Synthesis of gold(I) phosphine complexes containing the 2-BrC6F4PPh2 ligand: Evaluation of anticancer activity in 2D and 3D spheroidal models of HeLa cancer cells

Article abstract of DOI:10.1016/j.ejmech.2017.12.048

Newly synthesised mononuclear gold complexes containing the 2-BrC₆F₄PPh₂ ligand have been fully characterised and their anticancer activity towards five human tumor [prostate (PC3), glioblastoma (U87MG), cervical (HeLa), fibrosarcoma (HT1080), ovarian (SKOV-3)] and normal human embryonic kidney (Hek-293T) cell lines investigated. Some of the synthesised gold complexes displayed higher cytotoxicity than cisplatin towards PC-3, HeLa and U87MG cells and inhibited the thioredoxin reductase (TrxR) enzyme, which is considered a potential target for new compounds in cancer treatment. The more physiologically relevant tumor spheroid assay demonstrated the superior potency of these gold phosphine complexes in inhibiting the growth of cervical carcinoma cell line HeLa (3D) spheroidal models. The mechanism of cell death was shown to be apoptotic cell death through cell cycle arrest, mitochondrial membrane depolarisation and increased ROS production.

Full text of DOI:10.1016/j.ejmech.2017.12.048

Accepted Manuscript
Synthesis of gold(I) phosphine complexes containing the 2-BrC F PPh ligand:
6
4
2
Evaluation of anticancer activity in 2D and 3D spheroidal models of HeLa cancer cells
T. Srinivasa Reddy, Steven H. Privér, Nedaossadat Mirzadeh, Suresh K. Bhargava
PII:
S0223-5234(17)31080-2
10.1016/j.ejmech.2017.12.048
EJMECH 10026
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 November 2017
Revised Date: 12 December 2017
Accepted Date: 13 December 2017
Please cite this article as: T.S. Reddy, S.H. Privér, N. Mirzadeh, S.K. Bhargava, Synthesis of gold(I)
phosphine complexes containing the 2-BrC F PPh ligand: Evaluation of anticancer activity in 2D
6
4
2
and 3D spheroidal models of HeLa cancer cells, European Journal of Medicinal Chemistry (2018), doi:
0.1016/j.ejmech.2017.12.048.
1
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ACCEPTED MANUSCRIPT
Synthesis of gold(I) phosphine complexes containing the 2-BrC F PPh ligand:
6
4
2
Evaluation of anticancer activity in 2D and 3D spheroidal models of HeLa cancer cells
T. Srinivasa Reddy, Steven. H. Privér, Nedaossadat. Mirzadeh,* Suresh. K. Bhargava*
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne 3001, Australia

ACCEPTED MANUSCRIPT
Synthesis of gold(I) phosphine complexes containing the 2-BrC F PPh ligand:
6
4
2
Evaluation of anticancer activity in 2D and 3D spheroidal models of HeLa cancer cells
T. Srinivasa Reddy, Steven. H. Privér, Nedaossadat. Mirzadeh,* Suresh. K. Bhargava*
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne 3001, Australia
Abstract
Newly synthesised mononuclear gold complexes containing the 2-BrC F PPh ligand have
6
4
2
been fully characterised and their anticancer activity towards five human tumor [prostate
PC3), glioblastoma (U87MG), cervical (HeLa), fibrosarcoma (HT1080), ovarian (SKOV-3)]
(
and normal human embryonic kidney (Hek-293T) cell lines investigated. Some of the
synthesised gold complexes displayed higher cytotoxicity than cisplatin towards PC-3, HeLa
and U87MG cells and inhibited the thioredoxin reductase (TrxR) enzyme, which is
considered a potential target for new compounds in cancer treatment. The more
physiologically relevant tumor spheroid assay demonstrated the superior potency of these
gold phosphine complexes in inhibiting the growth of cervical carcinoma cell line HeLa (3D)
spheroidal models. The mechanism of cell death was shown to be apoptotic cell death
through cell cycle arrest, mitochondrial membrane depolarisation and increased ROS
production
Keywords: Gold(I) phosphine complexes; anticancer activity; spheroids; thioredoxin
reductase; apoptosis.
*
Corresponding authors: e-mail addresses: suresh.bhargava@rmit.edu.au (Suresh. K.
Bhargava), nedaossadat.mirzadeh@rmit.edu.au (Nedaossadat. Mirzadeh)
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1
.0 Introduction
Cancer is a major cause of death worldwide and new drugs are desperately needed in the fight
for the treatment of the disease, as almost all drugs used today suffer from severe side effects
due to rapid emergence of drug resistance and lack of selectivity towards tumors [1].
Recently, there has been a growing interest in the development of metal-based complexes for
the treatment of cancer, mainly because metals exhibit unique characteristics, such as redox
activity, variable coordination modes and reactivity toward organic substrates, and have
shown promising in vitro cytotoxic effects towards various cancer cells [2]. Cisplatin is one
of the most widely used metal-based complexes in the treatment of a variety of tumors,
including cervical, ovarian, non-small cell lung carcinoma, and testicular cancers. However,
the clinical use of cisplatin is hampered by its toxic side-effects, drug resistance and poor
solubility. These limitations have led to an intense search for new metal-based agents that
show greater selectivity, lower toxicity and improved anticancer activity [3, 4].
Over the last decade, gold complexes have gained attention as an alternative to platinum
drugs because of their potential applications in cancer treatment [5]. While the development
of anticancer gold(I) complexes has enjoyed an exponential growth, the high redox potential
and relatively poor stability of gold(III) complexes have hampered their medicinal
applications [6]. Studies on anticancer gold(I) phosphine complexes have continued to accelerate
since the discovery that auranofin, which was previously used for the treatment of rheumatoid
arthritis [7, 8], has been shown to induce cell death in several cancer cell lines with varying
degrees of cytotoxicity [9]. Despite the excellent in vitro cytotoxicity, the in vivo efficacy of
auranofin was limited due to decreased stability in the presence of natural thiols [10]. Intensified
research on the development of gold antitumor drugs has led to a number of several other gold(I)
phosphine complexes displaying a broad spectrum of anticancer activity, such as
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triphenylphosphine gold chloride, [AuCl(PPh )], which has been shown to exhibit toxicity
3
towards various cancer cells and induce autophagy [11, 12].
Following our previous studies on gold(I) complexes with anticancer activities [13,14], we
were inspired to extend our interest in developing new anticancer complexes based on
[
AuCl(PPh )] containing the 2-BrC F PPh ligand, a well-established ligand used in the
3 6 4 2
development of organogold chemistry [15,16]. In this study, we have investigated the
anticancer properties of the newly synthesised gold phosphine complexes (Fig. 1) towards a
comprehensive panel of human cancer cell lines and compared the results to those of
cisplatin. These complexes were evaluated for their capacity to inhibit the thioredoxin
reductase (TrxR) enzyme. Finally, the apoptosis inducing ability of the complexes was also
investigated.
<Fig. 1>
2
2
.0 Results and Discussion
.1 Synthesis of gold complexes:
Reaction of [AuCl(tht)] (tht = tetrahydrothiophene) with an equimolar amount of
-BrC F PPh afforded the neutral gold(I) chloride complex [AuCl(2-BrC F PPh )] (1), from
2
6
4
2
6
4
2
which the corresponding bromide (2) and iodide (3) complexes were prepared by metathetical
reaction with LiBr and NaI, respectively (Scheme 1). Abstraction of chloride in 1 by TlPF in
6
the presence of one equivalent of 2-BrC F PPh gave the cationic complex [Au(2-
6
4
2
BrC F PPh ) ]PF (4) (Scheme 2). The analogous, previously reported, complex
6
4
2 2
6
[
Au(PPh ) ]PF (6) was prepared similarly from [AuCl(PPh )] (5) and PPh (Scheme 3) [17].
3 2 6 3 3
<Scheme. 1>
<Scheme. 2>
<Scheme. 3>
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1
19
The new complexes 1-4 were characterised by multinuclear NMR spectroscopy ( H, F and
3
1
P, Figs. S1-S14) and their structures confirmed by single crystal X-ray diffraction. In all
1
cases, the H NMR spectra are unremarkable and each show the expected aromatic multiplet
1
9
resonances between δ 7.5-7.8. Similarly, the F NMR spectra each show the expected
aromatic fluorine resonances in the δ -120 to -150 region, split into multiplets due to complex
F-F and F-P coupling. In the case of complex 4, an additional resonance at δ -73 was also
3
1
present assignable to the PF counter ion (J = 714 Hz). The P NMR spectra for complexes
6
PF
1
-4 each show a single broad resonance with unresolved P-F coupling in the δ 30-39 region;
the spectrum for 4 also showed the characteristic septet (J = 712 Hz) at δ -144 due to PF .
PF
6
The molecular structures of complexes 1-4 were confirmed by X-ray diffraction. The
structures of 1 and 4 are shown in Figs. 2 and 3, respectively, and those of 2 and 3 in Figs.
S15 and S16, respectively. Selected bond distances (Å) and angles (°) are collected in Table 1
and crystal structure refinement details for complexes 1-4 are reported in Tables S1-S2. The
structures of 1-4 each consist of a gold(I) atom coordinated by a phosphorus atom of the
tertiary phosphine and a halide, or, in the case of 4, a second phosphorus atom. As expected,
the geometry about the gold atom is linear or close to linear (177-180°), with Au−P bond
lengths in the range 2.23-2.30 Å. The Au−X bond lengths are typical for gold(I) halide
complexes and increase in the order X = Cl (2.28 Å) < Br (2.39 Å) < I (2.54 Å). In all cases,
the metrical parameters in 1-4 are comparable to those observed for their triphenylphosphine
analogues [AuX(PPh )] [18, 19] and [Au(PPh ) ]PF [17].
3
3 2
6
<Fig. 2>
<Fig. 3>
<Table. 1>
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2
.2 Stability of gold phosphine complexes:
3
1
The P NMR spectra of the metal complexes 1-6 in DMSO-d were measured over a period
6
of 72 h to investigate their stability in this solvent. No changes in the spectra over time were
observed, indicating the complexes did not undergo reduction to metallic gold or participate
in ligand exchange reactions with DMSO (Figs. S17-S38, Supporting Information). The
stability of the complexes under physiological-like conditions was estimated by monitoring
the electronic spectra of all the synthesised complexes in a buffer solution (50 mM sodium
phosphate, 4 mM NaCl, pH 7.4) over 72 h. As shown in Fig. 4, no change in the electronic
spectra was observed over time, indicating the complexes are stable under physiological-like
conditions.
<Fig. 4>
2
.3 Cytotoxicity:
Given the stability of the gold(I) complexes under physiological-like conditions, the in vitro
anticancer properties were evaluated against five human tumor cell lines [prostate (PC3),
glioblastoma (U87MG), cervical (HeLa), fibrosarcoma (HT1080), ovarian (SKOV-3)] and
normal human embryonic kidney cells (Hek-293T) and the results compared to those of
cisplatin. Cells were incubated with the gold phosphine complexes for 72 h and cell
viabilities were measured using the MTT assay [20]. The results of this assay are expressed
as IC values and are shown in Table 2. Most of the synthesised gold phosphine complexes
5
0
investigated exhibited remarkable cytotoxic properties with some of the complexes
displaying better anticancer activity than cisplatin. Among the series, the newly synthesised
cationic gold complex 4 and [Au(PPh ) ]PF (6) displayed potent cytotoxicity towards all the
3
2
6
tested cancer cell lines with IC values ranging from 1.23 to 6.12 µM. Notably, lower IC
50
5
0
values were observed for these complexes towards HeLa cancer cells in comparison to
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cisplatin, indicating their higher potency. Among the neutral derivatives, the iodo-complex 3
displayed significant cytotoxicity with IC50 values of 1.96 and 2.62 µM towards PC3
(prostate) and U87MG (glioblastoma) cancer cell lines, respectively. The chloro- (1) and
bromo- (2) complexes showed moderated growth inhibition towards all the tested cell lines.
<Table. 2>
The metal complexes were also screened for their cytotoxicity against non-tumorigenic Hek-
2
93T cells to determine their selectivity toward cancer cells. As shown in Table 1, complexes
5
and 6 are, respectively, 5 and 2 times more selective towards the HeLa cancer cells
compared to healthy cells. Moreover, the iodo-complex 3 was 6 and 4 times more selective
towards PC3 and U87MG cancer cells, respectively, compared to Hek-293T cells. The
promising cytotoxic activity of the metal complexes towards HeLa cancer cells encouraged
us to study their effects at the cellular level.
2
.4 Cellular uptake of gold complexes
As it has been shown that the cellular uptake ability of gold complexes can have an influence
on their cytotoxicity [11], we used inductively coupled plasma–mass spectrometry (ICP-MS)
to analyse the cellular uptake of complexes 1-4 and 6. In this assay, the amount of gold taken
up by the HeLa cells was analysed after 18 h exposure to IC concentrations of the gold
5
0
complexes. As shown in Fig. 5, after 18 h incubation, the bromo complex 2 entered the cells
with similar efficiency to the iodo complex 3. In contrast, the cellular uptake of the chloro
complex 1 was 3 times lower than that of the bromo and iodo complexes. As expected, the
highly cytotoxic cationic complexes 4 and 6 were efficiently internalised in the cells with 5-
1
5 fold higher amounts than the neutral complexes. These results are clearly in agreement
with the anticancer activity of the metal complexes.
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<Fig. 5>
2
.5 Spheroid inhibition assay
3
D Multicellular spheroids closely reflect the in vivo tumor environment with respect to
nutrient and oxygen gradients, phenotypic heterogeneity, micrometastases and gene
expression profiles [21]. Therefore, we examined the effect of the gold phosphine complexes
on the 3D multicellular spheroids of HeLa. Spheroids grown in ultra-low attachment plates
for 3 days were treated with the gold phosphine complexes for a period of 96 h and were
examined for morphological changes. The results showed that all the gold phosphine
complexes significantly inhibited the growth of HeLa cell spheroids compared to the
untreated control cells. This effect was more prominent for complexes 2, 4 and 6, with
complex 4 being particularly effective at inhibiting tumor spheroid growth (Fig. 6).
<Fig. 6>
2
.6 TrxR/GR inhibition assay
The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and
NADPH (nicotinamide adenine dinucleotide phosphate), is associated with maintaining and
regulating cellular redox homeostasis [22]. Overexpression of TrxR in many tumor cells
contributes to increased drug resistance [23]. Thus, inhibition of TrxR has emerged as a new
target for anticancer drugs and gold complexes have been shown to be potent TrxR inhibitors
[
24-26]. Therefore, in order to investigate the TrxR inhibitory activities of the gold phosphine
complexes, the DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] assay was carried out [27]. In
this assay, DTNB is used as a TrxR substrate that is reduced to 2-nitro-5-thiobenzoic acid
(TNB) in the presence of NADPH. The rate of conversion of DTNB to TNB is reflective of
TrxR activity and was monitored by UV-Visible spectrometry at 412 nm. The calculated IC50
values are shown in Table 3. All of the gold phosphine complexes exhibited significant TrxR
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inhibitory activity with EC values of 0.08-0.28 µM, except for the cationic gold complexes
50
4
and 6, which showed moderate inhibition with EC values of 2.6-3.5 µM. A possible
50
explanation for the decreased TrxR inhibitory activity of the cationic gold complexes over the
neutral complexes may be due to the absence of labile halide ligands, which may participate
in ligand exchange reactions with the selenium-containing amino acid selenocysteine within
the active site of the TrxR enzyme. Indeed, these cationic complexes showed potent
cytotoxicity among the series, indicating a different cell growth inhibition mechanism other
than TrxR inhibition.
<Table. 3>
2
.7 Cell cycle analysis:
The effect of the metal complexes on the cell cycle distribution of HeLa cells was examined
by flow cytometric analysis of the cellular DNA content. HeLa cells were treated with IC50
concentrations of the gold complexes for 48 h. The cells were stained with propidium iodide
(PI) and the DNA content was analysed using flow cytometry. The results are expressed as
the percentage cell population in each phase of the cell cycle and are shown in Fig. 7. The
results show that after 48 h of treatment with the gold complexes, accumulation of cells in the
G0/G1 phase with a concomitant loss of cells in the S and G2/M phases had taken place. The
effect is more prominent after treatment with complexes 2 and 5, with approximately 73% of
cells in the G0/G1 phase compared to only 47% for the control cells, indicating G0/G1 phase
cell cycle arrest in HeLa cells. In stark contrast, treatment with the cationic gold complexes 4
and 6 resulted in accumulation of cells in the G2/M phases, further supporting a different
mode of action for these complexes.
<Fig. 7>
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2
.8 Hoechst staining:
Because arresting cell cycle progression can result in the initiation of apoptosis [28], we next
investigated the apoptosis inducing ability of complexes 1-6 in HeLa cells using Hoechst
3
3242 nuclear staining. To examine this, HeLa cells were treated with IC concentrations of
50
the complexes, stained with Hoechst 33242, and examined for nuclear morphological
changes by fluorescence microscopy. The results showed that treatment with complexes 1-6
resulted in condensation of nuclei (brightly stained), as indicated by the arrows in Fig. 8,
while the control cells exhibited uniformly dispersed chromatin. Moreover, fragmented nuclei
can also be seen in the cells treated with complexes 1, 4, 6 and cisplatin. These results
indicate that the metal complexes induce apoptosis in HeLa cells.
<Fig. 8>
2
.9 Assessment of mitochondrial membrane potential:
To investigate whether apoptosis induced by metal complexes was mediated through
mitochondrial damage, we measured the mitochondrial outer membrane potential in HeLa
cells using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide).
JC-1 is a mitochondrial-selective fluorescent dye which emits red fluorescence (J-aggregates)
in normal mitochondria and green fluorescence (J-monomers) in damaged mitochondria as
the membrane potential decreases due to apoptosis [29]. Fluorescence microscopic imaging
analysis using JC-1 showed that after 48 h, HeLa cells treated with the metal complexes (1-6)
exhibited higher JC-1 green fluorescence with lower red fluorescence compared to the
untreated cells (Fig. 9). The mitochondria in the control cells were intact and emitted red
fluorescence after JC-1 staining. These results indicate disruption of mitochondrial membrane
integrity and loss of mitochondrial membrane potential in the treated cells.
<Fig. 9>
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2
.10 Reactive Oxygen Species (ROS)
Several studies have demonstrated that mitochondrial damage leads to increased production
of reactive oxygen species (ROS) [30]. Therefore, it was of interest to investigate whether the
mitochondrial damage induced by the gold complexes would lead to increased ROS
generation. HeLa cells were treated with IC concentrations of the gold complexes for 48 h,
5
0
stained with a ROS-sensitive fluorogenic dye (dichlorodihydrofluorescein diacetate, DCF-
DA) and analysed by flow cytometry. The results, shown in Fig. 10, indicate that the cells
treated with the metal complexes generated 1.8 to 3.5 times more intracellular ROS compared
to the control cells, with the cationic complexes 4 and 6 being particularly effective.
<Fig. 10>
2
.11 Annexin V-FITC/propidium iodide double staining assay
Phosphatidylserine (PS) externalisation on the outer leaflet of the plasma membrane occurs
during the early stages of apoptosis and can be detected using fluorescein-labelled Annexin V
2
+
(
Annexin V-FITC), a Ca -dependent phospholipid binding protein with a high affinity for
PS [31]. To quantify the apoptosis induced by the metal complexes in HeLa cells, and to
differentiate the cell death induced by the metal complexes due to necrosis or apoptosis, the
Annexin V-FITC/PI dual staining assay was carried out. In this assay, HeLa cells treated with
the metal complexes were stained with Annexin V-FITC and PI and analysed for their
fluorescence by flow cytometry. As shown in Fig. 11, the percentage of early apoptotic cells
and late apoptotic cells increased after treatment with the metal complexes (19-33%)
compared to the control cells (12.3%). Treatment with the cationic complexes 4 and 6
resulted in higher amounts of cells stained with Annexin (early apoptotic cells) and PI-
positive (late apoptotic cells). The total percentage of apoptotic cells (sum of early apoptotic
cells and late apoptotic cells) in HeLa cells increased from 12.1% to 29.6 and 26.6% after
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treatment with complexes 4 and 6, respectively. From these results, it can be concluded that
the metal complexes induce extensive apoptosis in HeLa cells.
<Fig. 11>
3
.0 Conclusions
In this study we have synthesised a series of neutral and cationic gold phosphine complexes
containing the 2-BrC F PPh ligand and investigated their ability to inhibit the growth of five
6
4
2
human tumor cell lines and one normal cell line. The metal complexes displayed significant
stability under physiological-like conditions and anti-proliferative properties. The neutral
complexes (1-3) displayed moderate cytotoxicity towards all the tested cancer cell lines with
IC values in the range 6.51-22.5 µM. Remarkably, the cationic complexes 4 and 6 showed
5
0
potent and higher cytotoxicity than cisplatin, with IC50 values of 1.9 and 1.2 µM,
respectively, towards HeLa cancer cells. These cationic complexes tend to accumulate in
higher amounts in the cancer cells than the neutral complexes, leading to higher cytotoxicity.
The metal complexes displayed significant inhibitory activities on solid tumor cell
proliferation (HeLa spheroids). Treatment of HeLa cells with the neutral complexes resulted
in G0/G1 cycle arrest whereas the cationic complexes caused G2/M cell cycle arrest. Hoechst
3
3342 staining demonstrated that the metal complexes effectively induced apoptosis of HeLa
cells. In addition, these complexes enhanced the level of intracellular ROS and induced a
decrease of mitochondrial membrane potential. Taken together all these results suggest that
the metal complexes induce apoptosis in HeLa cells through mitochondrial membrane
depolarisation and increased ROS accumulation and ROS mediated DNA damage. Moreover,
the neutral metal complexes strongly inhibited thioredoxin reductase (TrxR) with lower
activity towards glutathione reductase (GR) while the cationic complexes displayed moderate
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TrxR inhibition, demonstrating their great potential in anticancer drug discovery and
development.
4
4
.0 Experimental
.1 General comments
The compounds 2-BrC F PPh [32, 33], [AuCl(tht)] [34], and [AuCl(PPh )] [35] were
6
4
2
3
prepared following literature methods, and [Au(PPh ) ]PF by a slightly modified literature
3
2
6
1
19
31
method [17], the details of which are given below. H (300 MHz), F (282 MHz) and P
(
121 MHz) NMR spectra were measured as CDCl solutions on a Bruker Avance 300
3
spectrometer at room temperature. For the stability studies, DMSO-d was used. Chemical
6
1
19
31
shifts are referenced to residual solvent signals ( H), CFCl ( F) or external 85% H PO ( P)
3
3
4
and coupling constant (J) are given in Hz.
.1.1 X-ray Crystallography
4
Crystals of complexes 1-4 suitable for single-crystal X-ray diffraction were obtained from
dichloromethane/hexane. Using a drop of inert oil (Nujol), crystals were mounted on a nylon
loop and transferred into a stream of cold nitrogen. The reflections were collected on a D8
Bruker diffractometer equipped with an APEX-II area detector using graphite‐
monochromated Mo Kα radiation (λ = 0.71073 Å) from a 1 µS microsource. The computer
programs SMART [36] and SAINT [37] were used for data collection in φ- and ω- scan
modes and data processing, respectively, and absorption corrections using SADABS
[
38]. The structures were solved using direct methods and refined with full-matrix least-
2
squares methods on F using the SHELX-TL package [39, 40]. The computer program
OLEX2 was used to generate the X-ray crystal structures [41]. The CCDC numbers for
complexes 1-4 are 1582742-1582745.
4
.1.2 Preparation of [AuCl(2-BrC F PPh )] (1)
6 4 2
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To a solution of [AuCl(tht)] (305 mg, 0.95 mmol) in CH Cl (20 mL) cooled to 0 °C was
2
2
added 2-BrC F PPh (393 mg, 0.95 mmol). After stirring for 15 min the solution was warmed
6
4
2
to room temperature and filtered through Celite. MeOH was added to the filtrate and the
volume of the solution was reduced, precipitating a white solid, which was filtered off,
1
washed with MeOH and dried in vacuo (535 mg, 87%). H NMR: δ 7.48-7.57 (m, 4H), 7.58-
3
1
19
7
.73 (m, 6H). P NMR: δ 30.2 (m). F NMR: δ -122.8 (m), -124.2 (m), -145.0 (m), -151.7
m).
.1.3 Preparation of [AuBr(2-BrC F PPh )] (2)
(
4
6
4
2
To a solution of [AuCl(2-BrC F PPh )] (200 mg, 0.31 mmol) in CH Cl (30 mL) was added a
6
4
2
2
2
solution of LiBr (45 mg, 0.52 mmol) in MeOH (15 mL) and the solution was stirred for 15
min. The solvent was removed in vacuo and the residue dissolved in CH Cl . After filtration
2
2
through Celite, MeOH was added to the filtrate and the volume of the solution was reduced in
vacuo. The white precipitate was filtered off, washed with MeOH and dried in vacuo (190
1
31
19
mg, 89%). H NMR: δ 7.48-7.57 (m, 4H), 7.58-7.73 (m, 6H). P NMR: δ 32.3 (m). F
NMR: δ -122.9 (m), -124.2 (m), -145.0 (m), -151.7 (m).
4
.1.4 Preparation of [AuI(2-BrC F PPh )] (3)
6 4 2
This was prepared analogously to the bromo complex above from [AuCl(2-BrC F PPh )]
6
4
2
(200 mg, 0.31 mmol) and NaI (70 mg, 0.47 mmol) to give the product as a white solid (201
1
31
19
mg, 88%). H NMR: δ 7.48-7.58 (m, 4H), 7.59-7.75 (m, 6H). P NMR: δ 36.1 (m). F
NMR: δ -123.0 (m), -124.2 (m), -145.2 (m), -151.8 (m).
4
.1.5 Preparation of [Au(2-BrC F PPh ) ]PF (4)
6 4 2 2 6
To a solution of [AuCl(2-BrC F PPh )] (100 mg, 0.15 mmol) and 2-BrC F PPh (64 mg, 0.15
6
4
2
6
4
2
mmol) in CH Cl (8 mL) was added TlPF (55 mg, 0.16 mmol) and the mixture was stirred
2
2
6
for 1.5 h in the dark. The suspension was filtered through Celite, hexane was added to the
filtrate and the volume of the solution was reduced in vacuo. The white solid that precipitated
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1
was filtered off, washed with hexane and dried in vacuo (156 mg, 86%). H NMR: δ 7.54-
3
1
19
7
.74 (m, 20H). P NMR: δ 38.8 (br m), -144.8 (sept, J = 712 Hz). F NMR: δ -73.4 (d, J
PF PF
=
714 Hz), -122.8 (m, 2F), -143.3 (m), -150.5 (m).
4
.1.6 Preparation of [Au(PPh ) ]PF (6)
3
2
6
To a solution of [AuCl(PPh )] (85 mg, 0.17 mmol) and PPh (45 mg, 0.17 mmol) in CH Cl
2
3
3
2
(
8 mL) was added TlPF (63 mg, 0.18 mmol) and the mixture was stirred for 1.5 h in the
6
dark. The suspension was filtered through Celite, hexane was added to the filtrate and the
volume reduced in vacuo. The white solid that precipitated was filtered off, washed with
1
31
hexane and dried in vacuo (146 mg, 97%). H NMR: δ 7.47-7.73 (m, 30H). P NMR: δ 45.1
(
s), -144.3 (sept, J = 713 Hz).
PF
4
.2 Stability of the metal complexes in DMSO and physiological-like conditions
The stability of the complexes in phosphate buffer saline (PBS) was monitored using UV-
Visible absorption spectroscopy. The UV-Vis absorption spectra of the gold complexes were
recorded on a Varian Cary 50 UV-Visible spectrophotometer. Stock solutions of the metal
complexes were freshly prepared in DMSO and diluted to 25 µM in PBS buffer. The
absorption spectra of 25 µM gold complexes in PBS were monitored over time, from 0 h to
1
31
7
2 h. H NMR and P NMR experiments of the gold complexes were carried out in DMSO-
d6 and monitored over time to determine their stability in DMSO.
4
.3 Cell Culture
Prostate (PC-3), glioblastoma (U87MG), cervical (HeLa), fibrosarcoma (HT1080), ovarian
SKOV-3) and human embryonic kidney (Hek-293T) cells were purchased from ATCC. PC-
and HeLa cells were grown in RPMI medium, whereas U87MG, HT1080, SKOV-3, and
(
3
Hek-293T were maintained in DMEM medium supplemented with 10% FBS and 1% PS. All
o
cells were maintained in a sterile incubator with 75% humidity and 5% CO at 37 C. Cells
2
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were harvested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA, Life
Technologies) for subculture and plating after reaching 80% confluence. For all the assays,
unless otherwise mentioned, stock solutions of the metal complexes were freshly prepared in
DMSO (10 mM) and 1% DMSO in complete medium was used as a control. Cisplatin
(positive control) was purchased from Sigma-Aldrich.
4
.3.1 MTT assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is based on
the conversion of a yellow coloured MTT solution to purple formazan crystals by the active
mitochondrial dehydrogenases of viable cells. Thus, the intensity of the produced purple
colour is proportional to the number of viable cells. The cells were seeded in 96 well plates
depending on cellular doubling time and were treated with solutions of the metal complexes
(100 µM, 10 µm, 1 µM, 0.1 µM, 0.01 µM) after overnight incubation. The cells were further
incubated for 48 h and the medium containing the test compounds was removed before
addition of 100 µL MTT solution (5 mg/mL) in serum-free medium. After 4 h incubation in
the dark at 37 °C, the excess MTT solution was removed from the wells and 100 µL of
DMSO was added to each well to solubilise the formed formazan crystals. The absorbance of
formazan solution from each well was recorded using a Spectramax micro titre plate reader at
5
70 and 630 nm. Growth inhibition of the compounds was calculated as IC50 values
(concentration of compound that causes 50% inhibition of cell growth) using Probit software.
Each experiment was repeated three time and the standard deviation values are reported in
Table 2.
4
.3.2 Spheroid assay. HeLa cells at low densities of 25000 per well were seeded in a
Corning® Costar® Ultra-Low attachment 24 well plate in complete growth medium and
allowed to incubate for 3 days to form spheroids. The cells were treated with IC50
1
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concentrations of metal complexes and the growth of the spheroids was monitored after 48
and 96 h using a phase contrast microscope.
4
.3.3 TrxR (thioredoxin reductase) inhibition assay
In this assay, 25 µL of the various concentrations of metal complexes (10 µM, 5 µM, 2.5 µM,
.25 µM, 0.62 µM, dissolved in DMSO: phosphate buffer) was incubated with 25 µL of TrxR
or GR enzyme (2.0 U/mL in milliQ water) in a 96 well plate. After incubation for 30 min at
1
3
7 °C, 200 µL of reaction mixture (1000 µL of mixture contains 500 µL phosphate buffer pH
.0, 80 µL of EDTA solution 100 mM pH 7.5, 20 µL of BSA [bovine serum albumin]
7
solution of 0.2%, 400 µL of distilled water) was added to each well and then 25 µL of 20 mM
NADPH (nicotinamide adenine dinucleotide phosphate) solution was added. Finally, 25 µL
of 20 mM ethanolic DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] was added and the increased
absorbance due to the formation of TNB (2-nitro-5-thiobenzoic acid) was recorded
immediately at 412 nm for 10 min with 30 s intervals using a SpectraMax plate reader. 1 unit
of enzyme activity was defined as 1 µmol of NADPH oxidised per min using the formula
∆A405nm × 1 / (13.6 × 2), where 1 mol of NADPH yields 2 mol of TNB. Data are the means
of triplicate of three independent experiments [27].
4
.3.4 Cell Cycle analysis
6
HeLa cells were plated in a 6 well plate at a density of 1×10 /well and allowed to adhere
overnight in an incubator. Cells were treated with IC concentrations of complexes 1-6 and
5
0
cisplatin for a period of 48 h and were collected by trypsinisation, washed with PBS and
fixed with 70% ethanol. After fixation, cells were stained with propidium iodide staining
solution and kept in ice till analysis. 10000 cells from each sample were analysed for
propidium iodide fluorescence in FL-2 channel mode using a BD Accuri C6 flow cytometer
[
42].
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4
.3.5 Hoechst staining
The nuclear morphological changes in the HeLa cells were assessed using Hoechst 33242. In
this assay, HeLa cells were grown on coverslips in 6 well plates and treated with IC50
concentrations of complexes 1-6 and cisplatin for 48 h. The culture medium was removed,
the cells washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min
and stained with 2 µg/mL Hoechst 33242. The excess dye was removed after 20 min
incubation and the cells were washed three times with PBS. The cells were observed for their
nuclear morphological changes under a confocal microscope using 20x lens (NIKON) [43].
4
.3.6 Assessment of mitochondrial membrane potential
5
HeLa cells were seeded in 24 well plates at a density of 5×10 cells/mL and treated with
complexes 1-6 and cisplatin. After 48 h incubation, the medium was removed and the cells
were washed with PBS and 500 µL of fresh medium containing 5 µg/mL JC-1 was added.
Cells were washed three times with PBS after incubating for 20 min and photographed at a
magnification of 20× using a fluorescence microscope (BIORAD) [44].
4
.3.7 Intracellular reactive oxygen species
The intracellular levels of reactive oxygen species were measured using DCFDA staining.
Briefly, HeLa cells were treated with IC concentrations of complexes 1-6 and cisplatin for
5
0
4
8 h. After treatment, the cells were collected by trypsinisation, washed with PBS, and
stained with Carboxy-DCFDA (10 µM). 5000 cells from each sample were analysed for the
green fluorescence of DCF using a BD Accuri C6 cytometer [45].
4
.3.8 Annexin-V FITC/propidium iodide double staining
6
HeLa cells (1×10 /well) were grown in 6 well plates and incubated with IC concentrations
5
0
of complexes 1-6. After 48 h, the cells were collected by trypsinisation and washed with PBS.
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The cells were resuspended in Annexin binding buffer and 5 µL of Annexin and 1 µL of PI
was added to each sample and incubated for 15 min at room temperature. 15000 cells from
each sample were analysed immediately using a BD Accuri C6 flow cytometer [46].
Acknowledgements
The authors acknowledge the Micro Nano Research Facility (MNRF), RMIT University for
providing the facilities to carry out these experiments.
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Figure/Schemes/Table captions
Fig. 1. Structures of the gold(I) phosphine complexes used in this investigation.
Scheme 1. Synthesis of neutral gold(I) complexes (1-3).
Scheme 2. Synthesis of a cationic gold(I) complex (4).
Scheme 3. Synthesis of a cationic gold(I) complex (6).
Fig. 2. Molecular structure of [AuCl(2-BrC F PPh )] (1). Ellipsoids show 50% probability
6
4
2
levels and hydrogen atoms have been omitted for clarity. Only one molecule in the
asymmetric unit is shown.
Fig. 3. Molecular structure of the cation in [Au(2-BrC F PPh ) ]PF (4). Ellipsoids show 50%
6
4
2 2
6
probability levels. Hydrogen atoms and PF counter ion have been omitted for clarity. Only
6
one molecule in the asymmetric unit is shown.
Fig. 4. UV-Vis absorption spectra of the gold(I) complexes (25 µM) in sodium phosphate
buffer (50 mM, pH 7.4) over 72h.
Table 1. Selected bond distances (Å) and angles (º) in complexes 1-4.
Table 2. IC values (µM)* of the gold phosphine complexes against selected human cancer
5
0
cell lines and normal cell line (Hek-293T).
Table 3. Thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibition activity
(
EC , µM)* of the gold phosphine complexes 1–6.
50
Fig. 5. The amount of gold taken up by cervical cancer cells (HeLa) after exposure to IC50
concentrations of the gold complexes for 24 h.
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Fig. 6. Effect of complexes 1-6 and cisplatin on HeLa spheroid formation. HeLa cells were
grown in ultra-low attachment plates and treated with IC50 concentrations of the metal
complexes for the spheroid assay. After 48 h and 96 h, the spheroids were photographed.
Fig. 7. Cell cycle distribution of HeLa cells after treatment with the gold complexes 1-6 and
cisplatin for 48 h. The percentage of cells in each phase was analysed using BDC6 accuri
software.
Fig. 8. Apoptosis in HeLa cells induced by complexes 1-6 and cisplatin, observed using
Hoechst 33242 staining. HeLa cells were incubated with IC50 concentrations of the
complexes for 48 h. The morphological changes of cell apoptosis such as condensation of
chromatin and nuclear fragmentations were observed by fluorescence microscopy after
staining with Hoechst 33242.
Fig. 9. Fluorescence microscopic images of mitochondrial membrane integrity disruption in
HeLa cells induced by treatment with complexes 1-6. HeLa cells were treated with IC50
concentrations of the metal complexes for 48 h before staining with JC-1 and were imaged by
fluorescence microscopy.
Fig. 10. Metal complex induced production of intracellular ROS in HeLa cancer cells. Cells
were treated with IC concentrations of the metal complexes for 48 h, stained with 10 µM
5
0
DCF-DA and analysed for green fluorescence using a FL-1 channel flow cytometer.
Fig. 11. Flow cytometric analysis of the apoptotic and necrotic cells induced by complexes 1-
6
and cisplatin. HeLa cells were stained with Annexin V-FITC and PI after 48 hours of
incubation with IC concentrations of the metal complexes. (LL: live; LR: early apoptotic;
5
0
UR: late apoptotic; UL: necrotic).
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Scheme 1. Synthesis of neutral gold(I) complexes (1-3)
Scheme 2. Synthesis of a cationic gold(I) complex (4)
Scheme 3. Synthesis of a cationic gold(I) complex (6)
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Table 1. Selected bond distances (Å) and angles (º) in complexes 1-4.
1
, X = Cl(1)
2, X = Br(2) 3, X = I(1)
4
Au(1)–P(1)
Au(1)–X
2.2307(11)
2.2817(11)
176.98(2)
2.2407(10)
2.3975(5)
178.07(3)
2.2468(8)
2.5413(3)
179.68(2)
Au(1)–P(1)
2.2976(7)
2.2976(7)
180.0
Au(1)–P(1*)
X–Au(1)–P(1)
P(1)–Au(1)–P(1*)
Table 2. IC values (µM)* of the gold phosphine complexes against selected human cancer
5
0
cell lines and normal cell line (Hek-293T).
Complex
PC3
U87MG
HeLa
HT1080
SKOV-3
Hek-293T
1
12.2±1.66 8.1±0.83
15.1±2.4
6.51±1.1
13.1±2.52 16.8±3.81
2
22.5±1.94 7.31±0.72 13.8±1.16 7.32±0.85 18.3±3.42 18.9±2.82
3
1.96±0.36 2.62±0.24 8.7±1.23
3.14±0.73 2.1 ±0.56 1.9±0.68
6.03±0.51 10.3±2.31 12.3±0.81
3.11±0.27 2.83±0.24 5.85±0.63
4
5
4.53±0.41 5.57±0.21 2.57±0.15 4.66±0.63 8.93±1.23 1.98±0.22
4.01±0.46 3.78±0.42 1.23±0.11 3.57±0.37 6.12±0.85 6.83±0.92
6.31±0.23 8.22±0.81 3.25±0.28 0.63±0.12 1.57±0.36 4.78±0.35
6
Cisplatin
*
IC values are the concentrations that cause 50% inhibition of cancer cell growth. Data
50
represent the mean values ± standard deviation of three independent experiments performed
in triplicate.
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Table 3. Thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibition activity
(
EC , µM)* of the gold phosphine complexes 1–6.
5
0
Complex
TrxR
GR
Selectivity
GR/TrxR (x fold)
1
2
3
4
5
6
0.15±0.05
0.27±0.11
0.11±0.03
2.6±0.31
0.28±0.09
3.5±0.27
2.65±0.16
1.93±0.24
6.9±1.12
15.6±0.36
1.5±0.07
10.7±0.54
17.6
7.1
62.2
6.0
5.3
3
*
The concentration of the metal complex required for 50% inhibition of enzyme activity.
Data represents the mean of triplicate runs of three independent experiments.
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