, B. J. Lu, S. C. Lin,
M. Q. Dong and J. Han, Science, 2009, 325, 332–336.
67 P. Vandenabeele, L. Galluzzi, T. Vanden Berghe and
G. Kroemer, Nat. Rev. Mol. Cell Biol., 2010, 11, 700–714.
68 W. B. Wang, L. X. Feng, Q. X. Yue, W. Y. Wu, S. H. Guan,
B. H. Jiang, M. Yang, X. Liu and D. A. Guo, J. Cell. Physiol.,
2012, 227, 2196–2206.
69 K. A. Elliget, P. C. Phelps and B. F. Trump, Cell Biol. Toxicol.,
1991, 7, 263–280.
70 G. Majno and I. Joris, Am. J. Pathol., 1995, 146, 3–15.
71 P. C. Phelps, K. A. Elliget and B. F. Trump, FASEB J., 1996, 10,
A1425.
72 G. Zhang, C. Jiang, Z. Wang, W. Chen, W. Gu and Y. Ding,
BioMed Res. Int., 2014, 2014, 682197.
73 R. Badugu, M. Garcia, V. Bondada, A. Joshi and J. W. Geddes,
J. Biol. Chem., 2008, 283, 3409–3417.
74 L. F. Liu, Z. H. Qian, Q. Qin, M. Shi, H. Zhang, X. M. Tao and
W. P. Zhu, Genet. Mol. Res., 2015, 14, 7481–7489.
75 T. R. O'Donovan, G. C. O'Sullivan and S. L. McKenna,
Autophagy, 2011, 7, 509–524.
76 F. R. Khuri, J. Nemunaitis, I. Ganly, J. Arseneau,
I. F. Tannock, L. Romel, M. Gore, J. Ironside,
R. H. MacDougall, C. Heise, B. Randlev, A. M. Gillenwater,
P. Bruso, S. B. Kaye, W. K. Hong and D. H. Kirn, Nat. Med.,
2000, 6, 879–885.
77 L. Ravagnan, S. Gurbuxani, S. A. Susin, C. Maisse, E. Daugas,
N. Zamzami, T. Mak, M. Jaattela, J. M. Penninger, C. Garrido
and G. Kroemer, Nat. Cell Biol., 2001, 3, 839–843.
78 L. Y. Li, X. Luo and X. Wang, Nature, 2001, 412, 95–99.
79 G. Kroemer, L. Galluzzi, P. Vandenabeele, J. Abrams,
E. S. Alnemri, E. H. Baehrecke, M. V. Blagosklonny,
W. S. El-Deiry, P. Golstein, D. R. Green, M. Hengartner,
R. A. Knight, S. Kumar, S. A. Lipton, W. Malorni, G. Nunez,
M. E. Peter, J. Tschopp, J. Yuan, M. Piacentini,
B. Zhivotovsky and G. Melino, Cell Death Differ., 2009, 16,
3–11.
This journal is © The Royal Society of Chemistry 2018