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J Biol Inorg Chem (2016) 21:263–273
DOI 10.1007/s00775-016-1337-z
ORIGINAL PAPER
Copper diethyldithiocarbamate as an activator of Nrf2
in cultured vascular endothelial cells
Tomoya Fujie1 · Masaki Murakami1 · Eiko Yoshida1 · Tadashi Tachinami2 ·
Yasuhiro Shinkai3 · Yasuyuki Fujiwara4 · Chika Yamamoto5 · Yoshito Kumagai3 ·
Hiroshi Naka2 · Toshiyuki Kaji1
Received: 28 October 2015 / Accepted: 8 January 2016 / Published online: 29 January 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract The interest in organic–inorganic hybrid
molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules
exhibit unique biological activities. Herein, copper(II)
bis(diethyldithiocarbamate) (Cu10) was found to activate
the transcription factor NF-E2-related factor 2 (Nrf2),
which is responsible for regulating antioxidant and phase
II xenobiotic enzymes, in vascular endothelial cells. The
copper complex rapidly accumulated within cells and
induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-016-1337-z) contains supplementary
material, which is available to authorized users.
* Hiroshi Naka
h_naka@nagoya‑u.jp
* Toshiyuki Kaji
t‑kaji@rs.tus.ac.jp
1
Department of Environmental Health, Faculty
of Pharmaceutical Sciences, Tokyo University of Science,
2641 Yamazaki, Noda 278‑8510, Japan
2
Graduate School of Science and Research Center
for Materials Science, Nagoya University, Chikusa,
Nagoya 464‑8602, Japan
3
Environmental Biology Laboratory, Faculty of Medicine,
University of Tsukuba, 1‑1‑1 Tennodai, Tsukuba 305‑8575,
Japan
4
Department of Environmental Health, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, 1432‑1
Horinouchi, Hachioji 192‑0392, Japan
5
Department of Environmental Health, Faculty
of Pharmaceutical Sciences, Toho University, 2‑2‑1 Miyama,
Funabashi 274‑8510, Japan
dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to
exhibit this activity. Intracellular accumulation of Cu10
was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was
reduced by small interfering RNA (siRNA)-mediated
knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that
Cu10 rapidly enters vascular endothelial cells via CTR1independent mechanisms. In addition, copper and iron
complexes with other ligands tested could not activate Nrf2,
suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression
of heme oxygenase-1, NAD(P)H quinone oxidoreductase
1, and γ-glutamylcysteine synthetase, downstream proteins
of Nrf2. It was suggested that Cu10-induced activation of
Nrf2 was due to proteasome inhibition as well as binding
to Kelch-like ECH-associated protein 1. Since the effects of
Cu10 on vascular endothelial cells are unique and diverse,
the copper complex may be a good molecular probe to analyze the functions of the cells.
Keywords Copper(II) bis(diethyldithiocarbamate) ·
Bio-organometallics · Nrf2 · Proteasome · Keap1 ·
Endothelial cell
Abbreviations
ARE Antioxidant response element
BPM Biotin-PEAC5-maleimide
Cu01 Bis(hexafluoroactylacetonato)copper(II)
Cu02
N,N′-Bis(2-methoxycarbonyl-3-oxobutylidene)
ethylenediaminatocopper(II)
Cu03 Bis(salicylidene)ethylenediaminatocopper(II)
Cu04 Copper(II) diacetate
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264
Cu07 Bis(1,3-propanediamine)copper(II) dichloride
Cu09 Copper(II) bis(2-hydroxyethyl)dithiocarbamate
Cu10 Copper(II) bis(diethyldithiocarbamate)
Cu15
N ,N′-Bis(3,5-di-tert-butyl-2-oxidobenzyl)
ethylenediaminatocopper(II)
Cu17 Copper(II) bis(dimethyldithiocarbamate)
Cu18 Copper(II) bis(dibutyldithiocarbamate)
Cu19 Copper(II) bis(dibenzyldithiocarbamate)
Fe01 Tris(acetylacetonato)iron(III)
Fe02 Iron(II) diacetate
Fe03 Iron(II) phthalocyanine
Fe04 Sodium iron(III) ethylenediaminetetraacetate
Fe05 Iron(III) tris(diethyldithiocarbamate)
Keap1 Kelch-like ECH-associated protein 1
MG132 Z-Leu-Leu-Leu-CHO
Na01 Sodium diethyldithiocarbamate trihydrate
Ni06 Nickel(II) bis(diethyldithiocarbamate)
Nrf2 NF-E2-related factor 2
Zn01 Zinc(II) bis(diethyldithiocarbamate)
Introduction
NF-E2-related factor 2 (Nrf2) is a transcription factor that
belongs to Cap‘n’Collar transcription factor family and
has a basic leucine zipper domain [1]. Under basal conditions, Nrf2 is bound to Kelch-like ECH-associated protein
1 (Keap1), which is an adapter protein to Cullin3-based E3
ubiquitin ligase, to prevent the proteasomal degradation of
Nrf2 in the cytoplasm [2]. Keap1 also functions as a sensor
protein against electrophiles and reactive oxygen species.
Modification of the reactive thiols of Keap1 by electrophiles results in the dissociation of Nrf2 from Keap1 and
its nuclear translocation, allowing it to bind antioxidant
response element (ARE) of the genes, thereby forming a
heterodimerized complex of Nrf2 with co-activators such
as small Maf [3].
Nrf2 mainly regulates the gene expression of antioxidant and phase II xenobiotic metabolizing enzymes such as
heme oxygenase-1, NAD(P)H quinone oxidoreductase 1,
and γ-glutamylcysteine synthetase, by binding to the ARE
of the promoter region of the genes. Induction of heme oxygenase-1 protects cells from oxidative injury by catalyzing
heme to biliverdin, carbon monoxide, and iron [4]. NAD(P)
H quinone oxidoreductase 1 catalyzes the detoxification of
quinones and their derivatives [5]. γ-Glutamylcysteine synthetase is a rate-limiting enzyme in glutathione synthesis
and consists of two subunits: the modifier subunit and the
catalytic subunit. We postulate that low-molecular-weight
molecular probes that activate Nrf2 and regulate cellular
functions will be useful in analyzing the involvement of the
transcription factor in the regulation of vascular endothelial
cell functions.
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J Biol Inorg Chem (2016) 21:263–273
Organic–inorganic hybrid molecules—organometallic
compounds and metal coordination compounds—consist
of metals and organic ligands in a common feature. These
compounds can exhibit unique biological activities, different from those of organic and inorganic compounds; their
activities are attributable to their unique three-dimensional
structures and electronic states [6–8]. It is most likely that
organic–inorganic hybrid molecules exhibit their activities by modifying the activities of ligand, those of metal,
or interaction between ligand and metal. We found that
bis(l-cysteinato)zincate(II) serves as a specific zinc donor
to the metal response element-binding transcription factor-1, a transcription factor containing six C2H2 zinc finger domains [6]. We have reported that an organobismuth
compound—tris[2-(N,N-dimethylaminomethyl)phenyl]bismuthane—exhibits vascular endothelial cell-specific toxicity [7] and the cytotoxicity disappears when the bismuth
atom is replaced with an antimony atom [8]. Recently,
it was found that an organoantimony compound—
tris(pentafluorophenyl)stibane—causes
transcriptional
induction of metallothionein (submitted).
There are many reports on low-molecular-weight compounds including toxic metal(loid)s that activate Nrf2; for
example, sulforaphane, curcumin, tert-butylhydroquinone,
1,2-naphthoquinone, methylmercury, and arsenite [9–13].
However, little is known about organic–inorganic hybrid
molecules. In the present study, to obtain a good molecular probe for analysis of vascular endothelial cell functions
that are regulated by Nrf2, we searched for organic–inorganic hybrid molecules that activate Nrf2 without cytotoxicity in cultured vascular endothelial cells. We found that
copper(II) bis(diethyldithiocarbamate) (Cu10) exerts such
a biological activity via proteasome inhibition as well as
Keap1 modification in the cells.
Materials and methods
Materials
Bovine aortic endothelial cells were purchased from Cell
Applications (San Diego, CA, USA). The following materials were purchased from the respective vendors: Dulbecco’s
modified Eagle’s medium and calcium- and magnesiumfree phosphate buffered saline from Nissui Pharmaceutical
(Tokyo, Japan); fetal bovine serum from HyClone Laboratories (Waltham, MA, USA); biotin-PEAC5-maleimide
(BPM) from Dojindo (Kumamoto, Japan); rabbit polyclonal anti-actin antibody (A5060) and copper(II) diacetate
(Cu04) from Sigma Aldrich Chemical (St. Louis, MO,
USA); horseradish peroxidase-conjugated anti-rabbit IgG
antibody (#7074) and anti-biotin, horseradish peroxidaselinked antibody (#7075) from Cell Signaling (Beverly,
J Biol Inorg Chem (2016) 21:263–273
MA, USA); anti-NAD(P)H quinone oxidoreductase 1 antibody (ab2346) and donkey polyclonal antibody to goat
IgG-horseradish peroxidase (ab6885) from Abcam (Tokyo,
Japan); rabbit polyclonal anti Nrf2 antibody (H-300), rabbit
polyclonal anti CTR1 antibody (FL-190), mouse polyclonal
anti Keap1 antibody (H-190), and rabbit polyclonal anti
γ-glutamylcysteine synthetase modifier subunit antibody
(FL-274) from Santa Cruz Biotechnology (Santa Cruz, CA,
USA); rabbit polyclonal anti-heme oxygenase-1 antibody
(ADI-SPA-895) and ubiquitin monoclonal antibody (ADISPA-203), and MG-132 from Enzo Life Sciences (Farmingdale, NY, USA); nitric oxide, hydrogen peroxide, copper(II)
bis(dimethyldithiocarbamate) (Cu17), and sodium diethyldithiocarbamate trihydrate (Na01) from Wako Pure
Chemical Industries (Osaka, Japan); 3,5-diaminobenzoic
acid, bis(hexafluoroactylacetonato)copper(II) (Cu01),
bis(1,3-propanediamine)copper(II) dichloride (Cu07),
copper(II) bis(2-hydroxyethyl)dithiocarbamate (Cu09),
copper(II) bis(diethyldithiocarbamate) (Cu10), iron(II)
phthalocyanine (Fe03), sodium iron(III) ethylenediaminetetraacetate (Fe04), iron(III) tris(diethyldithiocarbamate)
(Fe05), nickel(II) bis(diethyldithiocarbamate) (Ni06),
zinc(II) bis(diethyldithiocarbamate) (Zn01), zinc(II)
bis(dibutyldithiocarbamate), and zinc(II) bis(dibenzyldi
thiocarbamate) from Tokyo Chemical Industry (Tokyo,
Japan); iron(II) diacetate (Fe02) from Acros Organics
(Thermo Fisher Scientific, Geel, Belgium); Chemi-Lumi
One L and other reagents were from Nacalai Tesque
(Kyoto, Japan).
Synthesis
N , N ′ - B i s ( 2 - m e t h o x y c a r b o n y l - 3 - o x o bu t y l i d e n e )
ethylenediaminatocopper(II) (Cu02), bis(salicylidene)
ethylenediaminatocopper(II) (Cu03), and N,N′-Bis(3,5di-tert-butyl-2-oxidobenzyl)ethylenediaminatocopper(II)
(Cu15) were synthesized by employing literature procedures [14–16]. Copper(II) bis(dibutyldithiocarbamate)
(Cu18) was synthesized by mixing copper(II) diacetate
(3.63 g, 20 mmol) and zinc(II) bis(dibutyldithiocarbamate)
(9.48 g, 20 mmol) in a 1:1 molar ratio in a biphasic mixture of dichloromethane (1 L), water (100 mL), and 25 %
aqueous ammonia (200 mL) at room temperature for 1 h
under aerobic conditions. The organic layer was separated, washed with water, concentrated under reduced
pressure, and dried in vacuum to yield the desired product as a black solid (9.32 g, 99 %). Elemental analysis
calculated for [C18H36CuN2S4]: C, 45.78; H, 7.68; N,
5.93 and found: C, 46.04; H, 7.80; N, 5.65. Copper(II)
bis(dibenzyldithiocarbamate) (Cu19) was prepared analogously using zinc(II) bis(dibenzyldithiocarbamate) (92 %
yield). Elemental analysis calculated for [C30H28CuN2S4]:
C, 59.23; H, 4.64; N, 4.60 and found: C, 59.64; H, 4.68;
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N, 4.24. The elemental analyses were recorded on a Yanaco
CHN recorder MT-6 at the Chemical Instrumental Center,
Research Center for Materials Science, Nagoya University.
Western blot analysis
Confluent cultures of vascular endothelial cells in 35-mm
culture dishes were incubated at 37 °C for 1, 2, 3, 4, 6, 8,
12, or 24 h with Cu10 or other compounds at 0.1, 0.5, 1, 2,
5, or 10 µM. The cells were washed twice with ice-cold calcium- and magnesium-free phosphate buffered saline; total
cellular proteins obtained by lysis in sodium dodecyl sulfate sample buffer (50 mM Tris–HCl buffer solution containing 2 % sodium dodecyl sulfate and 10 % glycerol, pH
6.8) were incubated at 95 °C for 5 min. The protein concentration was determined using a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, Waltham,
MA, USA). 2-Mercaptoethanol and bromophenol blue
(1.67 % each) were added to the proteins (10 μg). The proteins were separated by SDS–polyacrylamide gel electrophoresis on 10 % polyacrylamide gel and transferred onto
a polyvinylidene difluoride membrane at 2 mA/cm2 for 1 h.
The membrane was blocked with 5 % skim milk in 20 mM
Tris–HCl buffer containing 150 mM NaCl and 0.1 %
Tween-20, pH 7.5 and incubated with primary antibodies
(1:200) at 4 °C overnight. The membrane was washed with
20 mM Tris–HCl buffer solution containing 150 mM NaCl
and 0.1 % Tween 20 (pH 7.5), and then incubated with
horseradish peroxidase-conjugated secondary antibodies
for 1 h at room temperature. Immunoreactive bands were
visualized by enhanced chemiluminescence and scanned
by LAS3000 (Fujifilm, Tokyo, Japan). Separately, vascular
endothelial cells were treated with Cu10 at 0.1, 0.5, 1, 5, or
10 µM for 3 or 6 h and the nuclear fraction was prepared
from the cell layer using the NE-PER Nuclear Cytoplasmic Extraction Reagents (Thermofisher Scientific). Nuclear
protein concentration was determined by a bicinchoninic
acid protein assay reagent kit (Thermo Fisher Scientific).
The samples (8 µg protein) were mixed with 50 mM Tris–
HCl solution containing 8 % glycerol, 2 % sodium dodecyl sulfate, 2-mercaptoethanol, and 0.005 % bromophenol
blue, pH 6.8 and incubated at 95 °C for 3 min. These samples were analyzed by western blotting as described above.
Intracellular accumulation of metals
Confluent cultures of vascular endothelial cells were
incubated in 6-well plates at 37 °C for 3 h in serum-free
Dulbecco’s modified Eagle’s medium in the presence of
CuSO4, sodium diethyldithiocarbamate trihydrate (Na01),
zinc(II) bis(diethyldithiocarbamate) (Zn01), iron(III)
tris(diethyldithiocarbamate) (Fe05), copper(II) bis(2hydroxyethyl)dithiocarbamate (Cu09), CuSO4 [Cu(II)],
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Keap1 protein (2 µg) was incubated with Cu10 (1, 10,
or 100 µM) at 37 °C for 30 min in 100 mM Tris–HCl
buffer solution (pH 7.5). After incubation, 25 µM biotin-PEAC5-maleimide was added to the samples and the
samples were incubated at 37 °C for 30 min. The samples
were electrophoresed on 10 % sodium dodecyl sulfate–
polyacrylamide gel in 50 mM Tris–HCl containing 2 %
SDS, 8 % glycerol, and 0.005 % bromophenol blue (pH
6.8) without 2-mercaptoethanol and were incubated at
37 °C for 30 min. They were then subjected to immunoblotting as described above.
CuSO4 with 1 mM ascorbate [Cu(I)] [17], or Cu10 (10 µM
each). In another experiment, subconfluent cultures of
bovine aortic endothelial cells were transfected with control or CTR1 small interfering RNA (siRNA) as described
below and incubated at 37 °C for 3 h in the presence of
Cu10, Cu17, Cu18, and Cu19 (10 µM each). After incubation, the medium was discarded and the cells were washed
twice with ice-cold calcium- and magnesium-free phosphate buffered saline. The cell lysates were prepared by
addition of 100 µL 50 mM Tris–HCl containing 2 % SDS
and 10 % glycerol (pH 6.8). The cell lysate was incubated
at 95 °C for 3 min and a portion was treated with nitric
acid-H2O2 at 130 °C for 1 day to degrade proteins and dissolved with 2 mL of 0.1 M nitric acid; the diluted samples were used for determination of zinc, copper, and iron
content by inductively coupled plasma mass spectrometry
(ERAN DRC II, PerkinElmer, MA, USA). Another portion
of the cell lysate was analyzed for DNA content by fluorometric method [18] to normalize the content of the metals
per µg DNA.
Results
Transfection
Cu10 activates Nrf2 in vascular endothelial cells
Vascular endothelial cells were cultured and siRNAs
(Bioneer, Daejeon, Korea) for the copper transporter CTR1
were transfected using RNAiMAX reagent (Invitrogen,
Crlsbad, CA, USA), as described previously [19]. Briefly,
the cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10 % fetal bovine serum
in 35-mm dishes until 70–80 % confluence. Separately,
siRNA duplex (35 pmol/mL) and transfection reagent (2
μL/mL) were mixed with Opti-MEM (Thermofisher Scientific) and incubated for 20 min at room temperature. The
mixture was added to the culture medium, and the cells
were incubated at 37 °C for 24 h and then treated with or
without Cu10, Cu17, Cu18, or Cu19 (10 µM each) for 3 h.
The sequences of the sense and antisense strands of siRNA
were as follows: bovine CTR1 siRNA, 5-AUAAGGAUGGUUCCAUUUGdTdT-3 (sense) and 5-CAAAUGGAACCAUCCUUAU-3 (antisense). A nonspecific sequence was
used as the siRNA negative control (Qiagen, Valencia, CA,
USA).
Figure 1 shows the activation of Nrf2 by Cu10 (the structure is shown in Fig. 1a) in vascular endothelial cells.
Cu10 at 10 µM or less increased the expression of Nrf2
in a concentration-dependent manner (Fig. 1b). Cu10 at
10 µM increased the expression of Nrf2 after 2 h or longer
in a time-dependent manner; the highest expression was
observed at 8 h and gradually reduced thereafter (Fig. 1c).
Nrf2 was detected in the nuclear fraction after 3 and 6 h in
vascular endothelial cells treated with Cu10 at 5 and 10 µM
(Fig. 1d). After a 24 h treatment with Cu10 at 0.1 µM or
higher, the expression of downstream proteins of Nrf2—
heme oxygenase-1, NAD(P)H quinone oxidoreductase 1,
and γ-glutamylcysteine synthetase modifier subunit—significantly increased in a concentration-dependent manner
(Fig. 1e).
Keap1 and biotin‑PEAC5‑maleimide‑labeling assay
in vitro
The mouse recombinant Keap1 construct was prepared
as described previously [10, 20]. The recombinant Keap1
protein was expressed as a C-terminal His-tagged fusion
protein in BL21(DE3)pLysS E. coli cells and purified using a ProBond nickel-resin. The BPM-labeling
assay was performed according to the method described
by Toyama et al. [20]. Briefly, mouse recombinant
13
Statistical analysis
The data were analyzed for statistical significance by Student’s t test when possible. P values less than 0.01 were
considered statistically significant.
Role of copper in the Cu10 molecule in Nrf2 activation
In order to examine whether copper in the Cu10 molecule
is critical to the activation of endothelial Nrf2, the effects
of zinc and iron complexes with the same ligand of Cu10
on Nrf2 activation were investigated. In this experiment,
copper sulfate, Na01 as the ligand of Cu10, and Cu09 were
investigated. The structures of the tested metal complexes
and Na01 are shown in Fig. 2a. As shown in Fig. 2b, Cu10
and Cu09 increased the expression of Nrf2; however, copper sulfate, Na01, Zn01, and Fe05 failed to exhibit such an
activity (Fig. 2b), indicating that copper is required for diethyldithiocarbamate complexes to activate Nrf2 in vascular
endothelial cells. Copper sulfate, Zn01, and Fe05 did not
J Biol Inorg Chem (2016) 21:263–273
267
(a)
(b)
(c)
(d)
(e)
Fig. 1 Activation of Nrf2 by Cu10 in vascular endothelial cells. a
The structure of Cu10. b The expression of Nrf2. Confluent cultures
of bovine aortic endothelial cells were incubated at 37 °C for 3 h in
the presence or absence of Cu10 (0.1, 0.5, 1, 5, or 10 µM). c Time
course of the effect of Cu10 on the expression of Nrf2. Confluent cultures of bovine aortic endothelial cells were incubated at 37 °C for 1,
2, 3, 4, 6, 8, 12, and 24 h in the presence or absence of Cu10 (10 µM).
d The expression of Nrf2 in the nuclei. Confluent cultures of bovine
aortic endothelial cells were incubated at 37 °C for 3 and 6 h in the
presence or absence of Cu10 (0.1, 0.5, 1, 5, or 10 µM). e The expression of downstream proteins of Nrf2. Confluent cultures of bovine
aortic endothelial cells were incubated at 37 °C for 24 h in the presence or absence of Cu10 (0.1, 0.5, 1, 5, or 10 µM). HO-1 heme oxygenase-1, NQO1 (upper bands) NAD(P)H quinone oxidoreductase 1,
GCLM γ-glutamylcysteine synthetase modifier subunit
accumulate within the cells after a 3 h treatment, whereas
significant accumulation of Cu10 and Cu09 was observed
(Fig. 2c), suggesting that Nrf2-activating activity of the
copper complexes may depend on their high intracellular
accumulation.
Since Cu10 and Cu09 highly accumulated within vascular endothelial cells and enhanced Nrf2 expression,
Cu(II) may have been reduced to Cu(I) by thiol groups
in the thiocarbamate ligands and Cu(I) ion released from
the Cu10 molecule efficiently entered the cells through
the copper transporter CTR1 that mediated Cu(I) uptake
[17]. To examine this hypothesis, we compared the intracellular accumulation of Cu(II), Cu(I), and Cu10 in vascular endothelial cells and determined the involvement
of CTR1 in the uptake of copper complexes with thiol
groups. The structures of the tested copper complexes are
shown in Fig. 3a. The intracellular accumulation of Cu10
was high in vascular endothelial cells compared with
Cu(II) and Cu(I) (Fig. 3b). The accumulation of copper(II)
bis(dimethyldithiocarbamate) (Cu17) was significantly
reduced by siRNA-mediated knockdown of CTR1; however, Cu10, copper(II) bis(dibutyldithiocarbamate)
(Cu18), and copper(II) bis(dibenzyldithiocarbamate)
(Cu19) accumulation was not affected by the knockdown
(Fig. 3c, d). Cu17 as well as Cu10 activated Nrf2, regardless of whether CTR1 was knocked down or not (Fig. 3e).
However, Cu18 and Cu19 highly accumulated in the cells
(Fig. 3d), regardless of CTR1 expression, but failed to activate Nrf2 in vascular endothelial cells.
Role of the ligand in the Cu10 molecule in Nrf2
activation
It is possible that copper complexes in general activate
endothelial Nrf2. To examine this possibility, we evaluated copper complexes with various ligands. In this experiment, compounds containing iron were also examined. The
compounds used in this experiment are shown in Fig. 4a.
Among the tested copper complexes, only Cu10 and Cu09
increased the expression of Nrf2 (Fig. 4b), suggesting that
the ligand as well as the copper ion is important for the
activation of Nrf2 by Cu10. Other compounds could not
activate the transcription factor. The expression of Nrf2 in
vascular endothelial cells treated with Cu01, Cu02, Cu03,
Cu04, Cu07, Cu09, Cu15, Fe01, Fe02, Fe03, Fe04, Fe05,
CuSO4, and Na01 is shown in S1.
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(a)
(b)
(c)
Fig. 2 Role of copper in the Cu10 molecule in Nrf2 activation in
vascular endothelial cells. a The structures of Na01, Zn01, Fe05,
Cu09, and Cu10. b The expression of Nrf2. Confluent cultures of
bovine aortic endothelial cells were incubated at 37 °C for 3 h in the
presence or absence of CuSO4, Na01, Zn01, Fe05, Cu09, and Cu10
(10 µM each). c Intracellular accumulation of CuSO4, Na01, Zn01,
Fe05, Cu09, and Cu10. Confluent cultures of bovine aortic endothelial cells were incubated at 37 °C for 3 h in the presence or absence
of CuSO4, Na01, Zn01, Fe05, Cu09, and Cu10 (10 µM each). White,
gray, and black bars indicate the content of zinc, iron, and copper,
respectively. Values are mean ± SE of three samples. *Significantly
different from the control, P < 0.01
Characterization of Nrf2 activation by Cu10
Keap1 with Cu10 decreased the signal of biotinylated Keap1
protein (Fig. 6), indicating that Cu10 was bound to Keap1.
As shown in the previous study [20], cadmium bound to
Keap1. Since Keap1 binds Nrf2 and protects it from proteasomal degradation, inhibition of proteasome can be a
mechanism through which Nrf2 is activated. To examine
this possibility, the proteasome inhibitory activity of Cu10
was investigated. In this experiment, Zn01 and nickel(II)
bis(diethyldithiocarbamate) (Ni06) were also evaluated (the
structures are shown in Fig. 7a). It was shown that Cu10
increased the ubiquitinated proteins in a concentration- and
time-dependent manner (Fig. 7b), indicating that the copper
complex inhibits proteasome; MG132, a typical proteasome
inhibitor also increased the level of ubiquitinated proteins.
Zn01 and Ni06 failed to increase both Nrf2 expression and
level of ubiquitinated proteins (Fig. 7c). Since cadmium, a
toxic heavy metal in vascular endothelial cells [22], did not
display proteasome inhibitory activity, it is suggested that
the proteasome inhibition by Cu10 was not a nonspecific
effect of the copper complex (Fig. 7d).
The activation of Nrf2 by Cu10 was compared with that
by sulforaphane (the structure is shown in Fig. 5a), an isothiocyanate that modifies Keap1 and activates Nrf2 [21].
It was shown that the Nrf2-activating activity of Cu10 and
sulforaphane is almost comparable (Fig. 5b); however,
the expression of Nrf2 downstream proteins induced by
Cu10 and sulforaphane differed. Specifically, Cu10 markedly increased the expression of heme oxygenase-1 and
γ-glutamylcysteine synthetase modifier subunit, whereas
the expression of NAD(P)H quinone oxidoreductase 1 was
markedly upregulated by sulforaphane (Fig. 5c), suggesting
that the mechanisms underlying Nrf2 activation by Cu10
may be different from that of sulforaphane.
Activation mechanisms of Nrf2 by Cu10
To examine whether Cu10 can bind Keap1, the BPM-labeling assay was performed. Incubation of recombinant mouse
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Fig. 3 Intracellular accumulation of Cu(II), Cu(I), and Cu10 in vascular endothelial cells, and involvement of CTR1 in the accumulation of copper complexes. a The structures of Cu10, Cu17, Cu18,
and Cu19. b Intracellular accumulation of Cu(II), Cu(I), and Cu10.
Confluent cultures of bovine aortic endothelial cells were incubated at 37 °C for 3 h in the presence or absence of CuSO4 [Cu(II)],
CuSO4 with 1 mM ascorbate [Cu(I)], and Cu10 (10 µM each). Values
are expressed as mean ± SE for the four samples. c CTR1 protein
expression after siRNA-mediated knockdown of CTR1. Subconfluent cultures of bovine aortic endothelial cells were transfected with
control or CTR1 siRNA and incubated at 37 °C in the presence or
absence of Cu10, Cu17, Cu18, and Cu19 (10 µM each) for 3 h. d
Intracellular accumulation of Cu10, Cu17, Cu18, and Cu19. Subconfluent cultures of bovine aortic endothelial cells were transfected with
control or CTR1 siRNA and incubated at 37 °C in the presence or
absence of Cu10, Cu17, Cu18, and Cu19 (10 µM each). Values are
expressed as mean ± SE for the four samples. *Significantly different from the control, P < 0.01. e Nrf2 expression. Subconfluent cultures of bovine aortic endothelial cells were transfected with control
or CTR1 siRNA and incubated at 37 °C in the presence or absence of
Cu10, Cu17, Cu18, and Cu19 (10 µM each)
Discussion
of CTR1 expression, and Nrf2 activation of copper complexes with thiol groups depended on the structures and
not on intracellular accumulation; (5) both proteasome
inhibition and binding to Keap1 are mechanisms through
which Cu10 activates Nrf2. In general, the relationship
between the biological activity and the structure of a certain organic–inorganic hybrid molecule could follow any
of the following three patterns. First, the ligand has biological activity and intramolecular metal intensifies the
activity. Second, the metal has biological activity and the
ligand intensifies the activity. Third, the biological activity of either the ligand or the metal is only slight but the
hybrid molecule exhibits activity owing to their discrete
molecular structures that originate from metal–ligand
coordination (i.e., intramolecular interaction). Since neither copper sulfate nor Na01 could activate Nrf2, we postulate that the activation of Nrf2 by Cu10 is induced by
the intramolecular interaction between copper atom and
Dithiocarbamates are metal-chelating compounds that
affect the activities of various metal-binding proteins such
as nuclear factor-kappa B and superoxide dismutase-1
[23, 24]. The present study revealed the previously unrecognized biological activities of metal diethyldithiocarbamate coordination compounds. The following results were
obtained: (1) copper(II) bis(diethyldithiocarbamate), Cu10,
activates Nrf2 in vascular endothelial cells without cytotoxicity, (2) the combination of copper with the diethyldithiocarbamate ligand is essential for the effect of Cu10, (3)
Cu10 rapidly enters the cells and induces nuclear translocation of Nrf2, resulting in the induction of downstream
proteins such as heme oxygenase-1, NAD(P)H quinone
oxidoreductase 1, and γ-glutamylcysteine synthetase modifier subunit, (4) high concentrations of Cu10 accumulated
within the cells compared with Cu(II) and Cu(I), regardless
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J Biol Inorg Chem (2016) 21:263–273
Fig. 4 Role of the ligand in Cu10 molecule in Nrf2 activation in vascular endothelial cells. a The structures of Cu01, Cu02, Cu03, Cu04,
Cu07, Cu09, Cu10, Cu15, Fe01, Fe02, Fe03, and Fe04. b The expression of Nrf2. Confluent cultures of bovine aortic endothelial cells
were incubated at 37 °C for 3 h in the presence or absence of Cu01,
Cu02, Cu03, Cu04, Cu07, Cu09, Cu10, Cu15, Fe01, Fe02, Fe03, and
Fe04 (10 µM each)
(a)
(b)
Fig. 5 Characterization of Nrf2 activation by Cu10 compared with
sulforaphane. a The structures of Cu10 and sulforaphane. b The
expression of Nrf2. Confluent cultures of bovine aortic endothelial
cells were incubated at 37 °C for 3 h in the presence or absence of
Cu10 (5 or 10 µM) or sulforaphane (1, 5, or 10 µM). c The expression
13
(c)
of downstream proteins of Nrf2. Confluent cultures of bovine aortic
endothelial cells were incubated at 37 °C for 24 h in the presence or
absence of Cu10 (5 or 10 µM) or sulforaphane (1, 5, or 10 µM). HO1 heme oxygenase-1, NQO1 NAD(P)H quinone oxidoreductase 1,
GCLM γ-glutamylcysteine synthetase modifier subunit
J Biol Inorg Chem (2016) 21:263–273
271
Fig. 6 Binding of Cu10 to Keap1. Recombinant mouse Keap1 protein (2 µg) was incubated with Cu10 (1, 10, or 100 µM) at 37 °C for
30 min in 100 mM Tris–HCl (pH 7.5) and then further incubated at
37 °C for 30 min after addition of 25 µM biotin-PEAC5-maleimide.
The samples were subjected to western blotting, which was performed using anti-biotin antibody (BPM) and anti-Keap1 antibody
(Keap1). Cadmium chloride (CdCl2) was used as the positive control
the diethyldithiocarbamate ligand. It is also suggested that
this interaction is required for rapid accumulation of Cu10
within vascular endothelial cells.
The mechanisms of high intracellular accumulation of
Cu10 are critical for understanding the mechanisms by
which Cu10 activates Nrf2 in vascular endothelial cells.
Copper complexes with thiol groups, such as Cu10 and
Cu09, markedly accumulate within the cells and activate
Nrf2, whereas copper complexes without thiol groups
failed to activate Nrf2, suggesting that Cu(II) may be
Fig. 7 Proteasome inhibition
by Cu10, Zn01, Ni06, and
CdCl2 in vascular endothelial
cells. a The structures of Cu10,
Zn01, and Ni06. b Proteasome
inhibitory activity. Confluent cultures of bovine aortic
endothelial cells were incubated
at 37 °C for 8 h in the presence or absence of Cu10, Zn01,
Ni06, or cadmium chloride
(CdCl2) (1, 5, 10 µM each).
MG132 was used as positive
control. The total cell lysates
were subjected to western
blotting, which was performed
using an anti-ubiquitin antibody
(a)
(c)
reduced to Cu(I) by thiol groups in thiocarbamate ligands,
and the Cu(I) ion released from the Cu10 molecule efficiently entered the cells via the copper transporter CTR1
[17]. In other words, Cu10 served as a donor of Cu(I) to
CTR1, and consequently, the copper ion, but not the Cu10
molecule, activated Nrf2 through modification of Keap1.
However, intracellular accumulation of Cu10 was much
higher than that of Cu(II) and Cu(I) and was not affected
by siRNA-mediated knockdown of CTR1. In contrast,
the accumulation of Cu17 was significantly reduced by
the knockdown, suggesting that CTR1 is at least partly
involved in the uptake of Cu17. Therefore, the possibility
that CTR1 partly mediates the uptake of copper complexes
as well as cisplatin [25], cannot be excluded, and it is suggested that CTR1 expression is not the major mechanism
of Cu10 uptake. In addition, Cu18 and Cu19 were also
highly accumulated within the cells, but failed to activate
Nrf2, thereby suggesting that activation of Nrf2 by copper complexes depends on the ligand structure rather than
the copper ion released from the molecule. Thus, although
the details are yet to be elucidated, an assumption can be
made that Cu10 was transported as a molecule, and modified Keap1 and activated Nrf2 in vascular endothelial cells.
Ubiquitin–proteasome system is responsible for the
degradation of numerous proteins, including Nrf2. The
26S proteasome consists of two complexes—the 20S
(b)
(d)
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272
proteolytic core and the 19S regulatory complex [26]. Previously, it was reported that Cu10 inhibits proteasomal
function by inhibiting both 20S chymotrypsin-like activity
and 19S complex in human breast cancer MBA-MD-231
cells [27]. In this report, Zn01 but not Ni06 showed proteasome inhibitory activity, suggesting that the type of
metal complexes that have similar biological activities may
depend on cell types. While the reason underlying this cell
type dependency is unclear, it is certain that Cu10 exhibits proteasome inhibitory activity and inhibits the degradation of Nrf2 in vascular endothelial cells. Proteasome
inhibition would be one of the major mechanisms by which
Cu10 activates endothelial Nrf2. Conversely, it was shown
that Cu10 binds Keap1 and this led to the release of Nrf2
from Keap1 followed by nuclear translocation of Nrf2.
There are several reactive cysteine residues in the Keap1
molecule and the residues that are involved in Nrf2 activation are Cys151 in the BTB domain and Cys273/Cys288 in
the IVR domain [28, 29]. The modified cysteine residues
that are employed for Nrf2 activation depend on the compounds that activate Nrf2 [30, 31]. For example, zinc ion
binds to both Cys273 and Cys288 and activates Nrf2 [32].
It is suggested that Cu10 binds to at least one of the three
reactive cysteine residues of the Keap1 molecule. While it
is unclear as to which cysteine residue(s) are modified by
Cu10, it is postulated that modification of Keap1 is one of
the major mechanisms by which Cu10 activates endothelial
Nrf2. It was shown that, among the downstream proteins
of Nrf2, heme oxygenase-1 and γ-glutamylcysteine synthetase modifier subunit were markedly induced by Cu10,
whereas sulforaphane, which is bound to Keap1 and activates Nrf2, strongly induced NAD(P)H quinone oxidoreductase 1. This difference in the induction of downstream
protein between Cu10 and sulforaphane may be attributable
to the difference of Nrf2 activation mechanisms. However,
this is yet to be elucidated.
A compound that has a specific target biomolecule is an
excellent tool to analyze the role of the biomolecule in the
regulation of some biological systems. However, a compound that has multiple targets is also useful to analyze the
relationship among the targets. Cu10 appears to belong to
the latter case. In fact, we have analyzed vascular endothelial cell functions using Cu10. Metallothionein is a lowmolecular-weight, cysteine-rich, metal-containing, inducible protein, which protects cells from heavy metals and
oxidative stress [33]. Since cadmium and zinc induce metallothionein, they have been used as tools to analyze mechanisms underlying metallothionein induction. However, the
metals cannot be good tools because vascular endothelial
cells are sensitive to cadmium [34] and zinc does not induce
metallothionein in the cells [35, 36]. Recently, we found
that this copper complex induces metallothionein in vascular endothelial cells; activation of Nrf2 and consequent
13
J Biol Inorg Chem (2016) 21:263–273
activation of ARE in the promoter region of metallothionein
genes contribute to the induction of specific metallothionein
isoform [19]. Further studies on Cu10 as a tool to analyze
vascular endothelial cell functions are ongoing.
Acknowledgments This work was supported by a Grant-in-Aid
for Challenging Exploratory Research #15K14992 from the Japan
Society for the Promotion of Science (to T. K.) and by the Nagoya
University Science Foundation (to H. N.). Masaki Shibata and Kiyotaka Mori (Nagoya University) are acknowledged for their technical
assistance. The authors are grateful to Dr. Kin-ichi Oyama (Chemical Instrument Center, Nagoya University) for his help in elemental
analyses.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts
of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
References
1. Ito K, Chiba T, Ishii T, Igarashi Y, Katoh Y, Oyake T, Hayashi N,
Satoh K, Hatayama I, Hatayama M, Yamamoto M, Nabeshima Y
(1997) Biochem Biophys Res Commun 236:313–322
2. Kobayashi A, Kang M-I, Okawa H, Ohtsuji M, Zenke Y, Chiba
T, Igarashi K, Yamamoto M (2004) Mol Cell Biol 24:7130–7139
3. Motohashi H, O’Conner T, Katsuoka F, Engel JD, Yamamoto M
(2002) Gene 294:1–12
4. Baumer M, Baumer I (2002) Antioxid Redox Signal 4:749–758
5. Riley RJ, Workman P (1992) Biochem Pharmacol 43:1657–1669
6. Kimura T, Yoshida K, Yamamoto C, Suzuki M, Uno T, Isobe M,
Naka H, Yasuike S, Satoh M, Kaji T, Uchiyama M (2012) J Inorg
Biochem 117:140–146
7. Fujiwara Y, Mitani M, Yasuike S, Kurita J, Kaji T (2005) J
Health Sci 51:333–340
8. Kohri K, Yoshida E, Yasuike S, Fujie T, Yamamoto C, Kaji T
(2015) J Toxicol Sci 40:321–327
9. Eggler AL, Kelly AG, Mesecar AD (2008) Mol Nutr Food Res
52:S84–S94
10. Cortese-Krott MM, Suschek CV, Wetzel W, Kröncke KD, KolbBachofen V (2009) Am J Physiol Cell Physiol 296:C811–C820
11. Miura T, Shinkai Y, Jiang H-Y, Iwamoto N, Sumi D, Taguchi K,
Yamamoto M, Jinno H, Tanaka-Kagawa T, Cho AK, Kumagai Y
(2011) Chem Res Toxicol 24:559–567
12. Toyama T, Sumi D, Shinkai Y, Yasutake A, Taguchi K, Tong KI,
Yamamoto M, Kumagai Y (2007) Biochem Biophys Res Commun 363:645–650
13. Abiko Y, Shinkai Y, Sumi D, Kumagai Y (2011) J Toxicol Sci
35:419–423
14. Rybka A, Koliński R, Domagała S, Kłak J, Mroziński J,
Woźniak K, Korybut-Daszkiewicz B (2006) Inorg Chim Acta
359:4526–4534
15. Ribeiro da Silva MDMC, Gonçalves JM, Silva ALR, Oliveira
PCFC, Schröder B, Ribeiro da Silva MAV (2004) J Mol Catal A:
Chem 224:207–212
J Biol Inorg Chem (2016) 21:263–273
16. Saint-Aman E, Ménage S, Pierre JL, Defrancq E, Gellon G
(1998) New J Chem 22:393–394
17. Lee J, Penã MMO, Nose Y, Thiele DJ (2002) J Biol Chem
277:4380–4387
18. Kissane JM, Robins E (1958) J Biol Chem 233:184–188
19. Fujie, T., Segawa, Y., Yoshida, E., Kimura, T., Fujiwara, Y.,
Yamamoto, C., Satoh, M., Naka, H., Kaji, T.: J Toxicol Sci, in
press (2016)
20. Toyama T, Shinkai Y, Kaji T, Kumagai Y (2013) J Toxicol Sci
38:477–484
21. Kobayashi M, Li L, Iwamoto N, Nakajima-Takagi Y, Kaneko
H, Nakayama Y, Eguchi M, Wada Y, Kumagai Y, Yamamoto M
(2009) Mol Cell Biol 29:493–502
22. Fujiwara Y, Yamamoto C, Yoshida E, Kumagai Y, Kaji T (2014)
Arch Toxicol. doi:10.1007/s00204-014-1420-6
23. Schreck R, Meler B, Männel DN, Dröge W, Baeuerle PA (1992)
J Exp Med 175:1181–1194
24. Heikkila RE, Cabbat FS, Cohen G (1976) J Biol Chem
251:2182–2185
25. Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) Proc Natl Acad
Sci USA 99:14298–14302
26. Gerards WLH, de Jong WW, Boelens W, Bloemendal H (1998)
Cell Mol Life Sci 54:253–262
273
27. Cvek B, Milacic V, Taraba J, Dou QP (2008) J Med Chem
51:6256–6258
28. Zhang DD, Hannink M (2003) Mol Cell Biol 23:8137–8151
29. Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang
M-I, Kobayashi A, Yamamoto M, Kensler TW, Talalay P (2003)
Proc Natl Acad Sci USA 101:2040–2045
30. Kumagai Y, Kanda H, Shinkai Y, Toyama T (2013) Oxid Med
Cell Longev. doi:10.1155/2013/848279
31. Wang WJ, Sun J, Chen W, Li Y, Villeneuve NF, Zhang DD
(2008) Toxicol Appl Pharmacol 230:383–389
32. Dinkova-Kostova AT, Holtzclaw WD, Wakabayashi N (2005)
Biochem 44:6889–6899
33. Kägi JH (1991) Methods Enzymol 205:613–626
34. Kaji T, Suzuki M, Yamamoto C, Imaki Y, Miyajima S, Fujiwara
Y, Sakamoto M, Kazuka H (1996) Toxicol Lett 89:131–137
35. Kaji T, Mishima A, Koyanagi E, Yamamoto C, Sakamoto M,
Kazuka H (1992) Toxicology 76:257–270
36. Fujie, T., Segawa, Y., Uehara, A., Nakamura, T., Kimura, T.,
Yoshida, E., Yamamoto, C., Uchiyama, M., Naka, H., Kaji, T.: J
Toxicol Sci, in press (2016)
13