Sulfur-Coordinated Organoiridium(III) Complexes Exert Breast Anticancer Activity via Inhibition of Wnt/β-Catenin Signaling

Article abstract of DOI:10.1002/anie.202015009

The sulfur-coordinated organoiridium(III) complexes pbtIrSS and ppyIrSS, which contain C,N and S,S (dithione) chelating ligands, were found to inhibit breast cancer tumorigenesis and metastasis by targeting Wnt/β-catenin signaling for the first time. Treatment with pbtIrSS and ppyIrSS induces the degradation of LRP6, thereby decreasing the protein levels of DVL2, β-catenin and activated β-catenin, resulting in downregulation of Wnt target genes CD44 and survivin. Additionally, pbtIrSS and ppyIrSS can suppress cell migration and invasion of breast cancer cells. Furthermore, both complexes show the ability to inhibit sphere formation and mediate the stemness properties of breast cancer cells. Importantly, pbtIrSS exerts potent anti-tumor and anti-metastasis effects in mouse xenograft models through the blockage of Wnt/β-catenin signaling. Taken together, our results indicate that pbtIrSS has great potential to be developed as a breast cancer therapeutic agent with a novel mechanism.

Full text of DOI:10.1002/anie.202015009

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: Sulfur-coordinated organoiridium(III) complexes exert anti-breast
cancer activity via inhibiting Wnt/β-catenin signaling
Authors: Qi Sun, Yi Wang, Qiuxia Fu, Ai Ouyang, Shanshan Liu,
Zhongyuan Wang, Zijie Su, Jiaxing Song, Qianling Zhang,
Pingyu Zhang, and Desheng Lu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202015009
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
Sulfur-coordinated organoiridium(III) complexes exert anti-breast
cancer activity via inhibiting Wnt/β-catenin signaling
Qi Sun,[a] Yi Wang,[b, c] Qiuxia Fu,[a] Ai Ouyang,[b] Shanshan Liu,[a] Zhongyuan Wang,[a] Zijie Su,[a] Jiaxing Song,[a] Qianling
Zhang,[b] Pingyu Zhang,[b] and Desheng Lu[a]
Abstract: The sulfur-coordinated organoiridium(III) complexes
pbtIrSS and ppyIrSS, which contain C,N and S,S (dithione) chelating
ligands, are found to inhibit breast cancer tumorigenesis and
metastasis via targeting Wnt/-catenin signaling for the first time.
Treatment with pbtIrSS and ppyIrSS induce the degradation of LRP6,
thereby decreasing the protein levels of DVL2, β-catenin and
activated β-catenin, resulting in downregulation of Wnt target genes
CD44 and survivin. Additionally, pbtIrSS and ppyIrSS can suppress
cell migration and invasion of breast cancer cells. Furthermore, both
complexes show the ability to inhibit sphere formation and mediate
stemness properties of breast cancer cells. Importantly, pbtIrSS
exerts potent anti-tumor and anti-metastasis effects in mouse
xenograft models through the blockage of Wnt/β-catenin signaling.
Taken together, our results indicate that pbtIrSS has great potential
to be developed as a breast cancer therapeutic agent with a novel
mechanism.
organism development, stem cell function and tissue
homeostasis.[3] The abnormal activation of Wnt signaling pathway
is closely related to the maintenance of CSCs and the
development of multiple tumors, including breast cancer.[4] The
multifunctional protein β-catenin is a key component of this
pathway, and its protein levels are controlled by a destruction
complex, which consists of scaffolding protein Axin,
Adenomatous polyposis coli (APC), Casein kinase 1 (CK1) and
Glycogen synthase kinase 3β (GSK3β). CK1 and GSK3β
phosphorylate β-catenin, promoting degradation of β-catenin
through the ubiquitin-proteasome pathway. Wnt ligands bind to a
Frizzled receptor (Fzd) as well as the co-receptors low density
lipoprotein receptor-related protein 5/6 (LRP5/6), resulting in the
recruitment of the β-catenin destruction complex to the LRP
receptors and subsequent phosphorylation of one or more
cytoplasmic motifs of LRP5/6. This event induces the activation of
Dishevelled
(DVL)
and
the
inhibition
of
GSK3β.
Unphosphorylated β-catenin accumulates in cytoplasm and
translocates into the nucleus. By interacting with T-cell
factor/lymphoid enhancing factor (TCF/LEF) transcription factors,
β-catenin displaces the corepressors and recruits the
transcriptional Kat3 coactivators p300 and/or CREB-binding
protein (CBP), finally resulting in the expression of Wnt target
genes, such as CD44, cyclin D1, c-Myc, survivin, and
fibronectin.[5]
Metal-based drug cisplatin is one of the most widely used
chemotherapeutic agent for various types of cancers. The
anticancer activity of cisplatin is associated with its ability to
crosslink with the guanine residues on the DNA, leading to DNA
damage in cancer cells.[6] However, its clinical efficacy is largely
limited by numerous side effects, drug resistance and limited
spectrum of activity.[7] This has prompted the exploration of other
metal-based anticancer drugs and their potential targets in cancer
cells.[8] The main challenge concerning the improvement of
therapy is to develop more metal complexes with ability to
selectively target cancer-associated signaling pathways. As far as
we know, very little is known about the effects of metal-based
complexes on the Wnt signaling pathway.
Recently, iridium(III)-based complexes have attracted
increasing attention because of their potent anticancer activities,
limited side effects and improved selectivity towards cancer
cells.[9] Iridium(III) complexes elicit their biological activity through
different mechanisms from platinum-based drugs. Previous
studies have demonstrated that iridium(III) complexes could
trigger the production of intracellular reactive oxygen species
(ROS) levels, reduce the mitochondrial membrane potential and
cause a series of cell death-related events mediated by
mitochondria.[10f,g] Sadler et al. reported on a luminescent
iridium(III) complex inducing NADH depletion, ROS generation,
Introduction
Breast cancer is the most commonly diagnosed cancer and the
second leading cause of cancer death in women worldwide.[1]
Although advanced therapeutic regimens have significantly
improved overall survival in patients with breast cancer, there are
still 20%-30% of patients with recurrence and 60% recurrent
patients with metastasis. Increasing evidences suggest that the
survival of cancer stem cells (CSCs) is a major cause of
recurrence and metastasis in breast cancer. CSCs are a small
subpopulation which possess self-renewal and differentiation
potential.[2] The development of targeting therapy against breast
CSCs may be a promising strategy for the treatment of breast
cancer.
The Wnt signaling pathway is a highly conserved signaling
system in biological evolution and plays an important role in
[a]
Dr. Q. Sun, Q. Fu, S. Liu, Z. Wang, Z. Su, J. Song, Prof. Dr. D. Lu
Guangdong Key Laboratory for Genome Stability & Disease
Prevention, International Cancer Center, Department of
Pharmacology, Shenzhen University Health Science Center
Shenzhen, 518060, China
E-mail: delu@szu.edu.cn
[b]
[c]
Dr. P. Zhang, Y. Wang, A. Ouyang, Prof. Dr. Q. L. Zhang, College of
Chemistry and Environmental Engineering, Shenzhen University
Shenzhen, 518060, P. R. China
E-mail:p.zhang6@szu.edu.cn
Y. Wang, Key Laboratory for Advanced Materials of MOE, School of
Chemistry & Molecular Engineering, East China University of
Science and Technology
Shanghai, 200237, P. R. China
Supporting information for this article is given via a link at the end of
the document.
1
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
intracellular redox imbalance and immunogenic apoptotic cancer
cell death.[10] Mao et al. showed that cyclometalated iridium(III)
complexes served as anion transporters mainly through an anion
exchange mechanism to regulate lysosomal pH, leading to the
inhibition of autophagic flux.[11] Moreover, the Ca2+-binding protein
calmodulin (CaM) has been identified as a target protein of iridium
complexes. Several cationic amphiphilic tris-cyclometalated
iridium(III) complexes were studied to interact with Ca2+-CaM
complex and then induce cell death.[12] In addition, some
cyclometalated iridium(III) complexes have shown to induce
apoptosis in breast CSC‐enriched HMLER‐shEcad cells through
targeting mitochondria.[13] These compounds
inhibited
mammosphere formation to a similar extent as salinomycin.
Salinomycin, an antibiotic potassium ionophore, has been
reported to act as a selective breast CSC inhibitor. It inhibits the
Wnt signaling pathway and exerts anticancer activity in vitro and
in vivo.[14] So far, the mechanism underlying the anti-CSC
properties of iridium complexes remain unclear. In this study, we
synthesized four iridium(III) complexes, pbtIrSS, ppyIrSS,
pbtIrOO and ppyIrOO (Scheme 1A), and explored their effects
on Wnt/β-catenin signaling, cell viability, apoptosis, migration,
invasion, and stemness in breast cancer cells. Their effects on
breast tumor growth and lung metastasis were evaluated using
mouse xenograph models. Our results demonstrated that pbtIrSS
and ppyIrSS but not pbtIrOO and ppyIrOO exerted anti-tumor
and anti-metastatic activities in breast cancer cells via inhibiting
Wnt/β-catenin signaling and CSCs (Scheme 1B).
Scheme 1. (A) The chemical structures of the iridium(III) complexes
studied in this work. (B) pbtIrSS and ppyIrSS exhibits potent anti-breast
cancer activity in vitro and in vivo via blocking Wnt/β-catenin signaling.
inductively coupled plasma mass spectrometry (ICP-MS). MDA-
MB-231 cells were treated with 2.5 M iridium complexes for 1 h
and then subjected to confocal imaging. As shown in Figure S16A,
ppyIrSS and pbtIrSS showed
a wide range of strong
Results and Discussion
luminescence in the cells, while ppyIrOO and pbtIrOO exhibited
only a small amount of spot-like luminescence. The cellular
uptake amounts of these iririum(III) complexes were further
quantitatively studied by ICP-MS (Figure S16B). The results
showed that ppyIrSS and pbtIrSS had a six to ten-fold higher
uptake than ppyIrOO and pbtIrOO across the cells. Thus, the
cellular uptake of S,S compounds was more efficient than that of
the O,O compounds.
The compounds were obtained in a high yield from the reaction of
iridium dimer [(ppy)4Ir2Cl2] or [(pbt)4Ir2Cl2] (ppy: 2-phenylpyridine,
pbt: 2-phenyl-1,3-benzo-thiazole) with 2 equiv. of S,S dithione
ligand or 2,2,6,6-tetramethyl-3,5-heptanedione, respectively. As
shown in Scheme 1A, both ppyIrSS and pbtIrSS had a unit
positive charge, while ppyIrOO and pbtIrOO were neutral. This
probably makes a difference in biological properties. Details of the
synthesis were showed in the experimental section and Scheme
S1. These compounds were characterized by the high-resolution
We first examined the effect of these iridium(III) complexes on
biological behaviours of breast cancer cells. The cytotoxicity of
pbtIrSS, ppyIrSS, pbtIrOO, ppyIrOO and cisplatin was detected
by a MTT assay. The complexes pbtIrSS and ppyIrSS exerted
the strong cytotoxic potency compared with other compounds,
with IC50 values of pbtIrSS at 1.30 ± 0.02 M in MDA-MB-231
cells and at 0.89 ± 0.09 M in MDA-MB-468 cells, IC50 values of
ppyIrSS at 1.77 ± 0.13 M in MDA-MB-231 cells and at 1.70 ±
0.15 M in MDA-MB-468 cells (Figure 1A and B). Flow cytometry
was used to analyze the apoptosis in MDA-MB-231 and MDA-
MB-468 cells. The compounds pbtIrSS and ppyIrSS exhibited
the potent pro-apoptotic effect in both cell lines (Figure 1C and D).
These results revealed that pbtIrSS and ppyIrSS have more
potent ability to induce apoptosis and inhibit cell viability in breast
cancer cells than pbtIrOO, ppyIrOO and cisplatin. We further
tested the effects of pbtIrSS and ppyIrSS on the migratory and
invasive activities of MDA-MB-231 and MDA-MB-468 cells using
transwell migration and invasion assays. The results showed that
treatment with pbtIrSS and ppyIrSS (Figure S17A-D) significantly
reduced the numbers of migrated and invaded cells in both cell
lines, suggesting that pbtIrSS and ppyIrSS could attenuate the
migratory and invasive ability of breast cancer cells. As a positve
control, salinomycin also effectively induced the apoptosis and
1
mass spectrometry (Figures S1-S4), H NMR (Figures S5-S8),
13C NMR, (Figures S9-S12) and elemental analysis. In addition,
the structures of ppyIrOO and pbtIrOO were studied by X-ray
crystallography (Scheme S2). The crystallographic data, selected
bond lengths and angles were listed in Tables S1-S2. The crystal
structures showed that the Ir-O bond lengths ranged from 2.13 to
2.15 Å and the twist angles of O-Ir-O were around 87.
The stabilities of the iridium(III) complexes in the PBS solution
were monitored using UV-vis abosorption spectroscopy and high
performance liquid chromatography (HPLC). The results showed
that the iridium(III) complexes were highly stable for 72 h in the
PBS solution (Figures S13-S14). The logarithm of the octanol-
water partition coefficient (log Po/w) is a measure of lipophilicity.
As shown in Figure S15, ppyIrSS and pbtIrSS (log P = +2.05 for
ppyIrSS and log P = +2.61 for pbtIrSS) were more lipophilic than
ppyIrOO and pbtIrOO (log P = +0.63 for ppyIrOO and log P =
+0.68 for pbtIrOO). The higher positive log Po/w value implied that
this molecule might be more readily incorporated into cells.[9a]
The cellular uptake of the iridium complexes in living cells was
investigatied using confocal laser scanning microscopy and
2
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10.1002/anie.202015009
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RESEARCH ARTICLE
Figure 1. The iridium(III) complex pbtIrSS potently promotes
apoptosis and suppresses viability in breast cancer cells. MDA-MB-
231(A and C) MDA-MB-468 (B and D) cells were treated with iridium(III)
complexes or cisplatin, respectively. (A and B) The cell viability was
examined using the MTT assay. (C and D) Cell apoptosis was detected by
FACS. Statistical analysis was conducted using one-way ANOVA followed
by a Dunnett-t test. *P < 0.05 compared to vehicle control.
inhibited the viability, migration and invasion of breast cancer cells
(Figure S18A-F). The IC50 values of salinomycin for MDA-MB-231
and MDA-MB-468 cell lines were 1.11 ± 0.12 M and 1.35 ± 0.28
M, respectively, showing comparable potency to pbtIrSS and
ppyIrSS (Figure 1A and B, Figure S18A and B). However,
cisplatin, a commonly used metal cancer chemotherapeutic drug,
had little effect on these biological behaviour in breast cancer cells
at the same concentrations (Figure1A-D, Figure S17E and F).
Figure 2. The iridium(III) complex pbtIrSS suppresses Wnt/-catenin
signaling. (A-D) HEK293T cells were transfected with SuperTopFlash
reporter gene together with empty vector or expression plasmids encoding
Wnt1 (A), LRP6 (B), Wnt1/LRP6 (C), DVL2 (D), and -catenin (E). (F-G)
HEK293T cells were transfected with NFAT-Luc reporter (F) together with
NFATc expression plasmid, or AP-1-Luc reporter (G) along with
constitutively active Rasv12 expression plasmid. The transfected cells were
treated with vehicle control (DMSO) or pbtIrSS. Data were expressed as
fold induction relative to vehicle control. Statistical analysis was conducted
using one-way ANOVA followed by a Dunnett-t test. *P<0.05 versus
vehicle control. (H) The expression levels of indicated components of Wnt
signaling were detected by western blotting.
Cyclometalated iridium(III) complexes have been reported as
anti-CSC agents.[14] Concerning the crucial role of Wnt/β-catenin
signaling in the survival and maintenance of CSCs, we examined
the effect of these iridium complexes on Wnt/β-catenin signaling.
A SuperTopFlash reporter was transfected into HEK293T cells
along with Wnt1, LRP6, Wnt1/LRP6, DVL2 or β-catenin
expression plasmids, respectively. Treatment with 0.625-5 M
pbtIrSS (Figure 2A-D) and ppyIrSS (Figure S19A-D) dose-
dependently inhibited the transcriptional activity of the
SuperTopFlash reporter activated by Wnt1, LRP6, Wnt1/LRP6,
and DVL2. However, pbtIrOO (Figure S20A-D) and ppyIrOO
(Figure S20E-H) had little effect on the SuperTopFlash activity
induced by Wnt1, LRP6, DVL2 and β-catenin. Furthermore,
pbtIrSS and ppyIrSS did not inhibit the SuperTopFlash activity
induced by β-catenin, indicating that pbtIrSS and ppyIrSS may
act on the upstream elements of β-catenin (Figure 2E and Figure
S19E). To examine the effect of pbtIrSS and ppyIrSS on other
signaling pathways, we performed the transfection assays using
an NFAT reporter (NFAT-Luc) and an AP1 reporter (AP1-Luc).
The expression plasmids encoding NFATc and RasV12 were used
to activate the NFAT and AP1 signaling pathways in HEK293T
cells, respectively. As shown in Figure 2F and G, Figure S19F and
G, pbtIrSS and ppyIrSS had no inhibitory effects on the luciferase
activities of NFAT-Luc and AP1-Luc. Salinomycin, an inhibitior of
the Wnt signaling pathway, specifically inhibited the
transcriptional activity of the SuperTopFlash reporter activated by
the components of Wnt signaling (Figure S21A-G). In contrast, we
did not observe any inhibitory effect of cisplatin on the
transcriptional activity of the SuperTopFlash reporter at any of the
concentrations tested (0.625-10 M or 5-40 M) (Figure S22). To
further assess the effect of these iridium(III) complexes on the
components of the Wnt/β-catenin signaling pathway, a Wnt1
expression plasmid was transfected into HEK293T cells. After
transfection for 24 h, the cells were treated with the increasing
concentrations of pbtIrSS and ppyIrSS. Figure 2H and Figure
S19H showed that expression of Wnt1 enhanced the levels of β-
catenin, demonstrating the activation of the Wnt/β-catenin
signaling pathway in HEK293T cells. Similar to salinomycin
(Figure S21H), pbtIrSS or ppyIrSS significantly downregulated
the levels of phosphorylated LRP6, total LRP6, DVL2, total β-
catenin and activated β-catenin in the cells transfected with Wnt1
expression vector (Figure 2H and S19H). Taken together, our
results illustrate that the iridium(III) complexes pbtIrSS and
ppyIrSS can specifically inhibit Wnt/β-catenin signaling in
HEK293T cells.
Increasing evidences have demonstrated that Wnt/β-catenin
signaling is activated in different subtypes of human breast cancer.
Several Wnt ligands, FZD receptors and LRP6 have been
detected in various breast cancer cell lines and primary tumor
tissues. Multiple negative modulators of this pathway are
downregulated in many breast tumor tissues. These negative
modulators include SFRP1, SFRP2, SFRP5, WIF1, DKK1 and
DKK3.[15] Moreover, increased levels of β-catenin have been
3
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 3. The iridium(III) complex pbtIrSS inhibits Wnt/-catenin
signaling in breast cancer cells. MDA-MB-231 and MDA-MB-468 cells
were treated with pbtIrSS. (A and B) The expression levels of indicated
components of Wnt signaling were analyzed by western blotting. The real-
time PCR analysis was used to detect the mRNA expression of survivin(C)
and CD44 (D). Statistical analysis was conducted using one-way ANOVA
followed by a Dunnett-t test. *P < 0.05 compared to vehicle control.
Figure 4. The iridium(III) complex pbtIrSS induces the internalization
and degradation of LRP6 via a lysosome-dependent manner. The
relative mRNA levels of LRP6 in pbtIrSS-treated MDA-MB-231 (A) and
MDA-MB-468 (B) were detected by real-time PCR. MDA-MB-231 (C) and
and MDA-MB-468 (D) cells were incubated with 1.25 or 2.5 M pbtIrSS
for 18 h before 10 nM BAF A1 was added. After incubation for another 6
h, the cells were harvested and the protein levels of LRP6 were detected
by immunoblotting. (E) HEK293T cells were transfected with LRP6-GFP
plasmid for 24 h. Then the cells were treated with vehicle or pbtIrSS before
70 nM Lyso Traker TM Deep Red was added. After incubation for indicated
periods of time (0.5, 1 and 3 h), the cells were fixed and stained with DAPI.
observed in about 90% of breast tumors. To assess the effect of
the iridium(III) complexes on Wnt/β-catenin signaling in breast
cancer cells, MDA-MB231 and MDA-MB468 cells were
transduced with lentiviral vectors encoding the 7xTCF-
FFluc/SV40-mCherry (7TFC) reporter gene, which allow us to
monitor Wnt/β-catenin activity using
a luceferase assay.
Treatment with pbtIrSS, ppyIrSS and salinomycin effectively
inhibited the transcriptional activity of 7TFC Wnt reporter in MDA-
MB-231 cells (Figure S23A-F) and MDA-MB-468 cells (Figure
S23G-L). In contrast，pbtIrOO, ppyIrOO and cisplatin had no
effect on Wnt signaling in both breast cancer cell lines. These
results indicate that pbtIrSS and ppyIrSS could suppress Wnt/β-
catenin signaling in breast cancer cells.
J) and Wnt target genes (Figure S25E and F). These results
indicate that pbtIrSS, ppyIrSS and salinomycin but not pbtIrOO,
ppyIrOO and cisplatin have the ability to inhibit Wnt/β-catenin
signaling in breast cancer.
In breast cancer, the components of Wnt signaling at the cell
surface play an important role in the activation of this pathway.
LRP6 is an essential Wnt co-receptor, whose expression is
upregulated in various breast cancer cell lines and tumor tissues.
Depletion of LRP6 in breast cancer cells remarkably suppressed
Wnt/β-catenin signaling.[16] Overexpression of LRP6 in mammary
epithelial cells is sufficient to activate Wnt/β-catenin signaling and
induce mammary gland hyperplasia.[17] Our results showed
pbtIrSS and ppyIrSS markedly downregulated the expression of
LRP6 in HEK293T and breast cancer cells. We then test the effect
of pbtIrSS and ppyIrSS on mRNA level of LRP6 in MDA-MB-231
and MDA-MB-468 cells. The real-time PCR results showed that
treatment with pbtIrSS (Figure 4A and B) and ppyIrSS (Figure
S26A and B) did not affect the mRNA expression of LRP6 in both
cell lines, indicating that pbtIrSS and ppyIrSS-induced
downregulation of LRP6 was independent of transcriptional
regulation. To examine whether these two sulfur-coordinated
organoiridium(III) complexes could enhance the degradation of
LRP6, MDA-MB-231 and MDA-MB-468 cells were treated with
the protein synthesis inhibitor cycloheximide (CHX) alone or
combined with pbtIrSS or ppyIrSS. The results showed that
treatment with CHX reduced the levels of endogenous LRP6 in a
We further evaluated the effect of these iridium(III) complexes
on the components of Wnt/β-catenin signaling in human breast
cancer cells. MDA-MB-231 and MDA-MB-468 cells were treated
with the increased doses of pbtIrSS, ppyIrSS, pbtIrOO and
ppyIrOO for 24 h. The complexes pbtIrSS and ppyIrSS but not
pbtIrOO and ppyIrOO remarkebly reduced the protein levels of
phosphorylated LRP6, total LRP6, phosphorylated and
unphosphorylated DVL2, total β-catenin and activated β-catenin
in a dose-dependent manner (Figure 3A and B, Figure S24A-F).
Survivin and CD44 are well-established Wnt target genes and are
upregulated in breast cancer. Real-time PCR was employed to
detect the expression of survivin and CD44. pbtIrSS and ppyIrSS
at concentrations as low as 0.625 M decreased mRNA
expression of survivin and CD44 (Figure 3C and D, Figure S25A
and B) in MDA-MB-231 and MDA-MB-468 cells. As expected,
salinomycin not only decreased the expression of components of
Wnt signaling (Figure S24G and H), but also downregulated the
mRNA expression of survivin and CD44 (Figure S25C and D).
However, cisplatin at dosed to 5 M showed little inhibitory effects
on the expression of Wnt signaling components (Figure S24I and
4
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
time-dependent manner, and addition of pbtIrSS (Figure S27A
and B) or ppyIrSS (Figure S26C and D) further accelerated the
degradation of LRP6 protein in both cell lines. Next, the lysosome
inhibitor bafilomycin A1 (BAF) and the proteasome inhibitor
MG132 were used to determine the roles of lysosomal and
proteasomal pathways in pbtIrSS/ppyIrSS-induced degradation
of LRP6. In MDA-MB-231 and MDA-MB-468 cells, pbtIrSS-
induced downregulation of LRP6 was significantly restored by
treatment with BAF A1 (Figure 4C and D) but not by MG132
(Figure S27C and D). Similar to pbtIrSS, ppyIrSS also induced
degradation of LRP6 in a BAF A1-mediated fashion (Figure S26E-
H). Moreover, we observed that pbtIrSS and ppyIrSS could
induce the internalization of LRP6 in a time-dependent manner
(Figure 4E and Figure S26I). An increased lysosomal
accumulation of LRP6 was observed following pbtIrSS or
ppyIrSS treatment for 3 hours in HEK293T cells transfected with
LRP6-GFP plasmid (Figure 4E and Figure S26I). These results
indicated that pbtIrSS/ppyIrSS-induced degradation of LRP6 is
regulated by the lysosomal pathway but not the proteasome
pathway.
To explore the potential interaction between LRP6 protein and
sulfur-coordinated iridium(III) complexes pbtIrSS and ppyIrSS,
the luminescence intensities response of iridium(III) complexes
were investigated in the presence of increasing concentrations of
LRP6 protein. Figure S28A and B showed that the luminescence
intensities of pbtIrSS and ppyIrSS increased in a LRP6
concentration-dependent manner. However, the luminescence
intensities of ppyIrOO and pbtIrOO were almost kept unchanged
under elevated LRP6 concentrations (Figure S28C and D). These
results suggest that the sulfur-coordinated organoiridium(III)
complexes are able to interact with LRP6 protein. Comparing the
chemical structure of sulfur-coordinated complexes with pbtIrOO
and ppyIrOO, the lipophilicity and type of charge of sulfur-
coordinated complexes may contribute to their interaction with
LRP6 protein. We further examined the colocalization of GFP-
labeled LRP6 protein and iridium(III) complexes. LRP6-GFP
expression plasmid was transfected into HEK293T cells for 48 h.
Then cells were treated with 5 M iridium(III) complexes for 0.5 h
at 37 C. The colocalization of LRP6-GFP and iridium(III)
complexes was analyzed by confocal microscopy. The yellow
color in the merged images indicate obvious colocalizations of
LRP6-GFP with pbtIrSS or ppyIrSS, while no significant
colocalization of LRP6-GFP and pbtIrOO or ppyIrOO was
observed (Figure S28E), suggesting that LRP6 could interact with
pbtIrSS and ppyIrSS, but not pbtIrOO and ppyIrOO.
Breast CSCs were firstly identified based on the expression of
CD44 and CD24. A cell population with high expression of CD44
and low expression of CD24 (CD44+/ CD24−/low) has been
reported to have stem cell properties, which exhibits enhanced
tumorigenesis and metastasis.[18] The CD44+/CD24−/low cell
population was about a 1000 times more tumorigenic than other
populations. CD44 is a major target gene of Wnt/β-catenin
signaling and considered an attractive target for development of
CSC-directed therapeutics.[18c,19] To evaluate the effect of pbtIrSS
and ppyIrSS on breast CSCs, the surface expression of CD44
and CD24 in MDA-MB-231 and MDA-MB-468 cells was detected
by flow cytometry. The results revealed that pbtIrSS (Figure 5A-
F), ppyIrSS (Figure S29A-F) and salinomycin (Figure S30A-F)
treatment downregulated the surface expression of CD44,
enhanced the proportion of CD44+/CD24+ cells and decreased
Figure 5. The iridium(III) complex pbtIrSS represses expression of
stem cell markers in breast cancer cells. The surface protein expression
of CD44 and CD24 was detected by FACS in MDA-MB-231 (A-C) and
MDA-MB-468 (D-F) cells treated with pbtIrSS. The percentage of CD44
(B and E) and CD24 (C and F) positive subpopulations were presented.
Statistical analysis was conducted using one-way ANOVA followed by a
Dunnett-t test. *P < 0.05 compared to vehicle control.
CD44+/CD24−/low cells in both cell lines, whereas cisplatin had no
effect on the expression of CD44 and cell subpopulations (Figure
S31A-F). Moreover, CD44 protein expression was also
decreased in a dose-dependent manner following treatment with
pbtIrSS (Figure S32A and B), ppyIrSS (Figure. S29G and H) and
salinomycin (Figure. S30G and H) in both cell lines. These results
suggest that pbtIrSS and ppyIrSS may have the ability to
abrogate the stemness properties of breast CSCs.
We next performed a sphere formation assay to examine the
effect of pbtIrSS, ppyIrSS, salinomycin and cisplatin on CSC self-
renewal potential, respectively. The breast cancer Hs578T cells
were treated with each complex at 1.25 and 2.5 M for 10 days.
As shown in Figure S33A and B, treatment with pbtIrSS, ppyIrSS
and salinomycin significantly decreased the number and size of
tumor sphere, while cisplatin had no inhibitory effect on sphere
formation of breast cancer cells. Importantly, we noted that
sulfur-coordinated
organoiridium(III)
complexes
and
salinomycin have comparable inhibitory effects on Wnt
signaling and breast CSCs.
Concerning about pbtIrSS has more potent effects on the
biological behaviours of breast cancer cells compared with
ppyIrSS, pbtIrOO, ppyIrOO and cisplatin, this complex was
further evaluated its anticancer activity in vivo by using a MDA-
MB-231 cell xenograft model. MDA-MB-231 cells were injected
into nude mice subcutaneously. When the tumor volumes
reached about 50 mm3, mice were treated by i.p. injection with
saline, cisplatin or pbtIrSS at 3 mg/kg on days 0, 3, 6, 9, 12. After
treatment for 5 times, mice were sacrificed on the fifteenth day,
5
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 7. The iridium(III) complex pbtIrSS exerts anti-metastatic
activity in lung metastasis mouse model. Experimental lung metastasis
mouse model was established by using luciferase-labelled MDA-MB-231
cells. MDA-MB-231-Luc cells were intravenously implanted into the nude
mice. On day 25 after injection of breast cancer cells, mice were treated
with saline, cisplatin or pbtIrSS, respectively, at a dosage of 3 mg/kg for
twice a week for two weeks by i.p. Bioluminescence imaging of mice in
the control and treatment groups on day 25 (A), day 32 (C) and day 39 (E)
after cell injection. The tumor burden was quantitatively expressed as
luminescence signal intensity in the region of interest on day 25 (B), day32
(D) and day 39 (F) after cell injection. Statistical analysis was conducted
using one-way ANOVA followed by a Dunnett-t test. *P < 0.05 compared
to vehicle control.
metastasis by inhibiting CSC-like phenotype.[20] As an
antagonistic agent against Wnt/β-catenin signaling, pbtIrSS
exhibited potent inhibitory effects on the stemness properties,
migration and invasion of breast cancer cells. These results
strongly suggest that pbtIrSS may possess anti-metastasis
potential. To validate the anti-metastatic efficacy of pbtIrSS in
vivo, we generated a breast cancer-derived lung metastases
model in BALB/c-nu mice. MDA-MB-231 luciferase-expressing
Figure 6. The iridium(III) complex pbtIrSS inhibits tumor growth in a
breast cancer xenograft mouse model. MDA-MB-231 xenografts were
treated with saline, pbtIrSS or cisplatin at 3 mg/kg on days 0, 3, 6, 9 and
12 by i.p. injection. After treatment for 5 times, mice were sacrificed on the
fifteenth day, and tumors were excised and weighed. (A) Images of tumors
from control group and treatment group. (B) Mean tumor volumes. (C)
Mean tumor weight. (D) H&E staining of tumor section; scale bar, 200 μm.
(E) IHC staining of Ki-67, β-catenin and CD44; scale bar, 200 μm (F) The
expression levels of indicated components of Wnt signaling in tumor
samples were visualized by immunoblotting. (G) The mRNA levels of
survivin and CD44 were quantitated by real-time PCR. Statistical-analysis
was conducted using one-way ANOVA followed by a Dunnett-t test. *P <
0.05 compared to vehicle control.
cells were injected
intravenously into nude mice.
Bioluminescence imaging was used to monitor the lung tumor
burden. The tumor growth was first detected on day 25 after
injection (Figure 7A and B). Then the mice were divided randomly
into three groups and treated i.p. with saline, cisplatin or pbtIrSS
3 mg/kg twice a week for two weeks. In vivo bioluminescent
imaging revealed that a significant reduction in metastasis to the
lungs in the pbtIrSS-treated group compared with the control
group and cisplatin-treated group for one week (Figure 7C and D)
and two weeks (Figure 7E and F), suggesting that pbtIrSS has
great anti-metastasis activity in breast cancer in vivo.
and tumor volumes and weight were measured. The total RNAs
and proteins in the xenografts were extracted, and the histological
properties of the tumors were studied. pbtIrSS exhibited more
potent inhibitory effect on tumor growth in vivo than cisplatin
(Figure 6A-C). Histological studies indicated that treatment with
pbtIrSS significantly decreased the tumor cell density (Figure 6D)
and expression of the proliferation marker Ki-67 (Figure 6E)
compared with vehicle control and cisplatin. In addition, the
results of immunohistochemical staining indicated that pbtIrSS
effectively inhibited the expression of active β- catenin and CD44
in xenograft tumor tissues (Figure 6E). Furthermore, treatment
with pbtIrSS significantly decreased the protein levels of
phosphorylated LRP6, total LRP6, phosphorylated and
unphosphorylated DVL2, active β-catenin and CD44 (Figure 6F).
The inhibition of pbtIrSS towards Wnt signaling were further
confirmed by real-time PCR analyses, which exhibited an
effective reduction in mRNA expression of Wnt target genes
survivin and CD44 (Figure 6G). However, we did not observe any
inhibitory effect of cisplatin on the Wnt signaling pathway (Figure
6D-G), suggesting that cisplatin exerts its antitumor activity in a
Wnt-independent manner.
Conclusion
In summary, four organoiridium(III) complexes pbtIrSS, ppyIrSS,
pbtIrOO and ppyIrOO were designed and characterized. Among
these complexes, pbtIrSS and ppyIrSS exhibited a novel
mechanism for breast cancer therapy for the first time. Both
complexes blocked the Wnt/β-catenin signaling cascade by
inhibiting LRP6 degradation via a lysosome-dependent manner.
We demonstrated that pbtIrSS and ppyIrSS could potently
induce apoptosis and inhibit viability, migration and invasion in
breast cancer cells. Moreover, both complexes inhibited sphere
formation and mediated stemness properties of breast cancer
cells. Importantly, pbtIrSS exhibited potent anti-tumor and anti-
metastasis activities in breast cancer xenograft models.
Metastasis is a major cause of breast cancer-related death.
Blockade of Wnt/β-catenin signaling suppresses breast cancer
6
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10.1002/anie.202015009
Angewandte Chemie International Edition
RESEARCH ARTICLE
Collectively, these results demonstrate that pbtIrSS exhibits great
anti-breast cancer activity in vitro and in vivo. This work provides
a novel mechanism underlying the inhibition of the Wnt/β-catenin
pathway by sulfur-coordinated organoiridium(III) complexes.
Zhang, Y. Zheng, L. Hao, L. N. Ji, Z. W. Mao, Angew. Chem.
Int. Ed. 2020, 59,18755-18762; (h) J. Roque III, D. Havrylyuk,
P. C. Barrett, T. Sainuddin, J. McCain, K. Colon, W. T.
Sparks, E. Bradner, S. Monro, D. Heidary, C. G. Cameron,
E. C. Glazer, S. A. McFarland, Photochem. Photobiol. 2020
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Acknowledgements
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This work was supported by National Nature Science Foundation
of China (Grant No. 81802662, 22077085, 21671137 and
31970739), Nature Science Foundation of Guangdong Province
(Grant No.
2017A030310329, 2019A1515011958
and
2020A1515010340), the Shenzhen Peacock Innovation Team
Project (Grant KQTD20140630100658078), Shenzhen Basic
Research Program (Grant No. JCYJ20170817094611664,
JCYJ20190808153209537 and JCYJ20190808173601655),
Shenzhen Peacock Plan (Grant No.827000186 and 827000183),
SZU medical young scientists program (Grant No. 71201-000001)
and Medical Science and Technology Research Foundation of
Guangdong Province (Grant No. A2019475). We appreciate the
Instrumental Analysis Center of Shenzhen University.
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Organoiridium • Wnt/β-catenin signaling • Anti-breast cancer
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7
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Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Sulfur-coordinated organoiridium(III) complexes were explored their effects on Wnt/β-catenin signaling, apoptosis, migration, invasion
and stemness in breast cancer cells for the first time. The results demonstrated that pbtIrSS exerted great anti-tumor and anti-
metastatic activities in breast cancer cells via inhibiting Wnt/β-catenin signaling and CSCs.
8
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