Anticancer IrIII–Aspirin Conjugates for Enhanced Metabolic Immuno-Modulation and Mitochondrial Lifetime Imaging

Article abstract of DOI:10.1002/chem.201900851

The chemo-anti-inflammatory strategy is attracting ever more attention for the treatment of cancer. Here, two cyclometalated Ir^III complexes Ir2 and Ir3 formed by conjugation of Ir1 with two antiphlogistics (aspirin and salicylic acid) have bee

Full text of DOI:10.1002/chem.201900851

A Journal of
Accepted Article
Title: Anticancer Ir(III)-Aspirin Conjugates for Enhanced Metabolic
Immuno-Modulation and Mitochondrial Lifetime Imaging
Authors: Xiao-Wen Wu, Yue Zheng, Fang-Xin Wang, Jian-Jun Cao,
Hang Zhang, Dong-Yang Zhang, Cai-Ping Tan, Liang-Nian Ji,
and Zong-Wan Mao
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Anticancer Ir(III)-Aspirin Conjugates for Enhanced Metabolic
Immuno-Modulation and Mitochondrial Lifetime Imaging
Xiao-Wen Wu,†[a] Yue Zheng,†[a] Fang-Xin Wang,[a] Jian-Jun Cao,[a] Hang Zhang,[a] Dong-Yang
Zhang,[a] Cai-Ping Tan,*[a] Liang-Nian Ji[a] and Zong-Wan Mao*[a]
Abstract: The chemo-anti-inflammatory strategy is gaining more
and more attention in the treatment of cancer. Herein, two
cyclometalated Ir(III) complexes Ir2 and Ir3 formed by conjugation of
Ir1 with two antiphlogistics (aspirin and salicylic acid) have been
designed. Ir2 and Ir3 exhibit higher antitumor and anti-inflammatory
potencies than a mixture of Ir1 and aspirin/salicylic acid. We show
that they can be hydrolyzed, accumulate in mitochondria, and induce
mitochondrial dysfunction. Due to their intense long-lived
phosphorescence, Ir2 and Ir3 can track mitochondrial morphological
changes. Phosphorescence lifetime imaging shows that Ir2 and Ir3
can aggregate during mitochondrial dysfunction. As expected, Ir2
and Ir3 exhibit immunomodulatory properties by regulating the
activity of the immune factors. Both Ir2 and Ir3 can induce caspase-
dependent apoptosis and caspase-independent paraptosis and
inhibit several events related to metastasis. Moreover, Ir2 and Ir3
show potent tumor growth inhibition in vivo. Our study demonstrates
because of their high emission quantum yields and good
photostability.[10-15] Due to their triplet emissive states,
phosphorescent iridium complexes have large Stoke shifts that
can minimize the self-quenching effects.[16] They also have long-
lived phosphorescence that is much longer than the lifetimes of
cellular background fluorescence (less than 10 ns) and organic
dyes commonly used in cell imaging.[11-12, 14] Therefore, iridium
complexes can be applied in time-gate imaging to avoid the
interference from short-lived scattering and autofluorescence.[12,
14]
The long lifetime of iridium complexes can also applied in
phosphorescence lifetime imaging (PLIM) to quantitatively detect
the analytes more accurately.[12, 14]
Inflammatory responses play important roles at different
stages of tumor development.[17] It has been reported that 18%
of human malignancies are linked to infectious agents.[18] Aspirin
(As), a salicylic acid (SA)-based medicine, is widely used as an
analgesic, antipyretic and anti-inflammatory medicine. Many
clinical studies have shown that long-term daily As use has an
antitumor effect.[19-21] Some successful chemotherapeutics can
induce a type of immunogenic cancer cell death,[22] and in turn
anticancer immune responses may make effect to control
cancer.[23] A chemo-anti-inflammatory molecule that can release
active anticancer drugs and anti-inflammatory agents
concurrently upon cellular activation is a potent approach to treat
stubborn cancers. For example, the platinum(IV) prodrug Platin-
A designed by Shanta[24] and Liu[25-26] exhibits both anticancer
and anti-inflammatory properties.
that
the
combination
of
mitochondrial
targeting
and
immunomodulatory activities is feasible to develop multifunctional
metal-based anticancer agents.
Introduction
Though platinum drugs like cisplatin have achieved great
success, their development is hindered by drug resistance and
[1]
severe side effects. Other non-platinum complexes may be
able to overcome cisplatin resistance if they have different
mechanisms of actions. Among them, organometallic metal
complexes show promise for overcoming the platinum
Mitochondria show important functions in physiological and
pathological conditions.[27] Cancer cells display different degrees
of transformations in mitochondrial functions,[28-29] which
resistance,
e.g.,
Ru(II)/Ir(III)
arene
complexes
and
provides opportunities to target mitochondria for the treatment of
cyclometalated Ir(III) complexes.[2-4] Notably, iridium complexes
are getting wide attention for biological applications, especially
organometallic iridium complexes.[3] Cyclometalated Ir(III)
complexes demonstrate interesting antitumor properties, e.g.,
inhibiting protein-protein interactions and targeting subcellular
organelles.[4] In immunotherapy for cancer, iridium complexes
have also shown potential applications in molecular imaging,[5]
protein inhibition[6-8] and inflammatory modulation.[9] On the other
hand, iridium complexes are promising bioimaging materials
30]
cancer.[27,
Moreover, accumulating evidence shows that
mitochondria also play important roles in the establishment and
functions of immune cell phenotypes.[31-34] As previous
researches have indicated that mitochondrial energetics and
metabolism are interconnected with immuno-response, we
purpose that the combination of mitochondria-targeting with
immunomodulatory properties may present an effective strategy
to obtain anticancer agents with novel mechanisms of action.
In this work, we designed two Ir(III)-antiphlogistics hybrid
complexes with both mitochondria-targeted capabilities and
immunomodulary activities. [Ir(ppy)2(L2)](PF6) (Ir2; ppy: 2-
phenylpyridine; L2: [2,2'-bipyridine]-4,4'-diylbis(methylene) bis(2-
hydroxybenzoate)) and [Ir(ppy)2(L3)](PF6) (Ir3; L3: [2,2'-
bipyridine]-4,4'-diylbis(methylene) bis(2-acetoxybenzoate)) were
obtained by conjugating SA or As with the cyclometalated Ir(III)
complex [Ir(ppy)2(L1)](PF6) (Ir1; L1: 4,4'-bis(hydroxymethyl)-2,2'-
bipyridine) by ester bonds (Figure 1A). Due to their positive
charge and lipophilicity, Ir1‒Ir3 were anticipated to accumulate
in mitochondria. The hydrolysis of ester bonds, the cytotoxicity
[a]
X. Wu, Y. Zheng, F. Wang, J. Cao, H. Zhang, Dr. D. Zhang, Dr. C.
Tan, Prof. L. Ji, Prof. Z. Mao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry, Sun Yat-Sen University
Guangzhou 510275 (P.R. China)
Email: cesmzw@mail.sysu.edu.cn; tancaip@mail.sysu.edu.cn
These authors contributed equally to this work.
†
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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and cellular uptake properties of Ir2 and Ir3 were investigated.
Their anti-inflammatory properties in inhibition of pro-
inflammatory cytokines were evaluated. Their anticancer
mechanisms, e.g., cell death modes, the impact on
mitochondrial functions and several events related to metastasis
including migration, colony-formation and angiogenesis, were
illustrated. Finally, the in vivo anticancer efficacy was evaluated
in a human tumor xenograft model. The applications of Ir2 and
Ir3 in tracking of mitochondrial morphology and lifetime imaging
were also demonstrated. In all, our study shows that the
combination of mitochondrial targeting and immunomodulary
activities is a feasible strategy to develop novel metal-based
anticancer agents.
Results and Discussion
Synthesis, Characterization and Hydrolysis of
the Ester Bonds
Figure 1. (A) The chemical structures of complexes Ir1‒Ir3. Time-dependent
emission spectra of Ir2 (B) and Ir3 (C) in the presence of PLE measured in
PBS.
The structures of Ir1‒Ir3 are shown in Figure 1A. Ligands L2
and L3 were prepared by condensation of 4,4'-
bis(bromomethyl)-2,2'-bipyridine with SA/As under weak basic
conditions in dry N,N-dimethyl formamide (DMF). Ir1 was
prepared by the literature method.[35] Ir2 and Ir3 were
synthesized similarly by reacting the Ir(III) precursors with the
corresponding ligands in CH2Cl2/CH3OH (1:1, v/v). The ligands
(L2 and L3) and the complexes (Ir2 and Ir3) were characterized
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay after 48 h treatment (Table 1). These cell lines
were chosen because they were reported to be sensitive to As
treatment.[24, 36] The IC50 values of Ir2 and Ir3 fall between 2.0
and 4.5 μM. Both Ir2 and Ir3 show higher toxicity than cisplatin
(up to 10-fold). With IC50 values higher than 200 μM, both As
and SA show negligible cytotoxicity. The result is in accordance
with the literature report indicating that As needs millimolar
doses to elicit cytotoxicity.[37] Ir1 shows moderate cytotoxicity
with IC50 values in the range between 91.2 and 100 μM. In
contrast, the mixtures of 2-fold molar ratio of As or SA with Ir1
do not show much improved cytotoxicity as compared with Ir1
alone. These results indicate that the ligation of As/SA to Ir(III)
complexes can achieve a superior synergistic effect on killing
cancer cells.
1
by ESI-MS (Figure S1‒S4), H NMR (Figure S5‒S8), 13C NMR
(Figure S9‒S12) and elemental analysis.
The photophysical properties of Ir2 and Ir3 in CH3CN,
CH2Cl2 and phosphate buffered saline (PBS) were tested
(Figure S13, S14 and Table S1). The high-energy bands (< 350
nm) are the spin-allowed ligand-centered (1LC) π-π* transitions
for the cyclometalated (C–N) and the 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.
Ir2 and Ir3 exhibit orange red phosphorescent emissions upon
excitation at 405 nm. The phosphorescence lifetimes of Ir2 and
Ir3 in CH3CN, CH2Cl2 and PBS fall between 64 and 631 ns.
The responses of Ir2‒Ir3 towards esterase were monitored
by time-resolved emission spectra and ESI-MS. Time-dependent
emission studies show that the emission spectra of Ir2 and Ir3
display a decrease in the intensities upon treatment with porcine
liver esterase (PLE, Figure 1B and 1C). Also, the mass spectra
show that Ir2 and Ir3 are completely hydrolyzed within 4 h upon
treatment with PLE (Figure S15 and S16).
Immunomodulatory Properties
Pro-inflammatory cytokines including interleukin (IL)-6 and tumor
necrosis factor alpha (TNF-α), are of great importance for
malignant proliferation, cancer progression, metastasis and
evasion of immune surveillance.[24, 38] Meanwhile, they are also
considered to be key growth-promoting and antiapoptotic factors
that can protect cancerous cells from apoptosis.[39] The effects
of Ir1−Ir3, a combination of Ir1 with As/SA, and As/SA alone
on IL-6 and TNF-α levels in lipopolysaccharide (LPS)-activated
RAW 264.7 macrophage-like cells mimicking the inflammatory
environment in cancer were evaluated by ELISA (Enzyme
Linked Immunosorbent Assay). A concentration-dependent
inhibition on IL-6 and TNF-α is observed for both preventative
(Figure 2A and 2B) and therapeutic anti-inflammatory treatment
(Figure 2C and 2D). The level of IL-6 is significantly
In Vitro Cytotoxicity
The cytotoxicity of Ir1–Ir3, As, SA, a 2-fold molar ratio of As or
SA mixed with Ir1, and cisplatin was determined against
prostate carcinoma (PC3), colon carcinoma (CT26) and human
colon adenocarcinoma (HT29) cells by the
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reduced by 2.1–19.4 fold in LPS-activated macrophages treated
with Ir2 or Ir3. Similar results are also observed for TNF-α
production, which is decreased by 2.1–21.1 fold in cells treated
with Ir2 and Ir3. However, Ir1, As, SA and the mixtures of Ir1
and As/SA show moderate effect on the levels of these
cytokines under the same conditions.
Table 1. IC50 values of tested compounds to different cellsa
IC50 (μM)
Compound
PC3
CT26
HT29
Cyclooxygenase (COX), a vital enzyme in prostaglandin
generation, is considered to be main target of As.[40] There are
two isoforms COX in which constitutive overexpression of COX-
2 is associated with resistance to apoptosis, which is the
hallmark of cancer cells.[41] In vitro COX-2 inhibitory screening
shows that Ir3 displays potent inhibition on COX-2, close to that
observed for As (Figure 2E). A much lower inhibitory activity is
observed for Ir2 and SA which is reasonable because As is
more effective than SA as an inhibitor of COX.[42] Since many
cancerous cells including PC3 cells over-express COX-2,[43] we
next investigated whether Ir3 could also influence the COX-2
expression in PC3 cells. An obviously down-regulated
expression level of COX-2 is observed in cells treated with Ir3
(Figure 2F), which is similar to that observed for As. The result
indicates that COX-2 inhibition may contribute to the
immunomodulatory properties of Ir3.
Ir1
> 100
> 100
91.2 ± 2.1
2.7 ± 0.3
2.8 ± 0.3
56.2 ± 3.1
85.1±6.6
> 200
Ir2
2.0 ± 0.4
4.5 ± 0.5
54.9 ± 2.6
63.1 ± 2.3
> 200
2.2 ± 0.3
4.4 ± 0.7
> 100
Ir3
Ir1+2 SA
Ir1+2 As
SA
> 100
> 200
As
> 200
> 200
> 200
cisplatin
25.1 ± 2.2
22.4 ± 2.6
70.8 ± 4.5
a Cell viability was evaluated after 48 h incubation.
Figure 2. (A‒D) Anti-inflammatory properties of Ir(III) complexes. The effects of Ir2, Ir3, As, SA, and mixtures of Ir1 with As/SA at molar ratio of 1:2 on the levels
of IL-6 and TNF-α in RAW 264.7 macrophages assessed by ELISA. (A and B) The cells were pretreated with LPS (100 ng/mL, 6 h) and then incubated with tested
compounds at the indicated doses for 12 hours. The concentration not mentioned in the figure is the highest dose of Ir2 and Ir3. (C and D) The cells were
incubated with test compounds for 6 hours and further treated with LPS (100 ng/mL, 12 h). The concentration not mentioned in the figure is the highest dose of Ir2
and Ir3. (E) Inhibition of human COX-2 by Celecoxib (100 nM), As (1 mM, 2 mM), SA (1 mM, 2 mM), Ir2 (0.5 mM, 1 mM) and Ir3 (0.5 mM, 1 mM) in vitro. (F)
Impact of Ir1 (2 μM), Ir3 (2 μM) and As (4 μM) on the expression of COX-2 in PC3 cells analyzed by immunofluorescence staining. Scale bar: 10 μm.
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Deep Red (MTDR, Figure S19A), indicating that Ir2 and Ir3 can
locate in mitochondria specifically. The pearson’s colocalization
coefficients of Ir2 and Ir3 with MTDR are 0.84 and 0.86,
respectively, while little overlap is found for Ir2/Ir3 with
LysoTracker Deep Red (LTDR, Figure S19B). As measured by
ICP-MS, the content of iridium in mitochondria is higher than that
in cytoplasm and nucleus (Figure S19C), indicating that both Ir2
and Ir3 can accumulate in mitochondria.
The morphology of the mitochondria can reflect the status of
the mitochondria.[44] Real-time tracking of the mitochondrial
morphology was carried out by monitoring the emission of Ir2
and Ir3 in PC3 cells using confocal microscopy. PC3 cells can
be effectively labelled by Ir2 and Ir3 after 0.5 h incubation, when
mitochondria show the normal tubular network and distribution.
As the incubation time increases, the normal tubular
mitochondrial network turns into swelling mitochondrial
aggregates, large perinuclear clusters (Figure 3 A and C).
Since iridium complexes have environmental-sensitive long
phosphorescence lifetimes, time-gate imaging can be used to
reduce the interference the background fluorescence.[14] More
importantly, PLIM technology is very advantageous for
phosphorescent metal complexes to accurately reflect the
changes in the microenvironments.[45-46] After 4 h of treatment,
the cellular lifetimes of Ir2 and Ir3 increase gradually (Figure 3B
and D). The lifetime of Ir2 changes from 240 ns to 388 ns, while
that of Ir3 changes from 300 ns to 360 ns. Because the
complexes could go through hydrolysis in cells, we first studied
the effect of hydrolysis on the lifetimes of Ir2 and Ir3. After
treatment with the hydrolase, the lifetime of Ir2 decreases from
393 ns to 142 ns, while that of Ir3 decreases from 631 ns to 249
ns (Figure S20). Therefore, hydrolysis is not the reason for the
time-dependent increase in the lifetimes of Ir(III) in cells. During
the process of mitochondrial damage, the polarity and other
physical properties of mitochondria will change significantly.[47]
Therefore, we studied the lifetimes of complexes in solvents of
different polarities. The lifetimes of Ir2 and Ir3 are indeed
affected by polarity, but no clear correlation is detected. The
lifetime (τ0(CH3CN) < τ0(DMSO) < τ0(CH2Cl2)) does not increase
with the increase of polarity (CH2Cl2 < CH3CN < DMSO, Figure
S21). Finally, we speculate that the increase in the lifetimes of
complexes in cells may be due to the aggregation of complexes.
It has been reported that the fluorescence intensities and
lifetimes of iridium complexes can be significantly influenced by
the aggregation states.[48-50] As in the solid state, the complexes
show relatively longer lifetimes (Table S1). Moreover, with the
increase of the aggregation degree of complexes, their lifetimes
tend to increase (Figure S22). Therefore, we reckon that the
changes in the aggregation state of the complexes in
mitochondria lead to the increase in the lifetimes of complexes in
cells.
Figure 3. Real-time tracking of the mitochondrial morphology by Ir2 (10 μM,
A) and Ir3 (10 μM, C) monitored by confocal microscopy over a period of 4 h.
λex = 405 nm; λem = 600 ± 20 nm. PLIM images of PC3 cells incubated with Ir2
(10 μM, B) and Ir3 (10 μM, D). λex = 405 nm; λem = 600 ± 20 nm. Scale bar: 20
μm.
Mitochondrial Real-Time Tracking and
Lifetime Imaging
The log Po/w values, representing the lipophilicity of the drug, of
Ir1 (0.21), Ir2 (0.70) and Ir3 (1.01) are determined by a shake-
flask method. After an incubation at 10 μM for 1 h, inductively
coupled plasma-mass spectrometry (ICP-MS) measurement
shows that the cellular uptake levels of Ir1, Ir2 and Ir3 are 0.22,
2.00 and 2.18 ng per μg protein, respectively. The results
indicate that the conjugation elevates the lipophilicity of the
complexes, thereby increases their uptake efficiency and
cytotoxicity. The emission of Ir2 and Ir3 in cells incubated at
4 °C or pretreated with carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) is weaker than that incubated at 37 °C
(Figure S17). However, chloroquine doesn’t show obvious effect
on the uptake of Ir2 and Ir3. The results indicate that Ir2 and Ir3
enter cells mainly by an energy-dependent mechanism instead
of endocytosis.
Induction Mitochondrial Dysfunction and
Metabolic Disorders
Confocal microscopy experiment shows that Ir2 (10 μM)
and Ir3 (10 μM) can effectively enter PC3 cells after an
incubation for 1 h (Figure S18). Ir2 and Ir3 are mainly scattered
in cytoplasm. Both Ir2 and Ir3 can overlap with MitoTracker
As Ir2 and Ir3 located in mitochondria, the influence on
mitochondria integrity was then monitored by detecting the
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Figure 4. (A) Detection of MMP stained with JC-1 on flow cytometry. λex = 488 nm. λem = 530 ± 30 nm and 585 ± 30 nm. (B) Intracellular ROS generation detected
by DCF (λex = 488 nm; λem = 530 ± 30 nm) with flow cytometry. For (A) and (B), PC3 cells were treated with vehicle (1% dimethyl sulfoxide (DMSO)), As, SA, Ir1,
mixtures containing Ir1 and two-fold molar ratio of As or SA, Ir2 or Ir3 in the mentioned concentrations for 6 h. (C) Respiratory curves of control, Ir2- and Ir3-
treated PC3 cells under basal conditions, and after the addition of oligomycin (1 mM), FCCP (0.8 mM) and the mixture of rotenone (0.5 mM) and antimycin A (0.5
mM) were recorded by
a Seahorse XF24 extracellular flux analyzer. (D) Basal respiration was computed by subtracting OCR values after adding
rotenone/antimycin A from basal OCR. (E) ATP production was computed by subtracting OCR values after adding oligomycin from basal OCR. (F) Maximum
mitochondrial respiration was computed by subtracting OCR values after adding rotenone/antimycin A from the OCR values after the adding FCCP. (G) Non-
mitochondrial respiration was the OCR value after adding rotenone/antimycin A. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. (H) Summary of
metabolic pathway analysis of PC3 cells treated with Ir3. The color of the circles means the pathway impact, and the area of the circles means p-value.
changes of mitochondrial membrane potential (MMP) using
5,5’,6,6’-tetrachloro-1,1’-3,3’-tetraethyl-
and Ir3-treated cells (Figure 4A). After 6 h treatment, the
proportion of cells with depolarized mitochondria increase from
6.7% (control) to 79.3% and 81.4% for Ir2 (8 μM) and Ir3 (8 μM),
respectively. The ability of these complexes to depolarize
benzimidazolylcarbocyanine
iodide
(JC-1)
staining.
A
concentration-dependent reduction in MMP can be found in Ir2-
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mitochondria correlates with their cytotoxicity. The impact of Ir1
in combination of 2-fold of As or SA on MMP is much lower than
the corresponding conjugates.
To further investigate the effects of Ir2 and Ir3 on
mitochondrial energetics, the intracellular ATP levels were
measured. Compared with the control, Ir2 and Ir3 treatment
causes dose-dependent declines in ATP levels (Figure S23). At
a concentration of 8 μM, the ATP levels decrease by about
46.8% and 47.6 % for Ir2 and Ir3, respectively.
acids metabolism, causing the accumulation of valine, leucine,
isoleucine, phenylalanine, lysine, aspartate and tyrosine. Similar
result is found in Ir2-treated samples. Ir2-treated cells showed
accumulation of pyruvate and citrate cycle metabolites, pyruvate,
citrate and malate. Ir2 impairs the amino acids metabolism,
causing the accumulation of glycine, valine, leucine, isoleucine,
threonine, serine, tyrosine and lysine. The result indicates that
Ir2 and Ir3 can impair mitochondrial biosynthesis leading to
mitochondrial dysfunction.
The increase of intracellular ROS levels caused by Ir2 and
Ir3 were then measured by the 2′,7′-dichlorodihydrofluorescein
diacetate (H2DCF-DA) staining assay. After treated with Ir2 and
Ir3, the cellular ROS levels are increased obviously (Figure 4B).
The emission of DCF increases by about 22.9-fold and 24.3-fold
in cells incubated with Ir2 (8 μM, 6 h) and Ir3 (8 μM, 6 h),
respectively.
We also evaluated the effect of Ir2 and Ir3 on mitochondrial
respiration by measuring the oxygen consumption rate (OCR).
Several key parameters were measured to assess mitochondrial
oxidative phosphorylation (OXPHOS)[51] by using respiration
modulators targeting electron transport chain components
(Figure 4C). Compared with control, cells treated with Ir2 and Ir3
show a concentration-dependent decrease in basal respiration
(Figure 4D), ATP production (Figure 4E), and maximal
respiration (Figure 4F). A slight decrease is also founded in non-
mitochondrial respiration (Figure 4G). These results imply that
Ir2 and Ir3 can damage the mitochondrial bioenergetic functions
by interfering with the mitochondrial respiration.
Cancer cells are reported to have altered or dysfunctional
mitochondrial metabolism.[52] The impact of Ir2 and Ir3 on
mitochondrial metabolism was investigated. The metabolites of
PC3 cells treated with Ir2 or Ir3 were extracted with methanol
and chloroform and analyzed by GC-TOF-MS (Gas
Chromatography Time-of-Flight Mass Spectrometry). The data
were analyzed using PCA (principal component analysis) to find
outliers (Figure S24). Then, candidate biomarkers were picked
out from S-plots following analysis of OPLS-DA (orthogonal
projections to latent structures discriminant analysis). The
OPLS-DA score plots for the Ir2-treated samples and controls
are shown in Figure S25. The R2Y (the goodness of fit) and Q2Y
(predictability) values are 0.989 and 0.91, respectively. The
cluster of the Ir2-treated group is located far away from that of
the controls, which indicates that the metabolic profile of Ir2-
treated samples is different from that of the controls. Similarly,
the OPLS-DA score plots of Ir3-treated samples and controls
(Figure S27, R2Y = 1, Q2Y = 0.98) show a clear discrimination.
The S-plots are shown in Figure S26 and S28. To find potential
biomarkers, 18 potential biomarkers (Table S2) between Ir2-
treated group and control group with the VIP >1 and p-value
<0.05 were selected for further analysis. Similarly, the 22
potential biomarkers (Table S3) between Ir3-treated group and
control group were selected for further analysis. To explore the
potential metabolic pathways, these metabolites were then
analyzed by MetaboAnalyst (Figure 4H and Figure S29). Ir3-
treated cells exhibit aberrant levels of pyruvate metabolites
(elevation of pyruvate and reduction of lactate) that is associated
with glycolysis/gluconeogenesis. Ir3 also impairs the amino
Induction of Caspase-Dependent Apoptosis
and Caspase-Independent Paraptosis
Based on morphological and biochemical criteria, cell death can
be divided into many categories, e.g., apoptosis, necrosis,
autophagy and paraptosis.[53] The morphological changes in
Ir(III)-treated cells were first analyzed by transmission electron
microscopy (TEM) (Figure 5A). Extensive cytoplasmic
vacuolation, widening of the membrane space with intact
nucleus are observed in cells treated with Ir2 and Ir3 at a lower
dose (2 μM). These morphological changes are the typical
paraptosis symptoms.[54] However, cells treated with Ir2 and Ir3
at a higher concentration (8 μM) display typical characteristics of
apoptosis including chromatin condensation and nuclear
fragmentation[55]
.
2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-1H,3'H-2,5'-
bibenzimidazole (Hoechst 33342) staining doesn’t show obvious
change in cell nuclei after Ir2- or Ir3-treatment at a relatively
lower concentration (2 μM) in PC3 cells, but typical apoptotic
alternations are observed in cells treated at a higher dose (8 μM)
(Figure 5B). Apoptosis is usually caspase-dependent while
paraptosis often occurs in the caspase-independent pathway.[56]
Compared with the control, little change of caspase-3/7 activity
is found in cells treated with Ir2 or Ir3 at 2 μM (Figure 5C).
However, the activity of caspase-3/7 is increased by
approximately 4.2- and 8.5-fold in Ir2- and Ir3-treated cells at 8
μM, respectively.
In order to further verify the cell death modes induced by Ir2
and Ir3, we studied the effect of different cell death inhibitors on
cell viability. Pretreatment of the pan-caspase inhibitor z-VAD-
fmk (50 μM) increases the cell viability for Ir2 (8 μM) and Ir3 (8
μM) treatment (Figure 5D). Treated with cycloheximide, an
inhibitor associated with paraptosis,[52] can effectively diminish
the cell death induced by Ir2 and Ir3 at a low concentration (2
μM) (Figure 5E). But cycloheximide fails to protect cells from the
cytotoxic effects of Ir2 and Ir3 at
a relatively higher
concentration (8 μM). The possibilities of autophagy and
necrosis are excluded based on the fact that 3-MA (3-
methyladenine, an autophagy inhibitor) and Nec-1 (a RIP1-
specific inhibitor that can prevent necrosis) show no obvious
effects on both Ir2- and Ir3-induced cell death (Figure S30 and
S31). These results imply that Ir2 and Ir3 can induce caspase-
independent paraptosis and caspase-dependent apoptosis at
lower and higher concentrations, respectively.
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Figure 5. (A) TEM graphs of PC3 cells treated with Ir2 and Ir3 at the indicated doses for 24 hours. PC3 cells were treated with vehicle (a), Ir1 (10 μM, b and c),
Ir2 (4 μM, d and e), Ir2 (8 μM, f), Ir3 (4 μM, g and h) or Ir3 (8 μM, i) for 24 h. Nuclei are labelled N. Black arrows: the nuclear membrane is affected, while the
nucleus is intact. The red dotted box represents the enlarged area. (B) PC3 cells staining with Hoechst 33342 after treated with Ir2 and Ir3 at the indicated doses
for 24 hours. (C) Caspase-3/7 activity in PC3 cells after incubated with Ir2, Ir3 or cisplatin for 6 hours. (D) The effect of z-VAD-fmk on the cytotoxicity of Ir2 and Ir3.
PC3 cells were incubated with Ir2 and Ir3 at varying concentrations for 24 hours in the absence or presence of z-VAD-fmk. (E) The impact of cycloheximide on
the cytotoxicity of Ir2 and Ir3. *p< 0.05, **p< 0.01, as compared with the group without z-VAD-fmk or cycloheximide treatment.
of the vehicle control cells is counted as 100%), respectively
(Figure 6B and 6D).
Inhibition of Metastasis-Related Events
Then, we evaluated the capability of inhibiting colony formation
by Ir2 and Ir3 at different concentrations. As shown in Figure 6E,
Cancer is a multistep and multifactorial process, and cancer
cells are different from their normal counterparts in many
aspects.[58] The effects of Ir1‒Ir3 on migration was measured by
the wound healing assay and transwell assay. After 24 h
incubation, Ir1‒Ir3 (0.5 μM) can effectively inhibit cell movement
with a wound closure ratio of 3.1‒4.6%, which is much lower as
compared with that of the vehicle control (18.6%) (Figure 6A and
6C). Transwell assay shows that Ir2 (0.5 μM) and Ir3 (0.5 μM)
decrease the cell migration ratios to 48.2% and 54.5% (the ratio
after 48
h exposure, both Ir2 and Ir3 demonstrate a
concentration-dependent anti-proliferative effect on colony
formation. Colonies can be completely restrained after treated
with Ir2 or Ir3 at 1 μM. The cytotoxic assay shows that the cell
viability is not obviously affected at the concentration tested
(Figure S32).
A tube formation assay was carried out to assess the anti-
angiogenesis properties of the Ir(III) complexes
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Figure 6. (A) Wound healing of PC3 cells after treated with Ir1‒Ir3 (0.5 μM) for 24 h. (B) Graphs of migrated cells. PC3 cells (1×105) were treated with Ir1‒Ir3 (0.5
μM) for 24 hours. Scale bars: 100 μm. (C) Wound closure rate. Wound closure (%) = [1 −(width at 24 h) /(width at 0 h)] × 100%. (D) Percentage of migrated cells
compared with the control. (E) Inhibition of colony formation by Ir2 and Ir3 at the indicated doses. PC3 cells were treated with Ir2 and Ir3 for 48 hours and further
incubated for a week. (F) Effect of Ir1‒Ir3 against the tube formation of HUVEC on matrigel. HUVEC seeded on matrigel in medium were treated with As (2 μM),
SA (2 μM), Ir1 (1 μM), Ir2 (1 μM) or Ir3 (1 μM) for 4 h. scale bar：200 μm. (G) Quantification of tube formation or intersections between cells counted manually.
(Figure 6F and 6G).Human umbilical vein endothelial cells
(HUVEC) can connect as network after plated on the matrix.
Compared with the control cells, much less tubes are formed
and the network disappears in Ir(III)-treated samples. MTT
assay shows that the compounds has low cytotoxicity at this
concentration (Figure S33). Similar phenomena are also
observed for SA and As, which is also consistent with the
literature reports that anti-angiogenesis is involved in the
anticancer properties of As.[59]
same conditions, the in vivo anticancer activity of Ir2/Ir3 is
approximate to that of cisplatin. No significant difference is found
in body weight among all groups of mice (Figure 7C), which
indicates Ir2 and Ir3 do not cause severe side effects in these
conditions.
Conclusion
In this study, two Ir(III) complexes with a cyclometalated Ir(III)
moiety for mitochondrial targeting and As or SA for immuno-
modulating, linked through ester bonds have been synthesized.
Conjugation of SA or As to the Ir(III) moiety can improve the
cellular uptake efficacy of the Ir(III) complexes and achieve a
high synergetic effect. Mechanism studies indicate that Ir2 and
Inhibition of Tumor Xenografts Growth In Vivo
To evaluate the antitumor effects of Ir2 and Ir3 in vivo, we used
PC3 cells to build subcutaneous tumor xenografts model in
female BALB/c nude mice. As shown in Figure 7A and Figure 7B, Ir3 accumulate in mitochondria and induce caspase-dependent
tumor growth was significantly slowed in mice intraperitoneally
injected with Ir2 and Ir3, compared with the control group. After
21 days treatment, the tumor volume decreases by 44 ± 13%
and 55 ± 7% for Ir2- and Ir3-treatment, respectively. Under the
apoptosis at lower doses and caspase-independent paraptosis
at higher concentrations. Using PLIM techniques, we observed
that Ir2 and Ir3 aggregate in the mitochondria over time. As
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Materials and Measurements
IrCl3•nH2O, ppy, DMF, SA, As, NH4PF6 were purchased from Alfa Aesar
(USA). L1 was purchased from TCI (China). Cisplatin, DMSO, MTT,
Hoechst 33342 and H2DCF-DA were purchased from Sigma Aldrich
(USA). MTDR, LTDR, and JC-1 were obtained from Life Technologies
(USA). LPS was purchased from Invitrogen (USA). Primary rabbit
polyclonal antibody for COX-2 and Alexa Fluor® 488 goat anti-rabbit
secondary antibody were purchased from Abcam (USA). Cellular ATP
quantification assay kit and caspase-3/7 activity kit were purchased from
Promega (USA). TNF-α and IL-6 were tested using eBiosciences ELISA-
Ready-Set Go® (USA).
ESI-MS was recorded on
a Thermo Finnigan LCQ DECA XP
spectrometer (USA). NMR spectra were recorded on a Bruker Avance III
400 MHz spectrometer or a Bruker Avance III 500 MHz spectrometer.
Elements analysis (C, H, and N) was done by an Elemental Vario EL
CHNS analyzer. UV/Vis spectra were conducted on a Varian Cary 300
spectrophotometer. Emission spectra and lifetimes were measured by a
fluorescence spectrometer (FLS 920, Edinburgh Instruments Ltd). Cell
imaging experiments were performed by a confocal microscope (LSM
710, Carl Zeiss, Göttingen). Flow cytometry was carried out by a BD
FACS Calibur™ flow cytometer (Becton Dickinson).
Synthesis and Characterization
4,4'-bis(bromomethyl)-2,2'-bipyridine.
4,4'-bis(bromomethyl)-2,2'-
bipyridine was prepared according to the literature method [60]. 4,4'-
bis(hydroxymethyl)-2,2'-bipyridine (4 g, 11.7 mmol) was dissolved in a
mixture of HBr (48%, 30 mL) and concentrated H2SO4 (10 mL). The
resulting solution was refluxed for 6 h and allowed to cool, and water
(100 mL) was added. The pH was adjusted to 7.0 by the addition of
NaOH saturated solution, and the resulting precipitate was filtered,
washed with water, and air-dried.
General procedures for the preparation of L2 and L3. 2 mmol
phenolic acid was dissolved in 3 mL dry DMF, and 2.4 mmol KHCO3 was
added and stirred for several minutes at room temperature. Then, 4,4'-
bis(bromomethyl)-2,2'-bipyridine (342 mg, 1 mmol) were added. The
reaction mixture was stirred for 6 h at room temperature. The reaction
mixture was added to 10 mL water and extracted with ethyl acetate. The
organic layer was subsequently washed with 5% NaHCO3 and 5% NaCl,
dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give the desired products.
Figure 7. Representative graphs of nude mice (A) after 21 days treated with
PBS, Ir2, Ir3 and cisplatin. Growth curve of tumor volume (B) and body weight
of mice (C) after intraperitoneal injection of PBS, Ir2, Ir3 and cisplatin.
[2,2'-bipyridine]-4,4'-diylbis(methylene) bis(2-hydroxybenzoate) (L2):
L2 was obtained as a white solid. Yield: 415 mg (91%). 1H NMR (400
MHz, CDCl3) δ 10.58 (s, 2H, OH-1), 8.72 (d, J = 5.0 Hz, 2H, CH-2), 8.50
(s, 2H, CH-3), 7.95 (dd, J = 8.0, 1.5 Hz, 2H, CH-4), 7.55–7.46 (m, 2H,CH-
5), 7.40 (d, J = 4.5 Hz, 2H, CH-6), 7.01 (d, J = 8.4 Hz, 2H, CH-7), 6.92 (t,
J = 7.6 Hz, 2H, CH-8), 5.49 (s, 4H, CH2-9). ESI-MS (CH3OH): m/z 457.6
[M+H]+. Elemental analysis calcd (%) for C26H20N2O6•0.25 H2O: C 67.75,
H 4.48, N 6.08; found: C 67.73, H 4.69, N 6.28.
expected, they exhibit immunomodulatory properties by
regulating the activity of some immune factors and inhibiting
COX-2. Moreover, they can inhibit migration, colony-formation
and angiogenesis. In vivo experiments also show that Ir2 and Ir3
show potent growth inhibitory effect on human solid tumor and
low systemic toxicity. Our study shows that a joint action of
mitochondrial targeting with immuno-modulating anticancer
strategies is a feasible strategy to develop metalloanticancer
agents with multiple anticancer effects.
[2,2'-bipyridine]-4,4'-diylbis(methylene) bis(2-acetoxybenzoate) (L3):
L3 was obtained as a white solid. Yield: 459 mg (85%). 1H NMR (500
MHz, CDCl3) δ 8.71 (d, J = 5.0 Hz, 2H, CH-1), 8.47 (s, 2H, CH-2), 8.12
(dd, J = 7.9, 1.6 Hz, 2H, CH-3), 7.60 (td, J = 7.8, 1.6 Hz, 2H, CH-4), 7.38
(dd, J = 4.9, 0.8 Hz, 2H, CH-5), 7.37 – 7.32 (m, 2H, CH-6), 7.13 (d, J =
8.0 Hz, 2H, CH-7), 5.42 (s, 4H, CH2-8), 2.26 (s, 6H, CH3-9). ESI-MS
(CH3OH): m/z 541.4 [M+H]+. Elemental analysis calcd (%) for
C30H24N2O8•0.8 CH3OH: C 65.34, H 4.84, N 4.95; found: C 65.93, H 4.94,
N 5.14.
Experimental Section
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Synthesis of Ir1‒Ir3. The dimeric iridium(III) precursors [Ir(ppy)2(μ-Cl)]2
were prepared according to the literature method. Complex Ir1 was
synthesized according to the literature methods [35]. Complexes Ir2 and
Ir3 were synthesized similarly. A mixture of [Ir(ppy)2(μ-Cl)]2 (0.2 mmol)
and L2 (0.4 mmol) or L3 (0.4 mmol) in CH2Cl2/CH3OH (1:1, v/v) was
heated to reflux under the protection by nitrogen in the dark for 6 h. The
solution was cooled to room temperature and evaporated to dryness
under reduced pressure. The oil-like substance was dissolved in CH2Cl2
and purified by column chromatography on silica gel eluted with
CH2Cl2/CH3OH. A 4-fold excess of NH4PF6 was added into the solution
and stirred for 1 h. The mixture was filtered and the filtrate was
evaporated under reduced pressure to obtain dry powder. The crude
product was washed with water to remove excess NH4PF6. The desired
product was further purified by recrystallization.
before Ir2 and Ir3 were added. Each experiment was repeated at least
three times to gain the mean values.
Anti-Inflammatory Properties by ELISA
RAW 264.7 cells were seeded in 96-well plates and allowed to grow
overnight. For the preventative anti-inflammatory treatment, RAW cells
were treated with Ir1 (10 µM), As (10 µM), SA (10 µM), mixtures
containing Ir1 (10 µM) and As (20 µM)/SA (20 µM), Ir2 (0.4, 0.8 or 1.6
µM), and Ir3 (0.4, 0.8 or 1.6 µM) for 6 hours. The cells were treated with
LPS (100 ng/mL) for another 12 hours. For the therapeutic anti-
inflammatory treatment, the cells were treated with LPS (100 ng/mL) for 6
hours. Ir1 (10 µM), As (10 µM), SA (10 µM), mixtures containing Ir1 (10
µM) and As (20 µM)/SA (20 µM), Ir2 (0.4, 0.8 or 1.6 µM), and Ir3 (0.4,
0.8 or 1.6 µM) were then added and incubated for 12 hours. ELISA was
performed on the supernatants against interleukin IL-6 and TNF-α,
according to the manufacturer's instructions.
[Ir(ppy)2(L2)](PF6) (Ir2): Ir2 was obtained as an orange red solid. Yield:
335 mg (76%). 1H NMR (500 MHz, [d6]-DMSO) δ 10.39 (s, 2H, OH-1),
8.96 (s, 2H,CH-2), 8.27 (d, J = 8.2 Hz, 2H, CH-3), 7.98 – 7.91 (m, 6H,
CH-4), 7.90 (d, J = 5.7 Hz, 2H, CH-5), 7.86 (d, J = 5.4 Hz, 2H, CH-6),
7.69 (d, J = 5.6 Hz, 2H), 7.57 – 7.50 (m, 2H), 7.17 (t, J = 6.2 Hz, 2H),
7.06 – 6.98 (m, 4H), 6.96 – 6.88 (m, 4H, CH-11), 6.21 (d, J = 7.5 Hz, 2H,
CH-12), 5.63 (s, 4H, CH2-13). ESI-MS (CH3OH): m/z 957.5 [M−PF6]+.
Elemental analysis calcd (%) for C48H36F6IrN4O6P•H2O: C 51.47, H 3.42,
N 5.00; found: C 51.30, H 3.31, N 5.03.
Mitochondrial Bioenergetics Analysis
The experiment was carried out as literature mentioned [35]. PC3 cells
were seeded 2 × 104 per well. The optimal concentrations of the
compounds were as following: oligomycin (1 μM), FCCP (0.8 μM) and a
mixture of antimycin A (0.5 μM) and rotenone (0.5 μM).
[Ir(ppy)2(L3)](PF6) (Ir3): Ir3 was obtained as a bright yellow solid. Yield:
323 mg (68%). ESI-MS (CH3OH): m/z 1041.4 [M−PF6]+. 1H NMR (400
MHz, [d6]-DMSO) δ 8.90 (s, 2H), 8.28 (d, J = 8.2 Hz, 2H), 8.11 – 8.08 (m,
2H), 7.94 (d, J = 8.0 Hz, 4H), 7.89 (d, J = 5.7 Hz, 2H), 7.82 – 7.79 (m,
2H), 7.79 – 7.69 (m, 4H), 7.68 (d, J = 5.8 Hz, 2H), 7.41 (td, J = 7.7, 1.1
Hz, 2H), 7.26 (dd, J = 8.1, 0.9 Hz, 2H), 7.19 – 7.14 (m, 2H), 7.06 – 7.00
(m, 2H), 6.92 (td, J = 7.4, 1.1 Hz, 2H), 6.20 (d, J = 6.8 Hz, 2H), 5.53 (s,
4H), 2.10 (s, 6H).Elemental analysis calcd (%) for C53H43F6IrN4O8P: C
53.00, H 3.61, N 4.66; found: C 52.96, H 3.64, N 4.57.
Metabolomics Analysis
Sample preparation: PC3 cells were plated in 15 cm dishes (Corning)
and cultured for 24 hours. After incubated with Ir2 and Ir3 for 6 hours, the
PC3 cells were trypsinised, washed 3 times with PBS and counted. The
centrifuged cell pellets were soaked in liquid nitrogen for at least 3 min
then stored at −80 °C before test.
Metabolites extraction：The sample were transferred to 2 mL EP tubes
and extracted with 1 mL extraction solvent (VMethanol:VChloroform = 3:1). 5 μL
of adonitol (internal standard, 0.5 mg/mL in dH2O) was then added.
Hydrolysis of Ir2 and Ir3 by PLE In Vitro
Time-dependent emission spectra and Luminescence decay signals.
Ir2 and Ir3 (20 μM) in degassed PBS were freshly prepared and added
into quartz cuvettes (3 mL), and then PLE (1 μL) in (NH4)2SO4
suspension was sequentially added. For each time, the emission spectra
were recorded after 5 min incubation of the reaction mixture at 310 K.
The luminescence decay curves were recorded for the indicated time
intervals.
GC-TOF-MS analysis： GC-TOF-MS analysis was done according to
literature conditions.[62]
Data processing and annotation: Peaks were exacted using Chroma
TOF 4.3X software and LECO-Fiehn Rtx5 database.[62]
Statistical analysis: Statistical analysis was performed by SIMCA-P
14.1 software. PCA was carried out to detect trends, and groupings. The
data were analyzed by orthogonal partial least squares (OPLS). The
variables with variable importance projection (VIP) > 1 in OPLS-DA score
plots were usually regarded as candidate biomarkers. Metabolism
pathways were noted by the Kyoto Encyclopedia of Genes and Genomes
(KEGG) database. Pathway enrichment analysis was conducted with
MetaboAnalyst.
ESI-MS. Ir(III) solutions (20 μM，3 mL) in degassed PBS were freshly
prepared and PLE (1 μL) was added. The samples were analyzed by
ESI-MS after incubation at 310 K for different time intervals.
Cell Lines and Culture Conditions
PC3, HT29, CT26, RAW 264.7 and HUVEC were purchased from
Experimental Animal Center of Sun Yat-Sen University (Guangzhou,
China). Cells were cultured with ATCC recommended media. The cells
were maintained in tissue culture flasks in an incubator at 37 °C under
5% CO2.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (21778078, 21837006, 21571196 and
21572282), the 973 program (2015CB856301), the Guangdong
Natural Science Foundation (2015A030306023), the Innovative
Research Team in University of Ministry of Education of China
Cytotoxicity Assay
The cytotoxicity of the compounds was measured as previously
described [61]. When necessary, cells were pretreated with cycloheximide
(1 μM), z-VAD-fmk (50 μM), 3-MA (1 mM) or Nec-1 (100 μM) for an hour
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Keywords: iridium complexes
•
aspirin
•
anticancer
•
mitochondrion • immunomodulation
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10.1002/chem.201900851
Chemistry - A European Journal
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We report here two iridium complexes
modified with anti-inflammatory drugs
show multiple anticancer effects by
disrupting the metabolism of cancer
cells and regulating their immune
functions. The aggregation of
Xiao-Wen Wu, Yue Zheng, Fang-Xin
Wang, Jian-Jun Cao, Hang Zhang,
Dong-Yang Zhang, Cai-Ping Tan,*
Liang-Nian Ji, and Zong-Wan Mao*
Page No. – Page No.
complexes in mitochondria can also
be observed by phosphorescence
lifetime imaging.
Anticancer Ir(III)-Aspirin Conjugates
for Enhanced Metabolic Immuno-
Modulation and Mitochondrial
Lifetime Imaging
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