Novel Copper Complexes That Inhibit the Proteasome and Trigger Apoptosis in Triple-Negative Breast Cancer Cells

Article abstract of DOI:10.1021/acsmedchemlett.9b00284

Five innovative ternary copper(II) complexes [Cu(OH-PIP)(Phe)Cl](1), [Cu(OH-PIP)(Gly)(H₂O)]NO₃·2H₂O (2), [Cu(OH-PIP)(Ala)(Cl)]·H₂O (3), [Cu(OH-PIP)(Met)]PF₆·2H₂O (4), and [Cu(OH-PIP)(Gln)(H

Full text of DOI:10.1021/acsmedchemlett.9b00284

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Letter
Novel copper complexes that inhibit the proteasome and
trigger apoptosis in triple-negative breast cancer cells
Dong-Dong Li, Ernesto Yagüe, Lu-Yao Wang, Lin-Lin Dai, Zi-
Bo Yang, Shuang Zhi, Na Zhang, Xiu-Mei Zhao, and Yun-Hui Hu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00284 • Publication Date (Web): 25 Jul 2019
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ACS Medicinal Chemistry Letters
Novel copper complexes that inhibit the proteasome and trigger
apoptosis in triple-negative breast cancer cells
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Dong-Dong Li 1*, Ernesto Yagüe3, Lu-Yao Wang1, Lin-Lin Dai1, Zi-Bo Yang1, Shuang Zhi1, Na
Zhang1, Xiu-Mei Zhao1*, Yun-Hui Hu2*
1Tianjin Institute of Medical and Pharmaceutical Sciences, Tianjin 300020, China
2The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060
3Cancer Research Center, Division of Cancer, Faculty of Medicine, Imperial College London, Hammersmith Hospital
Campus, London W12 0NN, UK
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Corresponding authors:
Dong-Dong Li, Tianjin Institute of Medical and Pharmaceutical Sciences, E-mail address: lidongdong2010@163.com;
Xiu-Mei Zhao, Tianjin Institute of Medical and Pharmaceutical Sciences, E-mail address: zxmmlg@163.com;
Yun-Hui Hu, The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, E-mail:
yunhuihu200408@163.com.
KEYWORDS: amino acid-polypyridine-copper complexes, proteasome inhibition, apoptosis induction,triple-negative
breast cancer therapy
ABSTRACT: Five innovative ternary copper(II) complexes [Cu(OH-PIP)(Phe)Cl](1), [Cu(OH-PIP)(Gly)(H2O)]NO3·2H2O (2),
[Cu(OH-PIP)(Ala)(Cl)]·H2O (3), [Cu(OH-PIP)(Met)]PF6·2H2O (4), [Cu(OH-PIP)(Gln)(H2O)](Cl)·3H2O (5) have been synthesized
and characterized by infrared spectroscopy, elemental analysis, and single crystal X-ray diffraction analysis. X-ray crystallography
indicates that all Cu atoms are five-coordinated in a square-pyramidal configuration. The complexes have been screened for
cytotoxicity against human breast cancer cell lines MCF-7, MDA-MB-231 and CAL-51. The best anticancer activity is obtained
with triple-negative breast cancer CAL-51 and MDA-MB-231 cell lines, with IC50 values in the range of 0.082-0.69 µM.
Importantly, the copper compounds were more effective than carboplatin at triggering cell death. Mechanistically, the complexes
inhibit proteasomal chymotrypsin-like activity, and docking studies reveal their 20S proteasome binding sites. As a consequence,
they cause the accumulation of ubiquitinated proteins, inhibit cell proliferation and induce apoptosis. In addition, these copper
complexes decrease the stemness of triple-negative breast cancer cells, and have synergistic effects with CBP on TNBC cells,
indicating their great potential as a novel therapy for triple-negative breast cancer.
The ubiquitin-proteasome system (UPS) plays an important role
in multitude of cellular processes including cell cycle progression,
apoptosis, angiogenesis, DNA damage and repair, drug resistance
and differentiation 1. The 20S proteasome is a high molecular
weight protease complex with a proteolytic core containing β1, β2
and β5subunits, which are responsible for its caspase-like, trypsin-
like and chymotrypsin-like (CT-like) activities, respectively. It is
well established that inhibition of the β5 proteasomal subunit is
development of proteasome as novel anticancer drugs has been
sought for some time 5.
Most proteasome inhibitors are short peptides that mimic
protein substrates 6. Bortezomib and carfilzomib, approved by the
US Food and Drug Administration, have proven to be effective
therapeutic agents for multiple myeloma and mantle cell
lymphoma7-12. However, these drugs have not been successful in
treating patients with solid cancers, probably due to poor in vivo
stability, undesirable pharmacokinetic properties and unwanted
toxicities arising from peptide backbones and electrophilic
pharmacophores 13-17. Thus, there is an urgent need to generate
new molecules that can inhibit the proteasome utilizing a non-
peptide scaffold which could overcome these drawbacks.
Metal-containing drugs have existed for decades and cisplatin,
a platinum containing compound, is known as one of the most
primarily associated with apoptosis induction in tumor cells 2-4
.
Proliferation and apoptosis pathways are tightly regulated in the
cell by the UPS and alterations in the UPS may result in cellular
transformation or other pathological conditions. As the
proteasome is often found to be overactive in cancer cells making
it more sensitive to proteasome inhibition than normal cells, the
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effective antitumor drugs. Nowadays, the platinum drugs are still
in the front line of metal-based cancer chemotherapy18,19
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2
.
However, their negative side-effects and the risk of resistance
remain a pressing matter in their clinical use. These issues drive
the research and development of new metallotherapeutics, in
many cases divergent from the platinum metals-based complexes
20. Copper is biocompatible and less toxic than non-endogenous
heavy metals. This makes it an interesting candidate for the
treatment of cancers due to its bioavailability and the observation
of increased copper levels in cancer tissue 21. Therefore, copper
complexes are regarded as one of the most promising alternatives
to cisplatin as anticancer substances22. Copper complexes called
Casiopeínas® have proved their significant anticancer activity
both in preclinical in vitro and in vivo testing and two of them
have entered Phase I clinical trials 23. J. Zuo 24 et al. have reported
two amino acid Schiff base-copper complexes which could inhibit
the chymotrypsin-like activity of 20S proteasome, cause
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accumulation of proteasome target proteins bax and IκB-α, and
induce growth inhibition and apoptosis in MDA-MB-231, MCF-7
and PC-3 tumor cells. Z.Y. Zhang 25 et al. have reported a L-
Ornithine Schiff base-copper complex with proteasome-inhibitory
activities and induction of apoptosis in MDA-MB-231 and
LNCaP cancer cells.
Figure 1 Molecular structures of complexes 1-5, dissociative
small molecules and non-essential H-atoms.
Complexes 1-5 were evaluated for their antiproliferative
activity against human breast tumor CAL-51, MDA-MB-231 and
MCF-7 cells by using an MTT assay, and comparing their
cytotoxicity to carboplatin (CBP). IC50 values for complexes 1-5
were lower than those of the free ligand and copper salt in all of
the tested cells, which suggests that the coordinated copper(II) ion
plays a major role in mediating the potency of the complexes
(Table 1). The complexes 1-5 had lower IC50 values than CBP
against the three breast cancer cells, indicating that these novel
complexes have stronger toxicity than carboplatin, a platinum
compound commonly used in the clinic. The complexes 1-5 had
extremely low IC50 levels on CAL-51 cells, with the order of the
cytotoxicity being 3 > 5 > 2 > 1 >4, indicating that the
configuration of the amino acid affects the activity of the
complexes. As complexes 3 (IC50 = 0.08 μM) and 5 (IC50 = 0.27
μM) were the most potent, their biological activity on TNBC
CAL-51 and MDA-MB-231 cells was evaluated further.
Previously, we have reported some polypyridine-copper
complexes as antitumor agents 26-28. These copper complexes are
toxic to HeLa and MCF-7 cells, with IC50 values in the range of
6.70-16.58 µM, inducing apoptosis in human cancer cells and
inhibiting tumor cell growth, although their molecular
mechanisms remain to be established. In order to improve the
targeting and solubility of this type of complex we introduced an
amino acid and generated five novel amino-polypyridine-copper
complexes. We found that they inhibit the proteasomal
chymotrypsin-like activity, cause accumulation of ubiquitinated
proteins, and induce growth inhibition and apoptosis in dose-
dependent manners. They also decrease the stemness of triple
negative breast cancer (TNBC) cells, indicating their great
potential as a novel therapy for triple-negative breast cancer.
The synthesis and characterizations of complexes 1-5 were
described in supporting information. They have been structurally
characterized by X-ray crystallography (Figure 1). The
crystallographic data and structure refinement parameters for the
complexes 1-5 were seen in Tables 1S and 2S.
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Table 1 The anti-proliferative effects of complexes 1-5 against
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3.1 
6.2 
12.5 
25 
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various breast cancer cells
Complex
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IC50 (µM)
MDA-MB-231
18.89±1.23
10.98±0.95
8.35±0.55
4.92±0.36
9.33±0.84
＞100
CAL-51
MCF-7
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0.52±0.02
0.37±0.04
0.08±0.004
0.69±0.04
0.27±0.02
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25.59±2.10
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18.99±1.54
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20.32±2.01
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copper salt ligand
complex 1 complex 2 complex 3 complex 4 complex 5
Copper salt
OH-PIP
Carboplatin
＞100
Figure 2 The copper complexes inhibit proteasomal CT-like
activity in vitro. Purified 20S proteasome was incubated in the
presence of increasing concentration of copper salt, ligand and
copper complexes 1-5 and the CT-like activity measured by the
production of fluorescent AMC groups from Suc-LLVY-AMC
substrate. Data represent the average ± SD of three independent
experiments.
18.51±1.5
1.05±0.1
22.84±2.1
15.02±1.4
68.08±3.2
36.65±2.5
To determine whether the complexes 3 and 5 have synergistic
effects with CBP on TNBC cells, MDA-MB-231 and CAL-51
cells were treated with 3 μM CBP and different concentrations of
either complex 3 or 5 for 48 h, and the proliferation was assessed
by MTT assay. MDA-MB-231 cells treated with 0.39-25.0 μM 3
and 0.19-12.5 μM 5 showed CI values in the range 0.50-0.93 and
0.03-0.92, respectively. Similarly, CAL-51 cells treated with 0.19-
6.25 μM 3 and 0.19-6.25 μM 5 showed CI values in the range of
0.37-0.96 and 0.43-0.86, respectively. In all cases CI values were
lower than 1.0 (Tables 3S and 4S), indicating synergistic effects
between the novel copper compounds and CBP on breast cancer
cells.
In order to ascertain that proteasome inhibition by the copper
complexes is associated with apoptosis induction, cellular
morphological changes were monitored in MDA-MB-231 and
CAL-51 cells after 24 h treatment. Cells treated with complexes 3
and 5 at a concentration double of their IC50 appeared with
characteristic dose-dependent apoptotic blebbing (Figures 1S).
These results suggest that complexes 3 and 5 have the ability to
inhibit the proteasome and induce apoptosis in a concentration-
dependent manner in MDA-MB-231 and CAL-51 cells.
To elucidate the mechanism by which copper complexes
cause cell death in human tumor cells, we performed apoptotic
assays by Annexin V-propidium iodide staining in cells after 24 h
treatment with the complexes at 1 × and 2 × their IC50. In MDA-
MB-231 cells, complex 3 at 8.35 and 16.70 μM triggered cell
death in a dose-dependent manner, with total apoptotic values of
7.5 % and 56.9 %, respectively. In a similar fashion, apoptotic
values of complex 5 at 9.33 and 18.66 μM were 56.5 % and 74.5
%, respectively. Importantly, most of the cell death reported was
due to cells in early apoptosis (Figure 3). CAL-51 cells responded
in the same way: complex 3 at 0.082 and 0.16 μM triggered cell
death in a dose-dependent manner, with total apoptotic values of
8.6 % and 15.5 %, respectively, whereas the apoptotic values after
treatment with complex 5 at 0.27 and 0.54 μM were 10.5 % and
14.4 %, respectively (Figure 4). Although 3 and 5 induced
apoptosis more efficiently in MDA-MB-231 than in CAL-51
cells, overall these results indicate that both compounds induce
apoptosis in a dose-dependent manner in TNBC cells.
To investigate whether the growth-inhibitory activity of the
complexes 1-5 was associated with their ability to inhibit the
proteasome activity, purified human 20S proteasome was treated
with a range of compound concentrations for 2 h at 37 °C in the
presence of fluorogenic Suc-LLVY-AMC, a specific substrate for
the CT-like activity. The results indicate that these copper
complexes did inhibit the proteasomal CT-like activity in a dose-
dependent manner and more efficiently than either the free ligand
or copper salt (Figure 2). Thus, complexes 1-5 target the 20S
proteasomal catalytic β5 subunit.
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Figure 4 The copper complexes induce apoptosis in TNBC CAL-
51 cells. Cells were treated with the copper complexes at 1 × and
2 × IC50 concentrations for 24 h and apoptosis detected by flow
cytometry using annexin V (x axis) –propidium iodide (y axis)
staining. Early apoptotic cells (annexin V-positive, propidium
iodide-negative) appear in the lower right quadrant and late
apoptotic cells (positive for both markers) in the upper right
quadrant. Percentage of cells in apoptotic quadrants is shown.
Cytometry plots are representative three independent experiments.
Histogram indicates average apoptosis (both early and late) ± SD
from three independent experiments.
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IC50
2IC50
Key regulators of the apoptotic pathway include Bax, Bcl-2 and
Caspase family proteins. When Bax expression is high, cells
proceed to apoptosis, whereas when Bcl-2 is produced in excess,
cells are protected from apoptosis 29. In order to validate the
functional results shown above, and to gain insights into the
molecular mechanisms of cell death triggered by the copper
complexes, we used western blots to detect Bax, Bcl-2 and
caspase-3 protein expression in MDA-MB-231 and CAL-51 cells
treated with 3 and 5 at the IC50 and 2 × IC50 for 24 h. Levels of
Bax protein increased, while Bcl-2 and caspase-3 protein
expression levels were downregulated, upon treatment with the
copper complexes (Figure 5). Furthermore, the PARP cleavage
fragment p89 appeared after 24 h treatment, indicating that the
cancer cells were undergoing apoptosis. In addition, and
consistent with the inhibition of proteasomal inhibiton under cell-
free conditions, increased level of ubiquitinated proteins were also
detected in a dose-dependent fashion upon copper complexes
treatment. These cellular data further indicate that the complexes
inhibit proteasome activity and induce apoptosis.
Control
Complex 3
Complex 5
Figure 3 The copper complexes induce apoptosis in TNBC
MDA-MB-231 cells. Cells were treated with the copper
complexes at 1× and 2 × IC50 concentrations for 24 h and
apoptosis detected by flow cytometry using annexin V (x axis)–
propidium iodide (y axis) staining. Early apoptotic cells (annexin
V-positive, propidium iodide-negative) appear in the lower right
quadrant and late apoptotic cells (positive for both markers) in the
upper right quadrant. Percentage of cells in apoptotic quadrants is
shown. Cytometry plots are representative three independent
experiments. Histogram indicates average apoptosis (both early
and late) ± SD from three independent experiments.
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% and 13.4 % when treated with 16.7 µM complex 3 and 18.66
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µM complex 5, respectively (Figure 6 and Table 5S).
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Figure 6 Complexes 3 and 5 decrease stem cell markers in
MDA-MB-231 cells. Breast cancer MDA-MB-231 cells were
treated with 3 and 5 at 0.5 ×, 1 × and 2 × IC50 concentrations for
24 h and the percentage of the CD44+/CD24- cell subpopulation
was determined by flow cytometry. Histogram indicates the
average percentage of the CD44+/CD24- cell subpopulation from
three independent experiments.
Figure 5 Complexes 3 and 5 induce accumulation of
ubiquitinated proteins and apoptosis. Breast cancer MDA-MB-
231 and CAL-51 cells were treated with 3 and 5 at 1 × and 2 ×
IC50 concentrations for 24 h and ubiquitinated proteins, Caspase-
3, PARP, Bcl-2 and Bax. proteins detected by western blotting.
Housekeeping GAPDH levels were used to confirm equal
protein loading. Arrow indicates molecular mass in kDa. Band
intensity was quantified using Image J and shown as the average
± SD (n=3), *P＜0.05.
As ALDH has also been identified as a CSC marker in different
type of cancers, and represents the CSC subpopulation better than
CD44+/CD24- in CAL-51 cells 34, we also tested ALDH activity
by ALDEFLUOR assay in both cell lines. Both complexes 3 and
5 reduced the percentage of ALDH-positive cells in a dose-
dependent manner. In MDA-MB-231 cells, that show a 22.7%
ALDH-positive cells, complex 3 treatment at 16.7 µM reduced
them to 0.72 %, whereas treatment with 5 at 18.6 µM reduced
them to 1.02 % (Figure 7). Similarly, CAL-51 cells, with 45.5 %
ALDH-positive cells, when treated with complex 3 had a
reduction in the percentage of ALDH-positive cells to 8.25 % and
with complex 5 to 5.50 % (Figure 8 and Table 6S).
Cancer stem cells (CSCs), a subpopulation of tumor cells
possessing the extensive self-renewal capability necessary to
successfully colonize distant organs, relate to highly aggressive
TNBC 30. The CD44+/CD24- breast cancer cell subpopulation has
a strong tumor forming ability and these markers are considered
one of the best in the field to determine breast CSC 31-33. In the
treatment of breast cancer, the elimination of breast cancer stem
cells is the key to completely cure the disease. To determine
whether 3 and 5 have the capacity to modulate stem cell
phenotypes, we determined the extent of CD44+/CD24- population
by flow cytometry. Indeed, MDA-MB-231 cells showed a dose-
dependent decreases in CD44+/CD24- stem cell population of 12.3
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Figure 8 Complexes 3 and 5 decrease stem cell marker ALDH in
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CAL-51 cells. Breast cancer CAL-51 cells were treated with 3 and
5 at 0.5 ×, 1 × and 2 × IC50 concentrations for 24 h and the
percentage of ALDH+ cells determined by flow cytometry. In
order to better visualize ALDH+ cells, two-dimensional plots were
obtained. SSC, side scatter channel. Histogram indicates the
average percentage of the ALDH+ cell subpopulation from three
independent experiments.
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Although CSC marker studies are informative, tumorsphere
assays were carried out to investigate whether 3 and 5 did affect
tumor forming characteristics in vitro and how they compared to
CBP. Seen in Figure 2S, compared with control, Both TNBC cell
lines produced robust tumorspheres that decreased in size after
CBP treatment. However, 3 and 5 completely disaggregated the
spheres and, at the highest dose, led to their depletion. Thus, both
3 and 5 decrease the stemness of TNBC cells.
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1 x IC50
2 x IC50
The docking analysis provided new insights on a possible
mechanism of proteasome inhibition. The molecular modeling
results may account for the data observed in the purified
proteasome studies. As shown in Figure 9, in addition to form
hydrophobic interaction with the hydrophobic pocket composed
of residues Ala-50, Ala-49, Ala-46, Met-45, Ala-31, Ala-20, Ala-
22 and Ala-27, complex 1 formed hydrogen bonding through
hydroxyl interaction with residue Ala-93, and formed hydrogen
bonding through imidazole-NH with residue Gly-94, and formed
hydrogen bonding through amino acid with residue Gly-47 and
Ala-49; complex 2 formed hydrogen bonding through hydroxyl
interaction with residue Lys-32, and formed hydrogen bonding
through imidazole with residue Ala-49 and Thr-1, and formed
hydrogen bonding through amino acid with residue Gly-23;
complex 3 formed hydrogen bonding through amino acid with
residue Thr-1, and formed a coordination bond between copper
atom and residue Gly-47; complex 4 formed hydrogen bonding
through imidazole-NH with residue Thr-1, and formed hydrogen
bonding through amino acid with residue Lys-23, and formed
hydrogen bonding through oxygen atom with residue Gln-53;
complex 5 formed hydrogen bonding through amino acid with
residues Thr-1 and Ser-129, and formed a coordination bond
between copper atom and residue Gly-47. The minimum relative
binding energy values were -7.51, -7.82, -6.51, -6.49 and -6.04
kcal/mol for 1-5, respectively, indicating the interaction between
the copper complexes and 20S proteasome.
0
Control
Complex 3
Complex 5
Figure 7 Complexes 3 and 5 decrease stem cell marker ALDH in
MDA-MB-231 cells. Breast cancer MDA-MB-231 cells were
treated with 3 and 5 at 0.5 ×, 1 × and 2 × IC50 concentrations for
24 h and the percentage of ALDH+ cells determined by flow
cytometry. In order to better visualize ALDH+ cells, two-
dimensional plots were obtained. SSC, side scatter channel.
Histogram indicates the average percentage of the ALDH+ cell
subpopulation from three independent experiments.
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Control
CAL-51
0.5 x IC50
1 x IC50
2 x IC50
Control
Complex 3
Complex 5
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Complex 1
labeled. Key H-bonds between the complexes and the protein are
1
shown as dashed yellow lines.
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ASSOCIATED CONTENT
Supporting Information
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Crystallographic data and structure refinement parameters,
selected bond lengths (Å) and angles (º) for the complexes 1-5.
The CI values of combination of 3 or 5 and CBP for MDA-MB-
231 and CAL-51 cells. The effect of 3 and 5 on CD44+/CD24-
phenotype cell subsets in MDA-MB-231 was detected by Flow
cytometry. The effect of the 3 and 5 on the ratio of ALDH1+ cell
population in MDA-MB-231 and CAL-51 was detected by Flow
cytometry. 3 and 5 abolish tumor forming capacity of TNBC cells
in vitro.
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Complex 2
AUTHOR INFORMATION
Corresponding Author
*Dong-Dong Li, Tianjin Institute of Medical and Pharmaceutical
Sciences, E-mail address: lidongdong2010@163.com;
*Xiu-Mei Zhao, Tianjin Institute of Medical and Pharmaceutical
Sciences, E-mail address: zxmmlg@163.com;
*Yun-Hui Hu, The Third Department of Breast Cancer, Tianjin
Medical University Cancer Institute and Hospital, E-mail:
yunhuihu200408@163.com.
Complex 3
Complex 4
Complex 5
Author Contributions
The manuscript was written through contributions of all authors
Funding Sources
This work was supported by the National Natural Science
Foundation of China (21501134).
Abbreviations
MCF-7
MDA-MB-231
CAL-51
TNBC
Human breast cancer cell line
Human breast cancer cell line
Human breast cancer cell line
triple-negative breast cancer
Carboplatin
CBP
CSCs
Cancer stem cells
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Figure 9 Schematic representation of the proposed binding modes
for complexes 1-5 with proteasome (PDB ID:3MG6). Only amino
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Molecular structures of complexes 1-5, dissociative small molecules and non-essential H-atoms.
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The copper complexes inhibit proteasomal CT-like activity in vitro. Purified 20S proteasome was incubated in
the presence of increasing concentration of copper salt, ligand and copper complexes 1-5 and the CT-like
activity measured by the production of fluorescent AMC groups from Suc-LLVY-AMC substrate. Data
represent the average ± SD of three independent experiments.
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The copper complexes induce apoptosis in TNBC MDA-MB-231 cells. Cells were treated with the copper
complexes at 1× and 2 × IC50 concentrations for 24 h and apoptosis detected by flow cytometry using
annexin V (x axis)–propidium iodide (y axis) staining. Early apoptotic cells (annexin V-positive, propidium
iodide-negative) appear in the lower right quadrant and late apoptotic cells (positive for both markers) in
the upper right quadrant. Percentage of cells in apoptotic quadrants is shown. Cytometry plots are
representative three independent experiments. Histogram indicates average apoptosis (both early and late)
± SD from three independent experiments.
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The copper complexes induce apoptosis in TNBC CAL-51 cells. Cells were treated with the copper complexes
at 1 × and 2 × IC50 concentrations for 24 h and apoptosis detected by flow cytometry using annexin V (x
axis) –propidium iodide (y axis) staining. Early apoptotic cells (annexin V-positive, propidium iodide-
negative) appear in the lower right quadrant and late apop-totic cells (positive for both markers) in the
upper right quadrant. Percentage of cells in apoptotic quadrants is shown. Cytometry plots are
representative three independent experiments. Histogram indicates average apoptosis (both early and late)
± SD from three independent experiments.
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Complexes 3 and 5 induce accumulation of ubiquitinated proteins and apoptosis. Breast cancer MDA-MB-231
and CAL-51 cells were treated with 3 and 5 at 1 × and 2 × IC50 con-centrations for 24 h and ubiquitinated
proteins, Caspase-3, PARP, Bcl-2 and Bax. proteins detected by western blotting. House-keeping GAPDH
levels were used to confirm equal protein load-ing. Arrow indicates molecular mass in kDa. Band intensity
was quantified using Image J and shown as the average ± SD (n=3), *P＜0.05.
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Complexes 3 and 5 decrease stem cell markers in MDA-MB-231 cells. Breast cancer MDA-MB-231 cells were
treated with 3 and 5 at 0.5 ×, 1 × and 2 × IC50 concentrations for 24 h and the percentage of the
CD44+/CD24- cell subpopulation was determined by flow cytometry. Histogram indicates the average
percentage of the CD44+/CD24- cell subpopulation from three independent experiments.
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Complexes 3 and 5 decrease stem cell marker ALDH in MDA-MB-231 cells. Breast cancer MDA-MB-231 cells
were treated with 3 and 5 at 0.5 ×, 1 × and 2 × IC50 concentrations for 24 h and the percentage of ALDH+
cells determined by flow cytometry. In order to better visualize ALDH+ cells, two-dimensional plots were
obtained. SSC, side scatter channel. Histogram indicates the average percentage of the ALDH+ cell
subpopulation from three independent experiments.
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Complexes 3 and 5 decrease stem cell marker ALDH in CAL-51 cells. Breast cancer CAL-51 cells were
treated with 3 and 5 at 0.5 ×, 1 × and 2 × IC50 concentrations for 24 h and the per-centage of ALDH+ cells
determined by flow cytometry. In order to better visualize ALDH+ cells, two-dimensional plots were
obtained. SSC, side scatter channel. Histogram indicates the average percentage of the ALDH+ cell
subpopulation from three indepen-dent experiments.
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Schematic representation of the proposed binding modes for complexes 1-5 with proteasome (PDB
ID:3MG6). Only amino acids located within 4 Å of the bound ligand are displayed and labeled. Key H-bonds
between the complexes and the protein are shown as dashed yellow lines.
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Novel copper complexes that inhibit the proteasome and
trigger apoptosis in triple-negative breast cancer cells
Dong-Dong Li 1, Ernesto Yagüe3, Lu-Yao Wang1, Lin-Lin Dai1, Zi-Bo Yang1, Shuang Zhi1, Na
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Zhang1, Xiu-Mei Zhao1*, Yun-Hui Hu2
1Tianjin Institute of Medical and Pharmaceutical Sciences, Tianjin 300020, China
2The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital,
Tianjin 300060
3Cancer Research Center, Division of Cancer, Faculty of Medicine, Imperial College London,
Hammersmith Hospital Campus, London W12 0NN, UK
Corresponding authors:
Dong-Dong Li, Tianjin Institute of Medical and Pharmaceutical Sciences, E-mail address:
lidongdong2010@163.com;
Xiu-Mei Zhao, Tianjin Institute of Medical and Pharmaceutical Sciences, E-mail address: zxmmlg@163.com;
Yun-Hui Hu, The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital,
E-mail: yunhuihu200408@163.com.
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