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MetallomicsView Article OnlineView Journal
1
Fluorescent silver(I) and gold(I) N-heterocyclic carbene
complexes with cytotoxic properties: mechanistic insights
Anna Citta,a Esther Schuh,b Fabian Mohr,b* Alessandra Folda,a Maria Lina Massimino,c Alberto
Bindoli,c Angela Casini,d* and Maria Pia Rigobelloa*
a. Dipartimento di Scienze Biomediche, Università di Padova, Viale G. Colombo 3, 35131 Padova, Italy, E-mail:
b. Fachbereich C, Anorganische Chemie, Bergische Universität Wuppertal, Gaußstr. 20, 42119 Wuppertal, Germany, E-
mail: [email protected]
c. Istituto di Neuroscienze (CNR) Sezione di Padova, c/o Dipartimento di Scienze Biomediche, Viale G. Colombo 3, 35131
Padova, Italy, E-mail: [email protected]
d. Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen, The Netherlands, Email: [email protected]
Abstract: Silver(I) and gold(I) N-heterocyclic carbene (NHC) complexes bearing a fluorescent
anthracenyl ligand were examined for cytotoxicity in normal and tumor cells. The silver(I) complex
exhibits greater cytotoxicity in tumor cells compared with normal cells. Notably, in cell extracts, this
complex determines a more pronounced inhibition of thioredoxin reductase (TrxR), but it is ineffective
towards glutathione reductase (GR). Both gold and silver complexes lead to oxidation of the
thioredoxin system, the silver(I) derivative being particularly effective. In addition, the dimerization of
peroxiredoxin 3 (Prx3) was also observed, demonstrating the ability of these compounds to reach the
mitochondrial target. The fluorescence microscopy visualization of the subcellular distribution of the
complexes shows a larger diffusion of these molecules in tumor cells with respect to normal cells.
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Keywords: gold and silver carbene complexes • thioredoxin reductase • thioredoxin • peroxiredoxin 3 •
fluorescence • cancer cells •
Abbreviations AIS, 4-acetamido-4′-((iodoacetyl) amino) stilbene-2,2′-disulfonic acid; GSH, glutathione; GR,
glutathione reductase; IAA, iodoacetic acid; NHC, N-heterocyclic carbene; PI, propidium iodide; ROS, reactive oxygen species; Trx, thioredoxin; TrxR, thioredoxin reductase; Prx3, peroxiredoxin 3.
Introduction
In the last years, the interest in exploiting the properties of metal ions to design new anticancer drugs
has constantly raised. A major aim is to develop new drug candidates with different mechanisms of
action and improved pharmacological properties with respect to existing drugs.1-3 In this respect,
important challenges must be faced to reach such a goal, among which the identification of the actual
sub-cellular targets for metal compounds, as well as the determination of their distribution in tissues,
cells and subcellular compartments.4,5 Among the various strategies to achieve metal compounds
imaging in biological environments, fluorescence microscopy is certainly one of the most explored, and
an increasing number of publications reporting on bifunctional metal compounds bearing fluorescent
moieties for both therapeutic and imaging applications (so called theranostic agents) has appeared.6-10
It is worth mentioning that gold complexes belonging to various families have drawn attention
in the last years as new generation experimental anticancer agents, and different families of gold(I) and
gold(III) compounds have been reported to possess anticancer properties in vitro and in vivo.11-14 In
particular, gold(I) phosphine complexes have been tested against a variety of human tumor cell lines.15-
19 Interestingly, the anticancer potential of metal N-heterocyclic carbene (NHC) complexes has also
been described. Thus, neutral NHC gold(I) halide complexes,20 cationic bis(carbene) gold(I) salts,21 and
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dinuclear gold(I)-NHC complexes22 have been proved to be anti-mitochondrial anti-tumor agents.18
Gold(I)-NHC complexes have also been designed as compounds that combine both selective
mitochondrial accumulation and selective thioredoxin reductase inhibition properties within a single
molecule.23 Indeed, the seleno-enzyme thioredoxin reductase (TrxR) is a major redox regulator in
mammalian cells, over-expressed in cancer cells, and inhibited by numerous anticancer compounds,
including metal complexes of gold(I/III),24,25 as well as silver complexes with phosphine ligands.26
Within this frame, a series of fluorescent gold(I) complexes bearing both phosphine and thiol-based
naphtalimide ligands, with TrxR inhibition properties and able to induce antiangiogenic effects, were
synthesized.27,28 Moreover, Barnard et al. exploited the “aurophilicity” of Au(I) ions to design
anticancer gold(I)-NHC compounds with photophysical properties, to study their distribution in cancer
cells.29 Concerning silver(I)-NHC complexes, various reports have described the cytotoxic properties
of different compounds in cancer cells in vitro,17,23 and very recently two papers have described the
effects of silver(I)-NHC complexes inducing mitochondrial damage,30 and TrxR inhibition properties in
vitro when evaluated directly against the purified protein.31
Following these promising results and with the purpose of obtaining metal-NHC compounds
with fluorescent properties to study their uptake and biodistribution in cells, we synthesized a new
gold(I)-NHC complex and its silver(I) precursor (Fig. 1), both bearing an anthracenyl unit anchored to
the N1 position of the NHC scaffold. The cytotoxic effects of the compounds were investigated in vitro
on different lines of normal and cancerous human cells. Afterwards, we evaluated the TrxR inhibition
properties of the NHC complexes both directly on the purified enzyme and in cell extracts, comparing
their ability to inhibit the enzyme glutathione reductase (GR), a pyridine-disulfide oxido-reductase
which maintains glutathione in its reduced state. The effects of the new complexes on the oxidation
state of the thioredoxin system and peroxiredoxins, were analysed. In particular, considering
mitochondrial peroxiredoxin 3 (Prx3), the redox state of the mitochondrial system was highlighted.
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Estimation of the glutathione content and reactive oxygen species (ROS) production was also
performed in cells treated with the gold and silver NHC compounds. Finally, preliminary fluorescence
microscopy studies allowed us to visualize the compounds uptake and biodistribution in cells.
Experimental
All manipulations were carried out without excluding air and moisture. Chemicals and solvents (HPLC
grade) were sourced commercially and used as received. The imidazolium salt 1-(anthracene-9-
ylmethyl)-3-methylimidazolium chloride as well as [AuCl(SMe)2] were prepared as described in the
literature.32 Concentrated (10 mM) fresh solutions of metal NHC complexes dissolved in DMSO and
protected from light were prepared prior any biological assays and appropriately diluted in water
solution.
Instrumentation
NMR Spectroscopy: 1H and 13C NMR spectra were recorded on Bruker Avance 400 or Bruker Avance
III 600 MHz spectrometers and are referenced to external TMS.
X- ray crystallography: Diffraction data were collected at 150 K using an Oxford Diffraction Gemini E
Ultra diffractometer, equipped with an EOS CCD area detector and a four-circle kappa goniometer. For
the data collection the Mo source emitting graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å)
was used. Data integration, scaling and empirical absorption correction was carried out using the
CrysAlis Pro program package.33 The structures were solved using Direct Methods or Patterson
Methods and refined by Full-Matrix-Least-Squares against F2. The non-hydrogen atoms were refined
anisotropically and hydrogen atoms were placed at idealized positions and refined using the riding
model. All calculations were carried out using the program Olex2.34 Full crystallographic and
refinement parameters as well as tables with bond lengths and angles are included in the supporting
information.
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Synthesis
1-(Anthracen-9-ylmethyl)-3-methylimidazol-2-ylidene silver chloride (1)
1.1 equiv Ag2O was added to a solution of 2 equiv imidazolium salt in 40 ml
MeOH and the suspension was stirred for 6 h at room temperature. 150 ml
CH2Cl2 was added, the suspension was filtered over Celite and the filtrate was
concentrated in vacuum. A red-brown solid was precipitated by addition of
hexane, isolated by filtration and washed with hexane. A yield of 85 % (0.8
mmol, 344 mg) was obtained. X-ray quality crystals were grown from a solution of CH2Cl2 and
hexane. 1H-NMR (CDCl3, 400 MHz) δ = 8.59 (s, 1H, H10), 8.26 (d, 2H, J = 8.8 Hz, H1 and H4 or H5
and H8), 8.09 (d, 2H, J = 8.3 Hz, H1 and H4 or H5 and H8), 7.63 - 7.57 (m, 2H, H2 and H3 or H6 and
H7), 7.57 - 7.50 (m, 2H, H2 and H3 or H6 and H7), 6.79 (d, 1H, J = 1.8 Hz, NCHCHN), 6.48 (d, 1H, J
= 1.8 Hz, NCHCHN), 6.28 (s, 2H, CH2), 3.89 (s, 3H, Me) ppm; 13C-NMR (CDCl3, 151 MHz) δ =
180.4 (C-Ag), 131.4 (C4a and C10a or C8a and C9a), 130.9 (C4a and C10a or C8a and C9a), 129.9
(C10), 129.5 (C1 and C4 or C5 and C8), 127.7 (C2 and C3 or C6 and C7), 125.4 (C2 and C3 or C6 and
C7), 124.0 (C9), 123.0 (C1 and C4 or C5 and C8), 122.0 and 120.6 (imidazole), 47.6 (CH2), 39.0 (Me)
ppm. Anal. calcd for C19H16N2AgCl: C, 55.07; H, 3.86; N, 6.76. Found: C, 54.56; H, 3.83; N, 6.62 %.
1-(Anthracen-9-ylmethyl)-3-methylimidazol-2-ylidene gold chloride (2)
1 equiv. [AuCl(SMe2)] was added to a solution of 1 (1 equiv) in 20 ml CH2Cl2
and the mixture was stirred for 2 h at room temperature. The suspension was
filtered over Celite and the filtrate was concentrated in vacuum. A pale yellow
solid was precipitated by addition of hexane, isolated by filtration and washed
with hexane. Yield 98 % (0.24 mmol, 120 mg). X-ray quality crystals were grown
from a solution of CH2Cl2 and hexane. 1H-NMR (CDCl3, 400 MHz) δ = 8.61 (s, 1H, H10), 8.31 (dd,
2H, J =0.7 Hz, J = 8.9 Hz, H1 and H4 or H5 and H8), 8.11 (dd, 2H, J = 0.7 Hz, J = 8.5 Hz, H2 and H3
or H6 and H7), 7.65 (dd, 1H, J = 1.3 Hz, J = 6.6 Hz, H1, H4, H5 or H8), 7.63 (dd, 1H, J = 1.5 Hz, J =
6.7 Hz, H1, H4, H5 or H8), 7.57 (dd, 1H, J = 1.1 Hz, J = 6.7 Hz, H2, H3 H6 or H7), 7.55 (dd, 1H, J =
0.9 Hz, J = 6.6 Hz, H2, H3, H6 or H7), 6.71 (d, 1H, J = 2.0 Hz, NCHCHN), 6.38 (s, 2H, CH2), 6.35 (d,
1H, J = 2.0 Hz, NCHCHN), 3.90 (s, 3H, Me) ppm; 13C-NMR (CDCl3, 151 MHz) δ = 171.4 (C-Au),
131.4 (C4a and C9a or C8a and C10a), 131.0 (C4a and C9a or C8a and C10a), 130.0 (C10), 129.5 (C1
and C4 or C5 and C8), 127.8 (C2 and C3 or C6 and C7), 125.5 (C2 and C3 or C6 and C7), 123.8 (C9),
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123.1 (C1 and C4 or C5 and C8), 121.5 and 119.8 (Imidazole), 47.1 (CH2), 38.4 (Me) ppm. Anal. calcd
for C19H16N2AuCl: C, 45.12; H, 3.39; N, 5.54. Found: C, 44.73; H, 3.16; N, 5.46 %.
Fluorescence spectroscopy: the luminescent spectra of the metal compounds were measured using a
Cary Eclipse Varian fluorescence spectrophotometer. Compound 1 and 2 (100 µM) were dissolved in
DMSO and than diluted in 0.1 mM phosphate buffer pH 7.4. The compounds’ fluorescence was
followed from 300 to 400 nm (excitation spectra) and from 380 to 600 nm (emission spectra) (Fig. S1
Electronic Supplementary Information).
Estimation of enzyme activities inhibition in vitro
Highly purified cytosolic thioredoxin reductase (TrxR1) was prepared from rat liver, according to
Luthman and Holmgren.35 Mitochondrial thioredoxin reductase (TrxR2) was purified from isolated rat
liver mitochondria following the procedure of Rigobello and Bindoli.36
Thioredoxin reductases activity was determined by measuring the ability of the enzyme to directly
reduce DTNB in the presence of NADPH.35 Aliquots of highly purified TrxR1 (60 nM) and TrxR2
(130 nM) in 0.2 M Na, K-phosphate buffer (pH 7.4), 5 mM EDTA, and 0.25 mM NADPH were pre-
incubated for 5 min with the gold complexes. Afterwards, the reaction was started with 1 mM DTNB,
and monitored spectrophotometrically at 412 nm for about 10 min.37 Yeast glutathione reductase was
obtained from Sigma (St. Louis Mo, USA) and used without further purification. Protein content was
assayed with the Lowry et al. procedure.38 Glutathione reductase activity was measured in 0.2 M Tris-
HCl buffer (pH 8.1), 1 mM EDTA, and 0.25 mM NADPH after 5 min pre-incubation with the silver or
gold complexes. The assay was initiated by the addition of 1 mM GSSG and followed
spectrophotometrically at 340 nm.
Cell cultures
Human ovarian carcinoma cell line A2780S (cisplatin sensible), A2780R (cisplatin resistant) and HEK-
293T (Human Embryonic Kidney) cells were used. A2780S/R cells were grown at 37 °C in 5% carbon
dioxide atmosphere using RPMI 1640 medium, containing 10% fetal calf serum and supplemented with
2 mM L-glutamine. HEK-293T cells were grown in DMEM (high glucose) with 10% fetal calf serum,
supplemented with 2 mM L-glutamine.
Cytotoxicity assay
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Cell viability was assayed with the MTT reduction assay. A2780S, A2780R and HEK-293T cells were
seeded (1x104) and treated for 24 h with increasing concentrations of gold and silver complexes. At the
end of incubation, cells were treated for 3 h at 37 °C with 0.5 mg/ml MTT dissolved in PBS.
Afterwards, MTT was removed and 100 µl of stop solution (90% isopropanol and 10% DMSO) were
added to each well. After 15 min of incubation at 37 °C, samples were estimated in a plate reader
(Multiskan EX, Labsystems, Finland) at 540-690 nm.
Determination of TrxR and GR activities in cell lysates
A2780S, A2780R and HEK-293T cells (1x106) were incubated for 24 h with the indicated
concentrations of gold and silver complexes. After incubation, cells were harvested and washed twice
with ice-cold PBS. Each sample was lysed with a modified RIPA buffer.39 After 40 min of incubation
at 0 °C, lysates were centrifuged at 14000 x g for 5 min. The obtained supernatants were tested for
enzyme activities. Aliquots (50 µg) of lysates were subjected to thioredoxin reductase determination in
a final volume of 250 µl of 0.2 M Na, K-phosphate buffer (pH 7.4), 5 mM EDTA, and 2 mM DTNB.
After 2 min the reaction was started with 0.3 mM NADPH.
In addition, cell lysate thioredoxin reductase was also estimated with a test based on insulin reduction. 40 Briefly, 12 µg of cell lysates were incubated in a final volume of 50 µl of 100 mM Hepes/Tris (pH
7.6), in presence of 12.5 mM EDTA, 1.5 mM NADPH, 0.25 mM insulin and 120 µM Trx from E. coli.
The reaction was stopped at fixed time (40 min) by adding 1 mM DTNB dissolved in 7.2 M guanidine
in Tris-HCl 0.1M (pH 8.1) and samples estimated at 412 nm (Fig. S2 in the Electronic Supplementary
Information). Glutathione reductase activity was estimated at 25 °C on 80 µg protein/ml as reported
above.
Redox Western blot analysis of Trx1, Trx2 and Prx3
To assess the redox state of thioredoxins we used the method described by Hansen et al.41 with
modifications. Trx1 redox state was measured by derivatizing thiols with 50 mM iodoacetic acid
(IAA). Cells (2x105) were plated (12-wells plate) and incubated with 6 µM gold and silver complexes
for 3 h in complete RPMI or DMEM, depending on the cell line. Then, cells were centrifuged in a rotor
plate at 500 x g for 5 minutes. Medium was removed and cells were washed with cold PBS. Samples
were added to 60 µl of a solution containing 6 M guanidine, 50 mM Tris-HCl buffer (pH 8.3), 3 mM
EDTA, 0.5% Triton X-100 and 50 mM IAA. Cells were rapidly scraped and maintained at 37 °C for 50
min. After incubation, samples were applied to MicrospinTM G-25 Columns (GE Healthcare, Little
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Chalfont, UK) to remove the excess of IAA. Proteins of eluted samples were measured with the Lowry
et al.38 procedure. Samples were treated with 0.5 M Tris-HCl buffer (pH 6.8), 50% glycerol (v/v),
bromophenol blue 0.05% (w/v), loaded onto a native gel (15%) and subjected to Western blotting using
a polyclonal primary antibody anti Trx1 (FL-105) (Santa Cruz Biotechnology, Santa Cruz, CA USA).
For the determination of Trx2 and Prx3 redox state, after incubation with gold and silver
complexes, cells were centrifuged in a rotor plate, at 500 x g, for 5 minutes, washed with cold PBS and
then treated with 1 ml of 10% trichloro-acetic acid. The scraped samples, transferred to Eppendorf
tubes and kept at 4 °C for 30 minutes, were centrifuged for 10 minutes at 10000 x g at 4 °C. Pellets
were washed with 0.5 ml of ice-cold acetone, then centrifuged twice at 10000 x g for 10 minutes at
room temperature. The pellets were dissolved in 62.5 mM Tris HCl (pH 8) and 1% SDS containing 8
mM AIS (4-acetamido-4'-((iodoacetyl) amino) stilbene-2,2'-disulfonic acid) (Invitrogen).
Derivatization lasted 90 min, at room temperature, followed by further 30 min at 37 °C. Samples were
loaded, without reducing agents, onto Bis-Tris Gel NUPAGE (12%) and blotted. To assess the redox
state of thioredoxin 2 and Prx3, a polyclonal antibody H-75 (Santa Cruz Biotechnology) and a
monoclonal antibody LF-MA0044 (Histoline) were used, respectively.
Glutathione content and redox state determination
A2780S, A2780R and HEK-293T cells (3x105) were plated in 6-wells plate and treated with 6 µM gold
and silver complexes for 3 h. After incubation, medium was rapidly removed and cells were washed
with PBS, deproteinized in each well with 6% meta-phosphoric acid and then scraped. The
deproteinized samples were centrifuged and the supernatant was neutralized with 15% of Na3PO4.
Aliquots of neutralized samples were tested for total glutathione 42 and 300 µl were treated with 6 µl of
2-vinylpyridine to derivatize the reduced glutathione in order to determine glutathione disulfide.43 In
addition, after deproteinization, the pellets were washed with 1 ml of ice-cold acetone, centrifuged at
11000 x g, dried, dissolved in 62.5 mM Tris/HCl buffer (pH 8.1) containing 1% SDS and utilized for
protein determination.
Estimation of ROS production
ROS generation in the various cell lines was assessed by the fluorogenic probe CM-DCFH2-DA
(Molecular Probes, Invitrogen). Cells (2x104) were seeded in 96-wells plate, and, after 24 h, washed in
PBS/10 mM glucose and loaded with 10 µM CM-DCFH2-DA for 20 min in the dark at 37 °C.
Afterwards, cells were washed with the same medium and incubated with gold and silver complexes (2
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-3 µM). Fluorescence increase was estimated in a plate reader (Fluoroskan Ascent FL, Labsystem,
Finland) at 485 nm (excitation) and 527 nm (emission) for 2 h.
Intracellular localization of gold/silver complexes by confocal microscopy
Cells (A2780S, A2780R and HEK-293T) were seeded (105 for each sample) and grown on a round
coverslips with a complete medium. After 24 h, cells were washed with PBS and then incubated with
10 µM complex 1 or 20 µM complex 2 in RPMI without FCS for different times (20 min, 30 min, 1 h,
1.30 h) at 37 °C. At the end of incubation, cells were rapidly washed with cold PBS and then fixed with
2% paraformaldehyde for 30 min at 4 °C. For the visualization of propidium iodide (PI, Sigma-
Aldrich), cells were permeabilized with 0.2% Triton X-100 for 20 min at 4 °C and treated with 1µg/µl
of PI for 10 min at room temperature. Cells were washed twice with PBS and then analyzed by
confocal microscopy.
Fluorescence was analyzed with a Leica Confocal SP5 microscope equipped with a diode, UV laser to
excite complexes 1, 2 at 405 nm (emission bandwidth 420-490 nm) and a HeNe visible laser to excite
PI at 543 nm (emission bandwidht 594-663 nm). Confocal stacks were acquired every 0.2 nm along the
z-axis (for a total of 40 images) with a 63X objective. Stacks were automatically thresholded and
deconvoluted using Image J 1.46 software.
Statistical analysis
All the values are the means ± SD of not less than five measurements. Multiple comparisons were
made by one-way analysis of variance followed by Tukey-Kramer multiple comparison test.
Results and Discussion Synthesis and structural characterization
The silver(I)-NHC chloro complex (1) containing the anthracenyl unit was obtained by stirring the
corresponding imidazolium chloride with Ag2O in MeOH solution (Scheme 1). The brown product was
isolated in pure form in 85% yield. The gold(I)-NHC chloro complex (2) was subsequently synthesized
via transmetalation of 1 with [AuCl(SMe2)] as a pale yellow solid in almost quantitative yield (Scheme
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1). The new compounds were characterized by spectroscopic methods including 1H and 13C NMR
spectroscopy (see Electronic Supplementary Information Figs. S3-S6 for NMR spectra) as well as X-
ray diffraction.
Scheme 1. Synthesis of the metal NHC complexes.
In the 1H NMR spectra of the two compounds the signal from the imidazolium proton is missing,
confirming formation of the carbene. The most diagnostic feature is however the chemical shift of the
carbene carbon signal in the 13C-NMR spectra. In the silver compound 1 this signal is observed at
180.4 ppm, whilst that of the gold compound 2 is observed at 171.4 ppm. The presence of a sharp
signal with no 107,109Ag-13C coupling in the 13C NMR spectrum of 1 is typical for Ag-NHC compounds,
which may exist as either NHC-Ag-X or [Ag(NHC)2][AgX 2]; the former species in solution, the latter
in the solid-state.44,45
The identity and structures of compounds 1 and 2 were further confirmed by X-ray diffraction analysis,
and the obtained molecular structures are shown in Fig. 1.
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Fig. 1. Molecular structures of 1 (left) and 2 (right). Thermal ellipsoids are drawn at 30% probability levels. Hydrogen atoms have been omitted for clarity.
The NHC silver chloride complex 1 crystallizes as colorless plates in the space group P21/c and
consists of polymeric chains of [bis(carbene)Ag]+ cations and [AgCl2]- anions. The Ag···Ag distances
of ca. 2.82 Å are less than the sum of the van-der-Waals radius of silver (3.44 Å). This observed
distance is at the shorter end of the range typically observed for ligand-unsupported Ag···Ag distances
(2.80 to 3.30 Å).46 The C-Ag-C axis is slightly bent (167.6°) allowing the anthracenylmethyl moieties
to approach each other although there are no direct intermolecular contacts. The Ag-C bond length of
2.09 Å is comparable to those typically found in silver(I)-NHC complexes. The [AgCl2]- anion is
almost linear (178.6°) and the average Ag-Cl bond lengths of 2.36 Å are within the range usually
observed in [AgCl2]- anions.47 The NHC gold(I) chloride complex 2 crystallizes as yellow needles in
the space group Pca21. The compound is, as expected for a gold(I) species, linear (178.9°) with a
carbene-carbon gold bond length comparable to that of other NHC gold(I) chlorides.48,49 In contrast to
the silver compound (1), the structure of 2 is monomeric without any intermolecular Au···Au contacts.
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Biological studies
The two NHC compounds were screened for their cytotoxic properties against human ovarian cancer
cell lines cisplatin sensitive (A2780S) and resistant (A2780R), as well as on the non-tumorigenic
human embryonic kidney cell line (HEK-293T). A dose-dependent inhibition of cell growth was
observed for both metal complexes in all cell lines with IC50 values ranging from 3 to 7 µM after 24 h
incubations as shown in Fig. 2A. However, while compound 1 (silver) is more effective on the
cancerous cell lines than on the HEK-293T cells, compound 2 (gold) is less selective and shows very
similar IC50 values in all cases.
Since TrxR is a potential target for gold complexes, including gold(I) N-heterocyclic carbenes,
in vitro inhibition of purified rat TrxR by the two NHC compounds was studied using established
protocols as described in the Experimental section. The two compounds inhibit both cytosolic (TrxR1)
and mitochondrial (TrxR2) thioredoxin reductases, but the silver derivative 1 is in all cases more
effective than the gold carbene 2, in particular on the mitochondrial enzyme (Table 1). Further studies
demonstrated that the NHC complexes are also able to inhibit the TrxR closely related, but Se-free,
enzyme glutathione reductase (GR) (Table 1), albeit at much higher concentrations (IC50 ≥ 60 nM).
To assess whether TrxR inhibition by the metal-NHC compounds could contribute to the
observed antiproliferative effects on cells, enzyme activity was also evaluated on protein extracts
obtained from A2780S and A2780R cancer cells, as well as from non-tumorigenic HEK-293T cells,
pre-treated with each compound at a fixed concentrations (4 and 8 µM) for 24 h. According to the
observed results (Fig. 2B) the silver complex 1 is again the most potent among the two NHC
compounds, being able to inhibit TrxR efficiently in the two cancerous cell lines, while practically
ineffective in the HEK-293T cells. Similarly, compound 2 shows some effects, in the cancerous cell
lines, although only at the highest concentration tested (8 µM). Notably, and at variance with the above
mentioned results on the purified protein, when GR activity was tested in the same cell lysates treated
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with the compounds, no significant changes in both non-tumorigenic (HEK-293T) and cancerous cell
lines (A2780S/R) were detected (Fig. 2C).
Table 1. IC50 values of 1-2 inhibiting rat TrxR1, TrxR2 and yeast GR, respectively.
IC50 (nM)
Compound 1 Compound 2
TrxR1 5.9 ± 0.8 12.6 ± 1.3
TrxR2 19.2 ± 1.5 67.1 ± 3.5
GR 65.1 ± 5.6 85.2 ± 4.2
Fig. 2. A: Cell viability of HEK-293T cells, A2780 cancer cells sensitive (S) and resistant (R) to cisplatin after 24 h incubation in presence of compounds 1 and 2. IC50 values for cytotoxicity are µM. B: Effects of compounds 1 and 2 on TrxR in cell lysates. HEK-293T, A2780S and A2780R cells, were incubated for 24 h with 4 and 8 µM compounds and lysed. TrxR activity was assayed by measuring NADPH-dependent reduction of DTNB at 412 nm. C: Effects of 1 and 2 on GR in cell extracts. (***p<0.001).
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In general, the inhibition of thioredoxin reductase by hindering the transfer of reducing equivalents
leads to a large oxidation of thioredoxin (Trx). Indeed, previous studies have demonstrated that the
redox conditions of Trx, can be profoundly altered by specific TrxR inhibitors or oxidants.50 Recently,
we have also reported on the inhibition of TrxR by a series of gold(I)-NHC complexes containing 1,3-
substituted imidazole-2-ylidene or benzimidazole-2-ylidene and chloro or 2-pyrimidinethiolato
ligands.39 Interestingly, the most effective TrxR inhibitors induced extensive oxidation of thioredoxins
(Trxs), which was more relevant in cancerous cells than in non-tumorigenic (HEK-293T) cells.
Therefore, we examined the redox state of cytosolic (Trx1) and mitochondrial (Trx2)
thioredoxins directly in cells after treatment with complexes 1 and 2 (6 µM, 3 h incubation) as
described in the Experimental Section. The obtained results are depicted in Fig. 3A, and show that Trx1
is oxidized by both compounds in all the tested cell extracts to different extents. In particular,
compound 1 is more efficient in oxidizing Trx1 in the cancerous cells (A2780S/R) than in non-
tumorigenic ones, while 2 causes Trx1 oxidation mainly in HEK-293T and A2780S cells.
Densitometric analysis of the bands for Trx1, are reported in Electronic Supplementary Information
(Fig. S7). Similarly, a non-reducing SDS-PAGE analysis was performed to determine the oxidation of
mitochondrial Trx2 (Fig. 3B) confirming the tendency of Trx2 to be oxidized after 24 h treatment of
the cells with both compounds (4 µM). The oxidized form of Trx2 is detectable as a shift in the band at
the lower molecular weight, using 4-acetamido-4’-(iodoacetyl) amino-stilbene-2,2’-disulfonic acid
(AIS) as the derivatizing agent.39
Furthermore, we investigated the influence of the metal NHC complexes on the redox state of
peroxiredoxin 3 (Prx3). In fact, one of the major antioxidant roles of Trx is to reduce a ubiquitous
family of thiol peroxidases known as peroxiredoxins (Prxs), enzymes decomposing peroxides using a
highly reactive cysteine thiolate in their active site. In the presence of peroxides the Prx active site
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cysteine forms a disulfide bond with a neighbouring cysteine residue, which Trx reduces to complete
the catalytic cycle.51 In mammals six Prxs were identified, with Prx3 localized to mitochondria.51 Prx3
is kept reduced by the mitochondrial thioredoxin system, thus playing a role in protecting mitochondria
from H2O2 produced by the respiratory chain complexes.52 Interestingly, auranofin-induced apoptosis,
is mediated by a Bax/Bak-dependent mechanism associated to an alteration of mitochondrial redox
homeostasis dependent on oxidation of Prx3.52 In HL-60 cells, it was also reported that TrxR is
strongly inhibited by auranofin, but the rapid oxidation of Prx3 requires an increased production of
oxidants by the respiratory chain.53 Thus, non-reducing SDS-PAGE analysis was performed to
determine the oxidation of mitochondrial Prx3, and according to the obtained results (Fig. 3C), Prx3
dimer formation (due to formation of disulfide bonds) takes place after incubation of the cells with the
compounds. Moreover, in accordance with the above mentioned trends of TrxR1 inhibition and Trxs
oxidation, 1 causes more pronounced oxidation of Prx3 in cancer cell lines compared to the HEK-293T
cells.
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Fig. 3. Trx1, Trx2, and Prx3 redox state in presence of silver (1) and gold (2) compounds. Redox Western blot of Trx1 (A), Trx2 (B) and Prx3 (C) were determined. A2780 cells sensitive (S) and resistant (R) to cisplatin, and HEK-293T cells were incubated and treated as indicated in the Experimental section. Cells were rapidly derivatized with IAA for Trx1 and with AIS for Trx2 and Prx3.
The glutathione redox pair (GSH/GSSG) is another fundamental component of the cell redox
regulation in cisplatin resistant cells. Therefore, our studies were oriented to the analysis of total
glutathione in normal cells and in cisplatin sensitive and resistant cancer cells, after treatment with gold
compounds. In addition, we examined the GSH/GSSG ratio. Cells were treated with 6 µM gold and
silver complexes for 3 h and the obtained samples were estimated both for total (reduced + oxidized),
and oxidized glutathione (GSSG) contents as described in the Experimental section. As shown in Fig.4
A the total glutathione content is slightly affected upon treatment with 1 in the case of the HEK-293T
and A2780S cells. Furthermore, the total glutathione content is not affected by the gold compound
treatment in any of the three cell lines examined.
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Fig. 4. A: GSH and GSSG levels in presence of compounds 1 and 2. Levels of total and oxidized glutathione were determined in HEK-293T, A2780S and A2780R cells, after incubation with compound 1 and 2 (6 µM). B: Effect of 1 and 2 on ROS formation in cancer and normal cells. A2780S/R and HEK-293T cells were pre-incubated in PBS/10 mM glucose medium for 20 min at 37 °C in presence of 10 µM CM-DCFH2-DA, and then treated with different concentrations of 3 µM metal NHC compounds. (***p<0.001; ** p<0.01). Subsequently, we evaluated the alterations of basal H2O2 production in cancer (A2780S and R) and
non-cancerous cells (HEK-293T) upon treatment with compound 1 and 2. Reactive oxygen species
(ROS) are products of the physiological mitochondrial cell metabolism and are involved in cellular
redox homeostasis. Their formation may perturb the cellular antioxidant defence system. In particular,
mitochondria generate hydrogen peroxide, that can play a crucial role in the apoptotic process. It has
been previously shown that TrxR inhibition alters cell conditions causing increase of hydrogen
peroxide concentration, as well as an imbalance in cell redox state leading to mitochondrial membrane
A
B
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permeabilization and swelling. Thus, metal NHC complexes (2 µM and 3 µM) were administered to
cells in presence of the peroxide-sensitive fluorescent probe CM-DCFH2-DA (see Experimental for
details). In HEK-293T cells no significant increase in ROS was observed with both compound 1 and 2,
while in A2780S and R, compound 1 significantly stimulates ROS formation (Fig. 4B).
Fluorescence confocal microscopy
It is particularly important to understand the subcellular distribution of the experimental anticancer
metal complexes in order to gain further mechanistic insights. Thus, the fluorescence properties of the
metal compounds were investigated in aqueous solution and the absorption and emission spectra
reported in the Electronic Supplementary Information (Fig. S1), showing substantially identical
patterns. Afterwards, we were able to visualize the gold and silver complexes directly in the cell. In
detail, cells (HEK-293T, A2780S and A2780R) grown onto a coverslip were treated with the
silver(I)/gold(I)-NHC luminescent compounds as described in the Experimental section, and
fluorescence of the carbene complexes was evaluated with a Leica Confocal SP5 microscopy. We also
used propidium iodide (PI) as nuclear marker. Fig. 5 shows representative images of the cells treated
with 10 µM complex 1 and 20 µM complex 2 for 1 h. It is worth mentioning that the fluorescence of
the compounds in cells was evaluated at different times (from 20 to 90 minutes), and it reached its
maximum in 30-60 min after treatment. We avoided longer incubation times due to the relatively high
tested concentrations of compounds which may have induced a rapid cell death. In any case, the
obtained results (Fig. 5) show that the complexes are largely distributed intracellularly. In addition,
both complexes appear to accumulate at the nuclear level, in accordance with previously reported
results by Wedlock et al. for a gold(I) phosphine complex.5
At variance with classical platinum(II) anticancer drugs, DNA is not expected to be a target for
gold(I) or silver(I) complexes, but they most likely interact with proteins, such as TrxR1 and Trx1 also
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present in the nuclear compartment.54,55 It is worth mentioning that other nuclear proteins have been
recently shown to be possible targets for gold(I) complexes, such as the zinc-finger enzyme PARP-1
involved in DNA repair,56 and presently we cannot exclude the possible role of these and other targets
in the metal NHC compounds’ mechanism of action.
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Fig. 5. Visualization of silver(I) and gold(I)-NHC compounds with confocal microscopy. HEK-293T, A2780S and A2780R cells were incubated for 1 h with 10 µM compound 1 and 20 µM compound 2. Column: (a) fluorescence of the compounds; (b) propidium iodide localization; (c) merge. The stack images were analyzed with “ImageJ software”.
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Conclusions
The synthesis and structural characterization of a new gold(I)-NHC complex and its silver(I) precursor,
both bearing an anthracenyl unit anchored to the N1 position of the NHC scaffold is herewith reported.
Both complexes showed antiproliferative effects in human ovarian cancer cell lines sensitive and
resistant to cisplatin, as well as in non-tumorigenic cells, with the silver compound being the most
selective for the cancer cells. TrxR inhibition upon treatment of cell extracts with silver(I)/gold(I)-NHC
compounds is more relevant in cancerous cell lines with respect to non-tumorigenic cells. This is in line
with our previously reported results on an other series of gold(I)-NHC complexes.39 An interesting
aspect of our studies concerns the observation that Trxs and Prx3 oxidation occur upon treatment with
both compounds. In particular, the silver(I) complex is more efficient in oxidizing Trx1 and Prx3 in the
cancerous cells with respect to the HEK-293T cells. In general, GSH and GSSG contents were poorly
affected by both compounds, suggesting that GSH does not affect their cytotoxic potency, as for
cisplatin in the case of certain resistant cancer cells. Conversely, ROS formation was stimulated by
both Au(I)/Ag(I)-NHC complexes in cancer cells, but not in HEK-293T.39 This behavior is somehow in
accordance with what has been already reported for gold(I) phosphine derivatives57, as well as for
gold(III) and gold(I)-NHC complexes, although caution has to be used in comparing different
experimental protocols.58,59
Furthermore, taking advantage of the fluorescence properties of the two new metal NHC
complexes, we were able to visualize their uptake and biodistribution in cells. Preliminary fluorescence
microscopy experiments showed that both compounds enter cells, and are particularly efficient in
penetrating tumor cells where they reach the nuclear compartment.
Overall, the obtained results indicate a correlation between the cytotoxicity of silver(I)/gold(I)-
NHC compounds and Trx/Prx3 oxidation via TrxR inhibition in cells. However, further studies are
necessary to validate our mechanistic hypothesis and to exclude other possible intracellular targets. As
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far as we are aware this is the first report on the effects of Ag(I)/Au(I)-NHC on the oxidation of Prx3. It
is worth mentioning that Prx3 appears to be a valuable marker of mitochondrial redox homeostasis, and
there is growing evidence that Prx3 coupled mitochondrial antioxidant enzyme systems may also play a
role in the regulation of apoptosis. In fact, overexpression of Prx3 provides protection against induction
of apoptosis by serum deprivation, hypoxia and cytotoxic drugs.60 The proposed mechanism is based
on the scavenging of H2O2 that otherwise may promote the release of pro-apoptotic factors from
mitochondria. In this context, our results shed light onto the mechanisms of action of metal-NHC
complexes and allow to further identify the pathways leading to oxidative mitochondrial damage
induced by these compounds.
Acknowledgements
The authors wish to thank the University of Wüppertal for support, as well as the DFG for a grant to
purchase the diffractometer. A.C. thanks the Rosalind Franklin program (University of Groningen). We
are grateful to the Ministero dell'Istruzione dell’Università e della Ricerca (PRIN 2010-2011-prot.
20107Z8XBW) and to the Consorzio Inter-universitario di Ricerca in Chimica dei Metalli nei Sistemi
Biologici CIRCMSB. COST Action CM1105 and CM0902 are acknowledged for financial support and
fruitful discussion.
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