Antitumor and Antiangiogenic Properties of Gold(III) Complexes Containing Cycloaurated Triphenylphosphine Sulfide Ligands

Article abstract of DOI:10.1021/acs.inorgchem.0c00423

A family of stable anticancer gold(III)-based therapeutic complexes containing cyclometalated triphenylphosphine sulfide ligands have been prepared. The anticancer properties of the newly developed complexes [AuCl₂{κ²-2-C₆H₄P(S)Ph₂}] (1), [Au(κ²-S₂CNEt₂){κ²-2-C₆H₄P(S)Ph₂}]PF₆ (2), [AuCl(dppe){κC-2-C₆H₄P(S)Ph₂}]Cl (3), and [Au(dppe){κ²-2-C₆H₄P(S)Ph₂}][PF₆]₂ (4) were investigated toward five human cancer cell lines [cervical (HeLa), lung (A549), prostate (PC3), fibrosarcoma (HT1080), and breast (MDA-MB-231)]. In vitro cytotoxicity studies revealed that compounds 2-4 displayed potent cell growth inhibition (IC₅₀ values in the range of 0.17-2.50 μM), comparable to, or better than, clinically used cisplatin (0.63-6.35 μM). Preliminary mechanistic studies using HeLa cells indicate that the cytotoxic effects of the compounds involve apoptosis induction through ROS accumulation. Compound 2 also demonstrated significant inhibition of endothelial cell migration and tube formation in the angiogenesis process. Evaluation of the in vivo antitumor activity of compound 2 in nude mice bearing cervical cancer cell (HeLa) xenografts indicated significant tumor growth inhibition (55%) with 1 mg/kg dose (every 3 days) compared with the same dose of cisplatin (28%). These results demonstrate the potential of gold(III) complexes containing cyclometalated triphenylphosphine sulfide ligands as novel metal-based anticancer agents.

Full text of DOI:10.1021/acs.inorgchem.0c00423

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Article
Antitumor and Antiangiogenic Properties of Gold(III) Complexes
Containing Cycloaurated Triphenylphosphine Sulfide Ligands
́
T. Srinivasa Reddy, Steven H. Priver, Nedaossadat Mirzadeh,* Rodney B. Luwor, Velma Ganga Reddy,
Shwathy Ramesan, and Suresh K. Bhargava*
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ABSTRACT: A family of stable anticancer gold(III)-based therapeutic
complexes containing cyclometalated triphenylphosphine sulfide ligands have
been prepared. The anticancer properties of the newly developed complexes
[AuCl2{κ2-2-C6H4P(S)Ph2}] (1), [Au(κ2-S2CNEt2){κ2-2-C6H4P(S)Ph2}]PF6
(2), [AuCl(dppe){κC-2-C6H4P(S)Ph2}]Cl (3), and [Au(dppe){κ2-2-C6H4P-
(S)Ph2}][PF6]2 (4) were investigated toward five human cancer cell lines
[cervical (HeLa), lung (A549), prostate (PC3), fibrosarcoma (HT1080), and
breast (MDA-MB-231)]. In vitro cytotoxicity studies revealed that compounds
2−4 displayed potent cell growth inhibition (IC50 values in the range of 0.17−
2.50 μM), comparable to, or better than, clinically used cisplatin (0.63−6.35
μM). Preliminary mechanistic studies using HeLa cells indicate that the
cytotoxic effects of the compounds involve apoptosis induction through ROS
accumulation. Compound 2 also demonstrated significant inhibition of
endothelial cell migration and tube formation in the angiogenesis process.
Evaluation of the in vivo antitumor activity of compound 2 in nude mice bearing cervical cancer cell (HeLa) xenografts indicated
significant tumor growth inhibition (55%) with 1 mg/kg dose (every 3 days) compared with the same dose of cisplatin (28%). These
results demonstrate the potential of gold(III) complexes containing cyclometalated triphenylphosphine sulfide ligands as novel
metal-based anticancer agents.
INTRODUCTION
■
In response to the adverse side effects of the clinically
approved anticancer drug cisplatin, gold complexes have been
investigated with the aim of delivering metal-based therapeu-
tics with minimal side effects.1−4 The promising outlook of
gold-based therapeutics lies in their ability to present effective
anticancer properties with high selectivity toward cancer cells,
however their poor stability toward reduction under
physiological conditions must be addressed in order to
advance their clinical translation. Gold(III) complexes are
particularly prone to reduction, therefore their potential in the
Figure 1. Bidentate C−N and tridentate C−N−C and C−N−N
ligands used in the development of cycloaurated anticancer gold(III)
complexes.
development of therapeutic agents has been explored to a
lesser extent compared to their gold(I) rivals.1−5 Despite these
limitations, mechanistic studies on several gold(III) complexes
have been recently reported.6
The inclusion of a cyclometalated ring for the structural
modification of gold(III) complexes has been proposed
recently as a strategy to enhance their stability under
physiological conditions, minimizing undesired intracellular
redox reactions as a result of the strength of the gold−carbon
σ-bond in the cyclometalated moiety.1 This strategy has been
applied to the preparation of cycloaurated gold(III) complexes
containing C−N, C−N−C, and C−N−N ligands shown in
Figure 1.1,2
Received: February 10, 2020
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Scheme 1. Synthesis of [AuCl2{κ2-2-C6H4P(S)Ph2}] (1)
Among these ligands, the iminophosphorane compound has
enabled the development of a unique class of gold(III)
complexes, shown in Figure 2, whose stability in solution can
be easily monitored by 31P NMR spectroscopy. Complexes A
and B exhibit high in vitro stability in DMSO, whereas C is
only stable for 24 h. In all cases, these gold(III) compounds
showed high toxicity against Jurkat T-cell acute lymphoblastic
leukemia cells and B-CLL cells.7,8
Scheme 2. Synthesis of the Cationic Gold(III) Complexes
2−4
Figure 2. Examples of cycloaurated gold(III) complexes containing an
iminophosphorane ligand.
In alignment with our current research in developing gold
complexes for the treatment of cancer8−16 and inspired by the
potential of iminophosphorane ligands in the medicinal
chemistry field, we have designed and extended a class of
cycloaurated gold(III) complexes derived from triphenylphos-
phine sulfide (Figure 3). In contrast to our previously
published work on ionic digold(I/III) complexes containing
two cyclometalated triphenylphosphine sulfide ligands,16 the
results herein are for a series of mononuclear gold(III)
complexes containing only one cyclometalated ligand. The
anticancer properties and mechanism of action of the gold(III)
complexes 1−4 containing a cycloaurated C−S moiety were
investigated toward a wide range of cancer cell lines, the results
of which are presented here.
terized by multinuclear NMR spectroscopy and, in the case of
complexes 1, 3, and 4, by single crystal X-ray diffraction.
1
The H NMR spectra for complexes 1−4 all showed the
expected aromatic multiplets in the range δ 7.3−7.9. In
addition, the spectrum for complex 2 showed two broad
multiplet resonances at δ 1.29 and 3.83 due to the methyl and
methylene protons of the dithiocarbamate ligand, respectively.
For complexes 3 and 4, the methylene protons of the dppe
ligand appeared as two broad multiplets between δ 3 and 4.
The 31P NMR spectra for complexes 1 and 2 showed a singlet
resonance at δ 56.1 and 61.1, respectively, the chemical shift of
which is typical of coordinated tertiary phosphine sulfide16,17
and similar to that of the cycloiminophosphorane complex
[AuCl2{κ2-2-C6H4P(NPh)Ph2}] (δ 65).18 The 31P NMR
spectra of 3 and 4 were more complex and showed three
resonances in a 1:1:1 ratio in the region δ 45−77, assignable to
the phosphorus nuclei in the dppe ligand and C6H4P(S)Ph2
group. Although the exact assignment of each resonance was
not possible, the resonances show fine structure due to P−P
RESULTS AND DISCUSSIONS
■
Synthesis of Gold Complexes. The reaction of [Bu4N]-
[AuCl4] with an equimolar amount of 2-Me3SnC6H4P(S)Ph2
in dichloromethane led to the formation of [AuCl2{κ2-2-
C6H4P(S)Ph2}] (1; Scheme 1) as a pale yellow solid in 94%.
This compound has been prepared previously in 42−46% yield
by the transmetalation of the organomercurials [HgCl{κC-
C6H4P(S)Ph2}] or [Hg{κC-C6H4P(S)Ph2}2] to [Me4N]-
[AuCl4] in acetonitrile.17 Treatment of 1 with NaS2CNEt2 or
1,2-bis(diphenylphosphino)ethane (dppe) afforded [Au(κ2-
S2CNEt2){κ2-2-C6H4P(S)Ph2}]PF6 (2) and [AuCl(dppe)-
{κC-2-C6H4P(S)Ph2}]Cl (3), respectively (Scheme 2). In the
presence of TlPF6, 1 reacted with dppe to give [Au(dppe){κ2-
2-C6H4P(S)Ph2}][PF6]2 (4). Complexes 1−4 were charac-
Figure 3. Cycloaurated gold(III) complexes derived from triphenylphosphine sulfide.
B
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coupling. The spectra of 2 and 4 also showed the expected
septet at δ −144 due to the PF6 anion.
of the dppe ligand, the carbon atom of the C6H4P(S)Ph2
ligand, and either a chlorine (complex 3) or sulfur (complex 4)
atom. As expected, the Au−P bond length trans to the carbon
atom of the C6H4P(S)Ph2 group (ca. 2.38 Å) is significantly
longer than the Au−P bond length trans to chloride or sulfur
(ca. 2.30 Å), consistent with the higher trans influence of σ-
carbon ligands.
̅
The structures of 1, 3 (shown in Figure 4), and 4 (shown in
Figure 5) were confirmed by X-ray diffraction. In all cases, the
geometry about the gold(III) atom was, as expected, square
planar. The metrical parameters of 1 are, within experimental
error, identical to those previously reported.17 In complexes 3
and 4, the gold(III) atom is bound by two phosphorus atoms
Interactions with Human Serum Albumin. Human
serum albumin (HSA) is a major plasma protein and comprises
∼55% of the whole blood plasma proteins. HSA increases the
solubility of drugs, including metal complexes, in plasma and
plays an important role in their cellular uptake and
pharmacokinetics. The intrinsic emissive properties of the
tryptophan residue in HSA is very sensitive to changes in the
microenvironment of the protein.19 Therefore, we investigated
the binding interactions of complexes 1−4 with HSA by
fluorescence spectroscopy. The fluorescence emission spectra
of HSA in PBS were acquired in the absence and presence of
different concentrations of complexes 1−4 (Figure 6A and
Figure S13), and a decrease in the characteristic emission
maximum of HSA at 346 nm was observed with increasing
concentrations of the complexes. Notably, the maximum
emission wavelength did not change during the interaction
with the complexes and combined with the decrease in
intensity, Indicated a reduction in the polarity of the
microenvironment as a result of the complexes binding to
the hydrophobic cavity of the protein.20 The interaction of
complexes 1−4 with HSA resulted in a quenching of the
fluorescence by 54.4, 19.7, 50.1, and 38.2%, respectively
(Figure 6B).
A better understanding of the HSA fluorescence quenching
mechanism by the gold compounds can be obtained using
Stern−Volmer equations. As shown in Figure 6C, the Stern−
Volmer plots (F0/F versus concentration of the compounds)
exhibited linear relationships and the Stern−Volmer quenching
constants (KSV) were obtained from the slope of these linear
plots using the equation F0/F = 1 + KSV[Q], where F and F0
are the fluorescence intensity of HSA in the presence and
absence of the gold compounds, respectively. The binding
constants (Kb) and number of binding sites (n) were obtained
from the intercept and slopes of scathard plots log (F0 − F)/F
vs log[Q] (Figure 6D) using the equation log (F0 − F)/F = log
Kb + nlog[Q]. The values are summarized in Table S1. The
results show that the calculated Kq values for the gold
compounds were higher than the Kq of quencher with
biopolymers (2.0 × 1010 M−1 s−1) suggesting a static
quenching mechanism is involved in the HSA fluorescence
quenching. Further, the values of Kb indicate a higher binding
affinity of compound 2 to HSA, followed by compounds 3, 1,
and 4. It was also observed that the values of n were
approximately equal to 1, suggesting a single binding site for
the metal complexes in the HSA molecule.
Figure 4. Molecular structure of [AuCl(dppe){κC-2-C6H4P(S)Ph2}]-
Cl·0.931(H2O)·0.069(CH2Cl2) (3). Ellipsoids show 50% probability
levels. Hydrogen atoms on the phenyl rings, chloride counterion, and
solvents of crystallization have been omitted for clarity. Selected bond
distances (Å) and angles (deg): Au(1)−C(1) 2.0815(16), Au(1)−
P(2) 2.3904(8), Au(1)−P(3) 2.3051(7), Au(1)−Cl(1) 2.3551(7),
P(1)−S(1) 1.9666(7), C(1)−Au(1)−P(2) 176.38(4), P(3)−Au(1)−
Cl(1) 175.913(14).
Stability of the Gold Complexes in Dimethyl
Sulfoxide (DMSO) and Physiological-Like Conditions.
Previous studies have reported that gold complexes are
vulnerable to ligand exchange and reduction reactions in
DMSO and physiological conditions.1,21 Therefore, the
stability of complexes 1−4 in d6-DMSO and cell culture
medium (Roswell Park Memorial Institute, RPMI medium)
was monitored by 31P NMR spectroscopy (Figures S9−S12)
and UV−vis spectroscopy (Figure 7), respectively. Complexes
1 and 2 were stable in DMSO solution, even after 72 h, and no
significant changes in their NMR spectra were observed. In
Figure 5. Molecular structure of [Au(dppe){κ2-2-C6H4P(S)Ph2}]-
[PF6]2·3(CH2Cl2) (4). Ellipsoids show 50% probability levels.
̅
Hydrogen atoms on the phenyl rings, PF6 counterions, and solvent
of crystallization have been omitted for clarity. Selected bond
distances (Å) and angles (deg): Au(1)−C(1) 2.090(4), Au(1)−
P(3) 2.3830(13), Au(1)−P(2) 2.3141(12), Au(1)−S(1) 2.3756(11),
S(1)−P(1) 2.0340(16), C(1)−Au(1)−P(3) 178.01(14), P(2)−
Au(1)−S(1) 171.71(6).
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Figure 6. Interaction between the gold(III) complexes and HSA studied by fluorescence spectroscopy. (A) The fluorescence emissions of HSA (10
μM) were recorded in the absence and presence of complex 1, with increasing concentrations (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μM) from a to
k. (B) Percentage of fluorescence quenching after treatment with different concentrations of complexes 1−4. (C) Stern−Volmer plots of quenching
of HSA fluorescence by the gold compounds 1−4. (D) Scathard plots of the fluorescence titrations of the gold compounds 1−4 with HSA.
contrast, complexes 3 and 4 were only stable for ca. 2 h before
evidence for the formation of new species was evident.
Complex 3 slowly but cleanly gave a new species, as shown by
the appearance of peaks at δ 42.3, 34.0, and 29.3 in the 31P
NMR spectrum, tentatively identified as [Au(DMSO)(dppe)-
{κC-2-C6H4P(S)Ph2}]Cl2. Attempts to obtain X-ray quality
crystals of this species were unsuccessful. In contrast to 3,
solutions of 4 slowly gave a complex mixture of species which
could not be identified. In agreement with the NMR
spectroscopic results above, complexes 1 and 2 showed no
significant time-dependent UV−vis spectral changes in cell
culture medium, indicating that the complexes are stable under
physiological-like conditions. Further, complexes 3 and 4
showed a slight decrease in absorbance over an incubation
period of 72 h (Figure 7).
In Vitro Cytotoxicity. Preliminary in vitro cytotoxicity
studies on the gold complexes were conducted to investigate
their anticancer activity against cervical (HeLa), lung (A549),
prostate (PC3), fibrosarcoma (HT1080), and breast (MDA-
MB-231) cancer cells. The compounds were also tested for
their cytotoxicity toward human umbilical vascular endothelial
cells (HUVEC), which were employed as a noncancerous cell
model. IC50 values were calculated from dose response curves
obtained after 72 h of exposure of the compounds using the
MTT assay, and the results were compared with the clinically
used drug cisplatin. As shown in Table 1, all the gold
compounds showed higher potency against HeLa, HT1080,
and MDA-MB-231 cells than cisplatin; compounds 2, 3, and 4
showed similar cytotoxicity and potency toward the three cell
lines with IC50 values in the range 0.17−2.50 μM and exhibited
approximately 8-, 3−6-, 3−5-, and 3−12-fold higher
cytotoxicity than cisplatin against HeLa, PC3, HT1080, and
MDA-MB-231 cancer cells, respectively. Among the series,
compound 2 showed the highest selectivity toward cancer cells
followed by compound 1. For instance, compound 2 exhibited
9-, 15- and 5-fold higher selectivity toward HeLa, HT1080, and
MDA-MB-231 cells, respectively, compared to noncancerous
HUVEC cells.
Cellular Uptake. As the biological activity of the
compounds is determined by the extent of cellular accumu-
lation,22 flow cytometric analysis of cells pretreated with
increasing concentrations (0.5, 1, and 2 μM) of complexes 1−
4 was undertaken. In all cases, a characteristic shift in the side
scatter pattern (SSC) owing to increased cellular granularity
was observed. The greatest shift was observed for compound 2
and is concentration-dependent, indicating its efficient uptake
(Figure 8A). We also investigated the amount of gold taken up
by the cells using ICP-MS. HeLa cells were treated with
equimolar concentrations of compounds 1−4 (1 μM) for 12 h,
and the total cellular accumulation of gold was determined. As
shown in Figure 8B, cells treated with complexes 2−4 showed
higher levels of gold uptake than those treated with 1 and
correlate with the cytotoxic potency of the compounds (Table
1).
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Figure 7. Electronic absorption spectra of gold complexes 1−4 (20 μM) in complete growth medium (RPMI) over a period of 72 h.
Table 1. In Vitro Anticancer Activity (IC50, μM) of the Newly Synthesized Gold Complexes 1−4 and Cisplatin against a Panel
a
of Cancerous and Noncancerous Cells
compound
HeLa
A549
PC3
HT1080
MDA-MB- 231
HUVEC
1
2
3
4
1.15 0.09
0.42 0.07
0.36 0.12
0.46 0.03
3.25 0.11
>50
2.41 0.14
1.01 0.21
1.65 0.16
2.45 0.11
6.31 0.06
9.11 0.78
0.26 0.06
0.21 0.05
0.17 0.04
0.63 0.06
17.41 1.63
0.73 0.05
0.54 0.08
2.50 0.09
6.35 0.51
5.26 0.15
3.89 0.32
0.93 0.16
2.13 0.19
6.89 1.23
6.97 0.32
2.60 0.47
6.08 0.48
5.69 0.37
cisplatin
a
HeLa, cervical cancer cells; A549, lung cancer cells; PC3, prostate cancer cells; HT1080, fibrosarcoma; MDA-MB-231, breast; HUVEC, human
umbilical vascular endothelial cells.
TrxR Inhibition. Although the exact mechanism of action
for the anticancer activity of many gold complexes has yet to
be elucidated, the high affinity of gold compounds to undergo
facile ligand exchange reactions with sulfur- and selenium-
containing groups in thioredoxin reductase (TrxR) makes this
enzyme a compelling molecular target.23 The thioredoxin
(TrxR) system plays a key role in cellular homeostatic
processes which neutralize reactive oxygen species (ROS)
and helps to overcome cellular oxidative stress.24 Therefore,
the inhibition of TrxR has recently emerged as an attractive
strategy in cancer treatment.25,26 In this regard, we investigated
the TrxR inhibition ability of compounds 1−4 using the
lipoate reduction assay.27 The results, shown in Figure 9,
indicate that treatment of HeLa cells with 1−4 leads to a
strong inhibition of TrxR in a concentration-dependent
manner. Relative to the vehicle control, the greatest inhibition
was observed for compound 1 followed by compounds 2, 4,
and 3. The higher TrxR inhibition in 1 may be due to
participation in ligand exchange reactions between the chloride
ligands and the selenocysteine residue in TrxR. These results
are in contrast with the growth inhibition values, where
compounds 2−4 are more cytotoxic than compound 1,
suggesting compounds 2−4 may have other cellular targets.
Gold Compound-Induced ROS Production in HeLa
Cells. It has been reported that inhibition of the antioxidant
TrxR enzyme may result in apoptosis induction in cancer cells
through increased ROS accumulation.28 Therefore, we
investigated whether TrxR inhibition led to ROS accumulation
in HeLa cells. The intracellular levels of ROS in HeLa cells
after treatment with the gold complexes were analyzed using
carboxy-H2DCFDA. After being taken up by cells, the
nonfluorescent DCFDA is oxidized to fluorescent dichloro-
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Figure 8. Determination of intracellular accumulation of gold compounds 1−4. (A) Side scatter analysis of HeLa cells after treatment with 0.5, 1,
and 2 μM concentrations of the gold complexes for 12 h. (B) HeLa cells were treated with 1 μM gold compounds for 12 h, and the amount of
intracellular gold was determined by ICP-MS. The data represent the mean the standard deviation of three independent experiments.
Figure 9. TrxR inhibition in HeLa cells treated with different
concentrations of compounds 1−4 for 6 h and monitored
colorimetrically for lipoate reduction.
Figure 10. Accumulation of reactive oxygen species in HeLa cells.
HeLa cells treated with gold compounds 1−4 (0, 0.25, 0.5, and 1 μM)
for 6 h were further incubated with carboxy-H2DCFDA (10 μM) for
30 min. The bar chart shows the fluorescence intensity values
compared to the untreated controls.
fluorescein (DCF) by the action of cellular ROS,29 and thus
the fluorescence intensity of DCF is directly proportional to
ROS levels.
As shown in Figure 10, treatment of HeLa cells with
compounds 1−4 led to increased intracellular ROS accumu-
lation. Among the series, treatment with compound 2 resulted
in the greatest accumulation of ROS in a concentration-
dependent manner. The intracellular ROS levels were
increased to significant levels when the cells were treated
with 1 μM of compound 2 (4.3-fold increase), correlating with
the high cytotoxicity of this compound.
Wound Healing Assay. The migration of endothelial cells
to form a blood supply network is essential for the progression
of tumors and metastasis.30 Therefore, the discovery of drugs
that can inhibit both cancer cell growth and endothelial cell
motility is essential for efficient cancer therapy. In this regard,
we investigated the ability of the gold complexes to inhibit the
migration ability of highly migratory human umbilical vascular
endothelial cells (HUVEC) using a wound healing assay.31
As shown in Figure 11, the linear scratch assay shows the
rapid migration of the control cells into the wounded area
while the wells treated with compounds 1−4 showed fewer
cells migrating to the scratched area. The wound closure
followed the order control > 1 > 4 > 3 > 2. These results thus
underpin the excellent migration inhibition abilities of
compound 2 in HUVEC lines.
Tube Formation and Angiogenesis Assay. It has been
reported that TrxR inhibition by metal complexes may
contribute to the inhibition of the angiogenesis process,
which plays an important role in the development and spread
of tumors from benign to malignant.32 Therefore, the
antiangiogenic effect of the synthesized compounds was
investigated by observing tube formation of HUVECs in
matrigel constructs.33 Elongated, robust networks of HUVECs
were clearly seen in wells seeded with untreated cells, whereas
network formation was significantly inhibited when cells were
treated with compounds 1−4 (Figure 12 and Figure S14).
Quantification of tube formation using WimTube showed that
all aspects of network formation were significantly affected in
complex-treated wells, as indicated by a decrease of cell-
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In Vivo Antitumor Activity. The excellent in vitro
cytotoxicity, cellular uptake, antimigratory, and angiogenesis
inhibition properties of compound 2 prompted us to
investigate the in vivo activity in nude mice. To test the in
vivo tumor growth inhibition efficacy, experiments were
conducted on female BALB/c nude mice bearing human
cervical tumor xenografts. Cisplatin was used as positive
standard. Nude mice bearing HeLa xenografts were treated
with intraperitoneal injections of compound 2 (1 mg/kg),
cisplatin (1 mg/kg), or vehicle control every 3 days for 42
days. As shown in Figure 13A and B, an exponential increase in
tumor volume over time was observed in the control mice, but
mice treated with the metal complexes showed tumor growth
inhibition. At the end of the experiment, tumor growth
inhibition was significant for mice treated with compound 2
(55%) compared to those treated with cisplatin (28%).
We also analyzed the tumor tissues for the expression of Ki-
67, a cell proliferation marker, by immunohistochemistry.34 As
shown in Figure 13C and D, treatment with the gold
compounds resulted in an approximately 50% reduction in
Ki-67 positive cells, whereas cisplatin treated mice showed 45%
lower Ki-67 positive cells, indicating the gold complexes
inhibited tumor proliferation. By comparison to the results of
the control mice, there were no significant morphological
changes in the kidney and liver tissues of the mice treated with
compound 2 (Figure 13C−E). In addition, no mouse death or
Figure 11. Wound healing assay. Wounds created in HUVEC
monocell layers. Cells were treated with IC50 concentrations of
compounds 1−4 for 24 h. (A) Images of wounds captured
immediately (0 h) and 24 h after treatment and viewed at 4×
magnification. (B) The number of cells migrated into the wound area
was counted manually. Data are the mean the standard deviation
from three independent experiments.
covered area, number of tubes and branching points, and total
length of tubes, highlighting the excellent antiangiogenic
properties of the complexes.
Figure 12. Tube formation of HUVEC cells. HUVECs cultured on matrigel coated plates were treated with IC50 concentrations of compounds 1−
4 for 16 h. (A) Morphological changes and tube formation were visualized by microscopy, and representative images are shown. The total lengths
of the tubes, loops, branching points, and covered area in the presence and absence of the gold compounds were analyzed and are shown in the bar
graphs B−E, respectively.
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Figure 13. In vivo antitumor activity of gold compound 2 and cisplatin. BALB/c nude mice bearing HeLa xenografts received intraperitoneal
injections of either metal complexes (1 mg/kg) or vehicle control (DMSO; n = 5) every 3 days. (A) Tumor volumes were measured at different
time points with vernier calipers and are expressed with
standard error. (B) Isolated tumor weights. (C) Representative images of the
immunohistochemical staining of the proliferation marker Ki-67. (D) Quantification of the percentage of cells positive for Ki-67 (as a % of control).
(E) Haematoxylin and eosin staining of isolated kidney, liver, and tumor tissues. A standard Student’s t test was used for statistical analysis to
determine statistically significant differences with respect to the control. *p < 0.01. **p < 0.001.
change in body weight was observed in any of the mice
throughout the experiments, suggesting no general toxicity.
compared to that of 1 (280 nmol Au/g protein). Additionally,
the bidentate dithiocarbamate ligand confers stability to the
complex due to significant contribution of the dithiolate
canonical form35 Et2N+CS2 and is more labile than the
2−
CONCLUSION
■
bidentate diphosphine ligand dppe for easier participation in
exchange reactions with cellular enzymes. The present work
highlights the promising prospects of cycloaurated gold(III)
complexes in the development of new drug molecules for
cancer treatment.
A series of cycloaurated gold(III) complexes derived from
triphenylphosphine sulfide have been prepared and their
anticancer properties investigated toward a panel of five
different human tumor cell lines. Among the complexes,
[AuCl2{κ2-2-C6H4P(S)Ph2}] (1) and [Au(κ2-S2CNEt2){κ2-2-
C6H4P(S)Ph2}]PF6 (2) demonstrated outstanding stability in
solution even after 72 h. Compounds 2−4 were found to be
the most cytotoxic against various cancer cell lines with high
selectivity and almost on par with the commercially used
anticancer drug cisplatin. The compounds also showed
excellent inhibition of endothelial cell migration with
antiangiogenic activity. In cervical cancer cells, the mechanism
of anticancer activity for complex 2 involved apoptotic cell
death through increased production of ROS. Compound 2 also
showed effective in vivo anticancer activity toward cervical
tumor xenografts in nude mice with no observable general
toxicity. The superior activity of 2 relative to the other
complexes studied may be due to several factors. For example,
replacement of the two chloride ligands in the coordination
sphere of [AuCl2{κ2-2-C6H4P(S)Ph2}] by a dithiocarbamate
ligand increases the lipophilicity of the complex, evidenced by
the greater cellular uptake of 2 (495 nmol Au/g protein)
EXPERIMENTAL SECTION
■
Chemistry. [Bu4N][AuCl4], 2-Me3SnC6H4P(S)Ph2, and
NaS2CNEt2·3H2O were prepared according to literature meth-
ods.36−38 1H (300 MHz) and 31P (121 MHz) NMR spectra were
acquired in d6-DMSO, unless stated otherwise, on a Bruker Avance
300 spectrometer at room temperature. Chemical shifts are referenced
to residual solvent signals (1H) or external 85% H3PO4 (31P), and
coupling constants (J) are given in Hz. Mass spectra were acquired on
a Bruker Autoflex Speed (MALDI) or PerkinElmer Axion 2 TOF
(ESI) spectrometer. Elemental analyses were performed by the
Microanalytical Unit of the Department of Molecular Sciences at
Macquarie University, Sydney.
X-ray Crystallography. Crystals of complexes 1, 3, and 4 suitable
for single-crystal X-ray diffraction were obtained from dichloro-
methane/hexane (1) or dichloromethane/ether (3 and 4). Using a
drop of inert oil (Paratone), crystals were mounted on a nylon loop
and transferred to a stream of cold nitrogen. The reflections were
collected on a D8 Bruker diffractometer equipped with an APEX-II
H
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area detector using graphite monochromated Mo Kα radiation (λ =
0.71073 Å) from a 1 μS microsource. The computer programs
SMART39 and SAINT40 were used for data collection in φ- and ω-
scan modes and data processing, respectively, and absorption
corrections using SADABS.41 The structures were solved using direct
methods and refined with full-matrix least-squares methods on F2
using the SHELX-TL package.42,43 The CCDC numbers for
complexes 1, 3, and 4 are 1883144−1883146.
solutions with or without metal complexes were incubated for 5 min
and their emission spectra recorded (λex = 295 nm, λem = 300−450
nm) using a cuvette of 1 cm path length.
Biological Methods. Cell culture supplements, media, Hoeschst
33242, and Carboxy-DCFDA were purchased from Invitrogen. Ten
millimolar stock solutions of the metal complexes were prepared in
DMSO.
Cell Culture. Human cervical (HeLa), prostate (PC3), lung
(A549), and breast (MDA-MB-231) cancer cell lines were grown at
37 °C in a humidified 5% CO2 atmosphere in RPMI medium
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
penicillin (100 U/mL), and streptomycin (100 μg/mL). Fibrosarco-
ma (HT1080) cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FBS, penicillin (100 U/
mL), and streptomycin (100 μg/mL). HUVEC cells were grown in
M200 medium supplemented with low serum growth supplement
(LSGS). 0.05% Trypsin-EDTA was used for detaching the cells from
the culture flasks.
Preparation of [AuCl2{κ2-2-C6H4P(S)Ph2}] (1). A bright yellow
solution of [Bu4N][AuCl4] (1.03 g, 1.77 mmol) and 2-Me3SnC6H4P-
(S)Ph2 (810 mg, 1.77 mmol) in CH2Cl2 (20 mL) was left to stir in
the dark overnight. Hexane was added to the resulting pale-yellow
solution, and the volume was reduced in vacuo, precipitating out a
pale yellow solid. The solid was isolated by filtration, washed
sequentially with hexane and MeOH, then recrystallized from
CH2Cl2/MeOH to give the title compound (933 mg, 94%). 1H
NMR: δ 7.36 (dd, J = 1.8, 11.7 Hz, 1H), 7.47 (ddt, J = 1.0, 4.4, 7.4
Hz, 1H), 7.54−7.61 (m, 1H), 7.72−7.94 (m, 10H), 8.21 (ddd, J =
1.0, 3.4, 8.2 Hz, 1H). 31P NMR: δ 56.1 (s). HR-ESI MS (m/z):
524.9856. Calcd for C18H14AuClPS: 524.9908 [M − Cl]+. Elem anal.
calcd for C18H14AuCl2PS: C, 38.52; H, 2.51; S, 5.71. Found: C, 38.46;
H, 2.33; S, 5.77.
Cell Viability Assay. The in vitro cytotoxicity of the metal
complexes was evaluated using the MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, 3000 to 5000
cells were seeded in 96 well plates, dependent upon the doubling time
of the cell line, and were allowed to adhere at 37 °C in a 5% CO2
atmosphere. After overnight incubation, the medium was removed
and replaced with fresh media containing different concentrations of
the metal complexes. Next, 0.5% DMSO was used as a vehicle control.
After 72 h of treatment, 10 μL of a 5 mg/mL MTT solution was
added to each well, and the cells were incubated for 4 h. Unreacted
MTT was removed, and 100 μL of DMSO was added to each well to
solubilize the obtained formazan crystals. The absorbance of each well
was recorded using a plate reader (Spectra Max) at 570 nm and is
proportional to the number of viable cells. Therefore, percentage
inhibition of cell growth at each tested concentration was determined
using the formula % inhibition = 100 × (control absorbance − sample
absorbance). Dose response curves were plotted, and IC50 values were
calculated using Probit software.
Preparation of [Au(κ2-S2CNEt2){κ2-2-C6H4P(S)Ph2}]PF6 (2). To
a solution of 1 (100 mg, 0.18 mmol) in CH2Cl2 (10 mL) was added
NaS2CNEt2·3H2O (50 mg, 0.18 mmol). The pale-yellow solution
darkened slightly and was stirred for 10 min. TlPF6 (130 mg, 0.37
mmol) was added to the solution, which was stirred for 1 h in the
dark. The suspension was filtered through Celite, and hexane was
added to the filtrate. The volume of the solution was reduced in vacuo,
precipitating out a pale yellow solid, which was filtered off, washed
with hexane, and dried in vacuo (126 mg, 90%). 1H NMR: δ 1.29 (br.
m, 6H), 3.83 (br. m, 4H), 7.42−8.00 (m, 14H). 31P NMR: δ 61.1 (s),
−144.2 (sept, J = 711 Hz). HR-ESI MS (m/z): 638.0431. Calcd for
C23H24AuNPS3: 638.0474 [M − PF6]+. Elem anal. calcd for
C23H24AuF6NP2S3·0.5H2O: C, 34.86; H, 3.18; N, 1.77; S, 12.14.
Found: C, 34.65; H, 2.86; N, 1.85; S, 13.02.
Preparation of [AuCl(dppe){κC-2-C6H4P(S)Ph2}]Cl (3). To a
solution of 1 (100 mg, 0.18 mmol) in CH2Cl2 (10 mL) was added
dppe (71 mg, 0.18 mmol). After stirring for 5 min, the volume of the
solution was reduced to ∼3 mL and Et2O added dropwise,
precipitating out a pale yellow solid. The solid was isolated by
Cellular Uptake. HeLa cells (1 × 106) were seeded in 25 cm2
flasks in 5 mL of complete growth medium and allowed to adhere for
24 h. The cells were treated with different concentrations (0.5, 1, and
2 μM) of gold complexes for 12 h and were collected by
trypsinization. The collected cells were washed with PBS and
analyzed by flow cytometry (BDC6-Accuri).
1
filtration, washed with Et2O, and dried in vacuo (157 mg, 92%). H
NMR: δ 3.60−3.89 (br. m, 2H), 6.84−8.11 (m, 34H). Two
methylene protons are obscured by the water peak at δ 3.4. 31P
NMR: δ 61.1 (d, J = 4.6 Hz), 52.1 (dd, J = 4.7, 20 Hz), 45.0 (d, J = 20
Hz). 1H NMR (CDCl3): δ 3.28−3.51 (br. m, 2H), 3.93−4.03 (br. m,
2H), 6.81−7.16 (m, 6H), 7.27−7.73 (m, 24H), 7.96−8.03 (m 4H).
MALDI MS (m/z): 919.2. Calcd for C45H41AuOP3S: 919.2 [M − 2Cl
+ OCH3]+ (from MeOH). HR-ESI MS (m/z): 914.1591. Calcd for
C45H38AuNP3S: 914.1603 [M − 2Cl + CN]+ (from MeCN). Elem
anal. calcd for C44H38AuCl2P3S·1.5H2O: C, 53.56; H, 4.19; S, 3.25.
Found: C, 53.47; H, 3.99; S, 3.33.
For ICP-MS measurements, cells treated with 1 μM concentrations
of the gold complexes were harvested with trypsin-EDTA. The
collected cell pellets were lysed with lysis buffer, and each sample was
divided into two portions. One aliquot was used to determine the
protein content in each sample using the bicinchoninic acid (BCA)
protein assay. The second aliquot was digested with HNO3 at 65 °C
for 3 h. After cooling, the mineralized sample was diluted to 10 mL
with ultrapure water. The gold content was determined by axial ICP-
MS (PerkinElmer).
TrxR Inhibition. In this assay, the ability of HeLa cells to reduce
the cell permeable cofactor (lipoate) was monitored colorimetrically
after 6 h of treatment with compounds 1−4. HeLa cells (10 000 cells/
well) seeded in 96 well plates were incubated with compounds 1−4
(0.312, 0.625, 1.25, 2.5, and 5 μM) for 6 h at 37 °C. After incubation,
the growth medium containing the gold compounds was replaced
with 100 μL of reaction mixture (20 mM lipoate and 1 mM DTNB)
in HBSS (Hanks balanced salt solution). The plates were monitored
immediately and after 180 min for a change in absorbance at 405 nm
due to the reduction of DTNB using a microplate reader
(SpectraMax).
ROS Production. The levels of intracellular ROS were determined
using the ROS-sensitive probe carboxy-DCFDA (Molecular Probes-
Invitrogen). Briefly, HeLa cells seeded in 24 well plates in RPMI 1640
were treated with IC50 concentrations of the gold compounds for 6 h.
The cells were collected with trypsin-EDTA, washed with PBS, and
incubated with 10 μM of carboxy-DCFDA for 30 min at 37 °C. After
incubation, the cells were washed with PBS, and the fluorescence
increase of DCFDA was measured utilizing the wavelengths of 485
Preparation of [Au(dppe){κ2-2-C6H4P(S)Ph2}][PF6]2 (4). To a
solution of 1 (100 mg, 0.18 mmol) in CH2Cl2 (10 mL) was added
dppe (71 mg, 0.18 mmol) followed by TlPF6 (140 mg, 0.40 mmol).
The suspension was stirred in the dark for 1 h, then filtered through
Celite. Hexane was added to the filtrate, and the volume of the
solution was reduced in vacuo, precipitating out a colorless solid. The
solid was isolated by filtration, washed with hexane, and dried in vacuo
(194 mg, 92%). 1H NMR: δ 3.18−3.30 (m, 2H), 3.59−3.77 (m, 2H),
6.67−6.80 (m, 1H), 7.14−7.23 (m, 1H), 7.33−7.88 (m, 30H), 7.92−
8.02 (m, 2H). 31P NMR: δ 77.0 (s), 65.0 (d, J = 39 Hz), 59.8 (d, J =
38 Hz), −144.2 (sept, J = 711 Hz). MALDI MS (m/z): 919.4. Calcd
for C45H41AuOP3S: 919.2 [M − 2(PF6) + OCH3]+ (from MeOH).
Elem anal. calcd for C44H38AuF12P5S: C, 44.84; H, 3.25; S, 2.72.
Found: C, 44.79; H, 3.29; S, 3.09.
Interactions with Human Serum Albumin. HSA solutions in
PBS (10 μM) were freshly prepared before performing the
experiments. The fixed concentration of HSA was titrated with
different concentrations of each gold complex (2−20 μM). The HSA
I
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Article
nm (excitation) and 527 nm (emission) in a plate reader
(Spectramax).
Accession Codes
CCDC 1883144−1883146 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Wound Healing Assay/Scratch Assay. HUVEC cells were
seeded in a 6 well plate (1 × 105 cells/well) in M200 medium
supplemented with low serum growth supplement (LSGS). After 24 h
to facilitate cell attachment, the confluent monolayer was scratched in
a single straight line using a sterile 200 μL plastic pipet tip to create a
wounded, cell-free area. The wells were washed with PBS and the
media containing either the vehicle (0.3% DMSO) or IC50
concentrations of the metal compounds was added. The cells were
constantly monitored, and images were taken at 0 and 24 h to observe
the migration of cells across the wounded area. The number of cells in
the wounded area was counted manually, and the total number of
cells was normalized against the untreated wells containing the highest
number of cell migration. All the experiments were repeated thrice
with triplicate samples in each set of experiments.
AUTHOR INFORMATION
■
Corresponding Authors
Suresh K. Bhargava − Centre for Advanced Materials and
Industrial Chemistry, School of Science, RMIT University,
Melbourne 3001, Australia; orcid.org/0000-0002-3127-
8166; Email: suresh.bhargava@rmit.edu.au
Tube Formation Assay. BD Matrigel (BD Bioscience, Heidel-
berg, Germany) was thawed at 4 °C overnight prior to the coating of
matrigel in a 96 well plate. The plates were placed on ice prior to the
coating, and 50 μL of matrigel was slowly added to each well with
constant, gentle agitation for an even layer formation. The coated
plates were incubated at 37 °C in a 5% CO2 atmosphere for 1 h. The
gels were overlaid with cells (10 000 HUVEC cells/well) in M200
media containing either the vehicle (0.3% DMSO) or the synthesized
compounds. The plates were visualized for live cells with the aid of
calcein-AM (4 μM) after 16 h of incubation. The images for both the
bright field and green channels were recorded using an inverted
microscope (BIORAD) to observe the subsequent effect of the
compounds on the tubular structure formation. Quantification of the
tube length formation was analyzed using WimTube analysis software
Nedaossadat Mirzadeh − Centre for Advanced Materials and
Industrial Chemistry, School of Science, RMIT University,
Melbourne 3001, Australia; orcid.org/0000-0002-3092-
2634; Email: nedaossadat.mirzadeh@rmit.edu.au
Authors
T. Srinivasa Reddy − Centre for Advanced Materials and
Industrial Chemistry, School of Science, RMIT University,
Melbourne 3001, Australia; orcid.org/0000-0002-2806-
4300
́
Steven H. Priver − Centre for Advanced Materials and
Industrial Chemistry, School of Science, RMIT University,
Melbourne 3001, Australia; orcid.org/0000-0001-5521-
9884
́
(Onimagin Technologies SCA, Cordoba, Spain).
In Vivo Antitumor Activity. The antitumor activity of the
synthesized compounds were tested in female BALB/c nude mice by
subcutaneous injection of 5 × 106 HeLa cells/100 μL RPMI medium
in both ventral flanks anterior to the hind legs using a 29-gauge insulin
syringe. The mice were constantly monitored for 48 h to observe any
changes in weight or adverse effects prior to treatment with the
compounds. Tumor volumes of 50 mm3 were established prior to the
random separation of mice into three groups comprising of 5 in each.
Intraperitoneal injections of either the vehicle control, 1 mg/kg of
complex 2, or cisplatin were supplied three times a week for 42 days.
The tumor volume was measured eight times during the 42 day
period before culling of the mice. All the tumors were collected and
weighed. The obtained tumors were also checked for the extent of
proliferation and hence the inhibitory effect of the gold complex by
subsequent staining for the Ki-67 proliferative marker. The tumor
xenografts were paraffin fixed and sectioned into 4 μm sections using a
microtome. The sections were further stained using the Ventana
BenchMark Immunostainer (Ventana Medical Systems, Inc., Tucson,
AZ). The sections were dewaxed, and the endogenous peroxidase
activity was blocked with Ventana EZ prep and Ventana Universal
3,3′-diaminobenzidine (DAB) inhibitor, respectively. The sections
were stained against the primary Ki-67 marker, which was
subsequently diluted according to the manufacturer’s instructions.
Counterstaining was carried out with Ventana hematoxylin and bluing
solution. Primary antibody staining was detected using the ultraView
Universal DAB Detection Kit (Roche, Basel, Switzerland). Negative
controls consisted of the tumor sections in the absence of the primary
antibody, while human tonsil cells were employed as positive controls.
Cells were visualized using an Aperio ImageScope (Leica Micro-
systems, Mount Waverley, Australia) and the related digital pathology
viewing software.
Rodney B. Luwor − Department of Surgery, Royal Melbourne
Hospital, University of Melbourne, Parkville 3050, Australia
Velma Ganga Reddy − Centre for Advanced Materials and
Industrial Chemistry, School of Science, RMIT University,
Melbourne 3001, Australia; orcid.org/0000-0001-5054-
2519
Shwathy Ramesan − School of Engineering, RMIT University,
Melbourne 3001, Australia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c00423
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the Micro Nano Research Facility
(MNRF), RMIT University for providing the facilities to carry
out cell culture experiments.
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