Gold(i) and gold(iii) phosphine complexes: synthesis, anticancer activities towards 2D and 3D cancer models, and apoptosis inducing properties

Article abstract of DOI:10.1039/C8DT01724G

A series of gold(i), gold(iii) and cationic gold(i) complexes of tris(4-methoxyphenyl)phosphine and tris(2,6-dimethoxyphenyl)phosphine were synthesised and fully characterised by spectroscopic methods. The molecular structures of selected complexes were also determined by X-ray diffraction analysis. The prepared complexes [AuX{P(C₆H₄-4-OMe)₃}] [X = Cl (1), Br (2), I (3)], [AuCl₃{P(C₆H₄-4-OMe)₃}] (4), [Au{P(C₆H₄-4-OMe)₃}₂]PF₆ (5), [AuX{P(C₆H₃-2,6-{OMe}₂)₃}] [X = Cl (6), Br (7), I (8)], [AuCl₃{P(C₆H₃-2,6-{OMe}₂)₃}] (9) and [Au{P(C₆H₃-2,6-{OMe}₂)₃}₂]PF₆ (10) were investigated for their anticancer activity against five human tumor cell lines [ovarian (SKOV-3), fibrosarcoma (HT1080), glioblastoma (U87MG), prostate (PC-3), and cervical (HeLa)] as well as against 3D spheroidal models of HeLa cells. The cationic complex 10 was found to exhibit a remarkably broad spectrum of anticancer activity with approximately 30-fold higher toxicity than cisplatin against PC-3 and U87MG cancer cells; this complex also showed the strongest inhibition of spheroid growth in 3D models of HeLa cells. The mechanism of anticancer activity of these gold complexes was found to be strong inhibition of thioredoxin reductase, increased ROS production and subsequent apoptosis induction as evidenced by the sub G1 cell accumulation, DNA fragmentation, and caspase-3 activation.

Full text of DOI:10.1039/C8DT01724G

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
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ARTICLE
Gold(I) and Gold(III) Phosphine Complexes: Synthesis, Anticancer
Activities Towards 2D and 3D Cancer Models, and Apoptosis
Inducing Properties
T. Srinivasa Reddy, Steven H. Privér, Vijay V. Rao, Nedaossadat Mirzadeh,* Suresh K. Bhargava*
Received 00th January 20xx,
Accepted 00th January 20xx
A series of gold(I), gold(III) and cationic gold(I) complexes of tris(4-methoxyphenyl)phosphine and tris(2,6-
dimethoxyphenyl)phosphine were synthesized and fully characterized by spectroscopic methods. The
molecular structures of selected complexes were also determined by X-ray diffraction analysis. The prepared
complexes [AuX{P(C6H4-4-OMe)3}] [X = Cl (1), Br (2), I (3)], [AuCl3{P(C6H4-4-OMe)3}] (4), [Au{P(C6H4-4-
OMe)3}2]PF6 (5), [AuX{P(C6H3-2,6-{OMe}2)3}] [X = Cl (6), Br (7), I (8)], [AuCl3{P(C6H3-2,6-{OMe}2)3}] (9) and
[Au{P(C6H3-2,6-{OMe}2)3}2]PF6 (10) were investigated for their anticancer activity against five human tumor
cell lines [ovarian (SKOV-3), fibrosarcoma (HT1080), glioblastoma (U87MG), prostate (PC-3), cervical (HeLa)]
as well as in 3D spheroidal models of HeLa cells. The cationic complex 10 was found to exhibit a remarkably
broad spectrum of anticancer activity with approximately 30 fold higher toxicity than cisplatin against PC-3
and U87MG cancer cells; this complex also showed the strongest inhibition of spheroid growth in 3D models
of HeLa cells. The mechanism of anticancer activity of these gold complexes was found to be strong
inhibition of thioredoxin reductase, increased ROS production and subsequent apoptosis induction as
evidenced by the sub G1 cell accumulation, DNA fragmentation, and caspase-3 activation.
DOI: 10.1039/x0xx00000x
www.rsc.org/
tumor tissue is by increasing the hydrophilic to lipophilic ratio of the
complex. This ratio can be altered by careful ligand design. Since
the influence of the phosphine ligand in terms of the cytotoxic
properties of phosphine-containing gold complexes is still not clear,
we chose to investigate what the effect of methoxy substituents on
the phosphine backbone would have on the anticancer activity of a
range of gold complexes. Recent reports on gold(I) and gold(III)
derivatives of the 2-(2′-pyridyl)benzimidazole (pbiH) ligand enabled
the exploration of different oxidation states of gold on anticancer
activity.18,19
Introduction
Gold phosphine complexes are ubiquitous in gold chemistry, and
they continue to be the subject of considerable intrigue owing to
their wide spectrum of anticancer properties.1-6 Since the discovery
that auranofin, the first clinically approved gold phosphine drug for
the treatment of rheumatoid arthritis,7-8 also possessed potent in
vitro anticancer activities,9 there has been growing interest in
developing gold(I) phosphine complexes of the type [AuX(PR3)] (X =
halogen, thiolate; R = alkyl, aryl). Recent reports on the anticancer
properties of triphenylphosphine gold chloride, [AuCl(PPh3)], and its
ability to induce autophagy has offered greater insights into the
biological properties of this class of gold complex.10 These gold(I)
phosphines appear to selectively accumulate in mitochondria,
which represent the most likely site for their biological action.
Further, gold complexes, including auronafin, proposed to induce
apoptosis by increasing free radical formation via inhibition of the
mitochondrial isoform of thioredoxin reductase.11-14
Herein, we report the synthesis of a number of gold(I) and gold(III)
complexes containing methoxy-substituted triaryl phosphine
ligands. The anticancer properties of the prepared complexes
(shown in Fig. 1) have been explored towards a wide range of
cancer cells, and the mechanism of cell death was investigated in
HeLa cancer cells.
_
+
PF6
1
)
MeO
P
Au
P
OMe
MeO
P
AuX
X = Cl (
2
Br (
)
3
3
3
To develop gold phosphine drugs, an important factor to consider is
the lipophilicity of the compound, since this plays a significant role
in cellular uptake, intracellular biodistribution, and pharmacokinetic
properties.15,16 It has been shown that highly lipophilic gold(I)
phosphine complexes display remarkable anticancer properties,
however, they also exhibit severe toxicity to healthy normal
tissues.17 One way to increase the bio-distribution and selectivity for
I (3)
5
(
)
4
Cl3
(
)
_
+
PF6
OMe
OMe
MeO
P
AuX
6
)
Br (7)
P
Au
P
X = Cl (
3
3
3
OMe
MeO
OMe
8
I (
)
Cl3 (9)
(10)
Fig. 1 Structures of the gold complexes containing methoxy-
substituted triaryl phosphine ligands used in this investigation.
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Results and discussion
Synthesis of gold complexes:
As shown in Scheme 1, reaction of [AuCl(tht)] (tht
=
tetrahydrothiophene) with an equimolar amount of tris(4-
methyoxyphenyl)phosphine afforded the desired gold(I) chloride
complex [AuCl{P(C6H4-4-OMe)3}] (1), from which the corresponding
bromide (2) and iodide (3) complexes were prepared by
metathetical reactions. Oxidation of 1 with chlorine (as PhICl2) gave
the gold(III) complex [AuCl3{P(C6H4-4-OMe)3}] (4), while the cationic
bis(phosphine) gold(I) complex [Au{P(C6H4-4-OMe)3}2]PF6 (5) was
prepared by treating 1 with one equivalent of phosphine in the
presence of TlPF6.
Fig. 2 The molecular structure of [Au{P(C6H4-4-OMe)3}2]PF6 (5).
Ellipsoids show 50% probability levels. Hydrogen atoms and PF6
counter ion have been omitted for clarity. Selected bond distances
(Å) and angles (°): Au(1)-P(1) 2.3151(6), Au(1)-P(2) 2.3145(6), P(1)-
Au(1)-P(2) 174.331(19).
Scheme 1 Synthesis of neutral gold(I), gold(III) and cationic gold(I)
complexes containing methoxy-substituted triaryl phosphine
ligands.
Analogous to the preparation of 1-3, [AuCl{P(C6H4-2,6-{OMe}2)3}] (6)
was synthesized from the reaction of [AuCl(tht)] with tris(2-6-
dimethylphenyl)phosphine, which was converted to the
corresponding bromide (7) and iodide (8) complexes. The gold(III)
complex [AuCl3{P(C6H4-2,6-{OMe}2)3}] (9) and cationic gold(I)
complex [Au{P(C6H4-2,6-{OMe}2)3}2]PF6 (10) were prepared
following an analogous method for the synthesis of 4 and 5,
respectively.The preparation of complexes 1-3,20
5 (as the
Fig.
3 Molecular structure of [AuCl3{P(C6H3-2,6-{OMe}2)3}] (9).
perchlorate salt)21 and 622 have been reported previously; all other
complexes are new. Complexes 1-10 were characterized by
multinuclear NMR spectroscopy (1H and 31P); complexes 4-10 were
also charecterised by single crystal X-ray diffraction.
Ellipsoids show 30% probability levels. Hydrogen atoms have been
omitted for clarity. Selected bond distances (Å) and angles (°):
Au(1)-P(1) 2.3261(6), Au(1)-Cl(1) 2.2941(6), Au(1)-Cl(2) 2.3508(6),
Au(1)-Cl(3) 2.2874(7), P(1)-Au(1)-Cl(2) 175.782(19).
In general agreement with the reported data (where available), the
1H NMR spectra for complexes 1-10 each showed the expected
aromatic multiplets between δ 6.5-7.5, together with a singlet
resonance around δ 3-4 due to the methoxy protons in the correct
integral ratio. Uniquely, in the case of the gold(III) complex
[AuCl3{P(C6H4-2,6-{OMe}2)3}] (9), three singlet resonances were
observed at δ 3.49, 3.62 and 3.94 arising from restricted rotation
about the P−AuCl3 bond. Similar restricted rotation about Au−P
bonds in gold(III) complexes containing phosphine ligands with
bulky ortho-substituents has been observed previously.23 The 31P
NMR spectra for the gold complexes 1-4 and 6-9 each showed a
singlet resonance in the expected region of ca. δ 35 and δ -25,
respectively. The gold(III) complexes 5 and 10 similarly showed a
singlet resonance, but are shifted downfield from their
corresponding gold(I) counterparts by approximately 10 and 30
ppm, respectively. In addition, the expected septet at δ -144 due to
PF6- counter ion was also observed.
The molecular structures of 4-10 were confirmed by X-ray
diffraction, and the molecular structures of 5 and 9 are shown in
Fig. 2 and 3, respectively. All other structures are shown in Fig. S11-
S15 (see Supporting Information). In the structures of 5-8 and 10,
the gold(I) atom is coordinated by a phosphorus atom of the
tertiary phosphine and a halide, or, in the case of 5 and 10, by a
second phosphorus atom. As expected, the geometry about the
gold atom is approximately linear (173-179°), with Au−P bond
lengths in the range 2.24-2.33 Å. The Au−X bond lengths in
complexes 6-8 are typical for gold(I) halide complexes and increase
in the order X = Cl (2.29 Å) < Br (2.42 Å) < I (2.56 Å). In the
structures of 4 and 9, the gold(III) atom is approximately square
planar, the Au−Cl bond lengths trans chloride being ca. 2.27-2.29 Å,
while those trans to phosphorus are significantly longer (ca. 2.35-
2.37 Å) as a consequence of the higher trans influence of tertiary
phosphines compared to chloride. In all cases, the metrical
parameters in 4-10 are comparable to those observed for their
2 | Dalton Trans., 2018, 00, 1-3
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triphenylphosphine analogues [AuX(PPh3)],24 [AuCl3(PPh3)]25 and Table 1 IC50 values (µM)* of the gold complexes 1-7, 9 and 10
[Au(PPh3)2]PF6.26
towards various cancer cells and one normal cell line after 72 h
It has been reported that metal complexes are vulnerable to exposure.
reduction in DMSO,27 therefore we monitored the stability of
complexes 1-10 in DMSO over a period of 72 h using 31P NMR
Complex
SKOV-3
HT1080
U87MG
PC-3
HeLa
Hek-293T
spectroscopy. The resulting spectral profiles are shown in Fig. S16-
S25 in the Supporting Information. In most cases there was no
observable shift in the characteristic peaks for the gold phosphine
complexes over 72 h, suggesting the gold complexes are stable in
DMSO. A change in the 31P NMR spectrum of the gold(III) complex
4 in DMSO was observed after I h indicating decomposition. The
stabilities of the synthesised gold complexes were also investigated
1
6.45±0.24
14.7±2.65
1.37±0.11
2.95±0.21
3.9±0.41
4.01±0.6
5.69±0.5
0.97±0.26
3.5±0.62
12.55±1.67
9.63±1.41
2
3.53±0.66
under physiological-like conditions by monitoring the electronic
absorption spectra of the complexes in a tris buffered saline
solution (50ꢀmM Tris, 150ꢀmM NaCl, pH 7.2) over 72ꢀh. As evident
from the electronic spectra (Fig. S26), gold(I), gold(III), and ionic
complexes under investigation displayed no change in the
absorbance over time, indicating the complexes are stable under
3
5.14±0.39
8.53±0.75
4.23±0.36
4.76±0.36
2.13±0.35
1.78±0.08
6.48±1.13
4.14±0.54
3.2±0.82
4.87±0.2
3.61±0.7
2.87±0.1
2.4±0.3
7.98±0.21
13.7±0.13
11.3±1.65
4
2.57±0.4
1.12±0.1
5
physiological-like condition over 72 h.
6
0.77±0.05
1.28±0.08
1.14±0.17
0.34±0.11
1.18±0.21
0.87±0.06
2.42±0.07
0.77±0.04
1.14±0.29
1.31±0.32
1.26±0.16
0.28±0.09
0.53±0.08
1.4±0.05
0.89±0.2
2.13±0.32
5.72±0.75
1.78±0.17
1.65±0.33
In vitro anticancer activity
7
0.26±0.08
1.17±0.06
0.25±0.05
The cell growth inhibitory effects of the gold complexes 1-7, 9 and
10 were evaluated towards a panel of five human tumor cell lines
[ovarian (SKOV-3), fibrosarcoma (HT1080), glioblastoma (U87MG),
9
0.57±0.03
0.19±0.02
prostate (PC-3), cervical (HeLa)] and normal human embryonic
10
kidney (Hek-293T) cells using the MTT assay.28 The gold iodide
complex 8 proved to be too insoluble for testing and was not
investigated further. The anticancer activities of the gold
complexes, reported as IC50 values, were compared to those
AuCl(PPh3)
8.93±1.21
1.57±0.36
4.66±0.63
0.63±0.1
5.57±0.28
8.22±0.8
4.53±0.74
6.31±0.2
2.57±0.16
3.25±0.28
1.98±0.23
4.78±0.35
Cisplatin
obtained for cisplatin, which was used as
a standard, and
[AuCl(PPh3)], the parent unsubstituted gold complex. As shown in
Table 1, nearly all of the newly synthesized complexes displayed
remarkable antiproliferative effects against all the tested cancer cell
lines. Notably, the presence of methoxy substituents on the
phosphine ligand in the gold complexes significantly effects their
anticancer activity and selectivity towards cancer cells. By
comparison to [AuCl(PPh3)], (IC50 2.57-8.93 µM), the 4-methoxy-
substituted complex 1 showed increased activity and selectivity
(IC50 0.97-6.45 µM), and an even more substantial increase was
observed for the 2,6-dimethoxy-substituted complex 6 (IC50 0.53-
1.18 µM). Despite the different oxidation state, the neutral gold(III)
complexes 4 and 9 displayed similar antiproliferative effects to their
gold(I) counterparts 1 and 6, respectively. Among all the complexes
in the series, the cationic dimethoxy-substituted complex 10
displayed a potent and broad spectrum anticancer activity with IC50
values in the submicromolar range (0.19-0.77 µM), significantly
lower than the IC50 values of cisplatin (0.63-8.22 µM). In particular,
complex 10 showed approximately 30 fold higher cytotoxicity than
cisplatin towards PC-3 and U87MG cells. The promising in vitro
anticancer activities of the neutral gold(I) chloride complexes 1 and
6 and cationic complexes 5 and 10 prompted us to investigate their
mechanism of cell death. We also investigated the effect of these
complexes on normal kidney cells (HeK-293T) to determine the
safety and selectivity against non-cancer cells. Interestingly, most of
the synthesized complexes displayed potent cytotoxicity towards
cancer cells in comparison to normal HeK-293T kidney cells. It was
observed that the highly active cationic gold complex 10 showed 5-
10 times higher selectivity towards various cancer cells.
*IC50 values are the concentrations that cause 50% inhibition of cell
growth. SKOV-3: ovarian; HT1080: fibrosarcoma; U87MG:
glioblastoma; PC-3: prostate; HeLa: cervical; Hek-293T: normal
human embryonic kidney cells. Data represent the mean values ±
standard deviation of three independent experiments performed in
triplicate.
3D Multicellular spheroid inhibition assay
3D Multicellular spheroids (MCSs) closely reflect the in vivo
pathophysiological situation in tumor tissues with respect to
phenotypic heterogeneity, nutrient and oxygen gradients and micro
metastases with gene expression closer to the clinical expression
profile.29-31 For instance, tumor cells growing in 3D spheroids
experience different adhesive, mechanical and topographical forces
compared to cells growing in 2D monolayers. Moreover, the cell-
cell, cell-ECM interactions of cells and permeability barrier in solid
tumors could be imitated by establishment of tumor cell 3D
spheroids. In a broad context, in vitro 3D tumor cell cultures could
emerge as appropriate preclinical models to screen the cancer drug
lead for solid tumors.32 Therefore, to validate the cytotoxicity of the
gold complexes in the in vivo tumor environment, 3D multicellular
spheroids were generated by growing the cells in ultra-low
attachment (ULA) plates for 2 days, which were then treated with
IC50 concentrations of the gold complexes, cisplatin, and auranofin.
Fig. 4A depicts representative morphological changes of the MCSs
after treatment with the gold complexes after 48 and 96 h. It is
noted that treatment with the vehicle (DMSO) did not cause any
growth inhibition and the volume of the spheroid increased over
time during the experiment. In contrast, treatment with the gold
complexes resulted in a reduction in surface area and volume of the
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spheroids and the effect is significant after 96 h. Treatment with the no longer capable of maintaining the characteristic broad
cationic complexes 5 and 10 lead to a significant reduction of lamellipodia. Collectively, these results indicate that gold complexes
spheroid volume and increased number of dead cells (propidium inhibit the migration of HeLa cancer cells through disruption of F-
iodide stained) in comparison to the control (Fig. 4B), consistent actin assembly and stress fibre formation.
with the results obtained from the 2D cell monolayer assays. These
results indicate that the cytotoxic effects of the cationic compounds
were equally potent in 3D spheroid cultures as they were in 2D
cultures.
Fig.
4 Antitumor potential of gold complexes in 3D HeLa
multicellular spheroids. (A) HeLa cell spheroids were generated in
96-well ULA round-bottomed plates and were examined for the
morphological changes immediately and after 48 and 96
h
incubation with IC50 concentrations of the gold complexes,
auranofin, and cisplatin. (B) Representative images of Calcein
AM/propidium iodide staining on HeLa MCSs after treatment with
IC50 concentrations of the metal complexes.
Effect of the gold complexes on cell migration
Migration of cancer cells plays an important role in the progression
of tumors and metastasis.33 Previous studies have demonstrated
that gold complexes inhibit the migration of cancer cells.34-35 To
investigate whether the selected gold phosphine complexes 1, 5, 6
and 10 could inhibit in vitro cell migration, a wound healing assay
was carried out using HeLa cervical cancer cells. The number of cells
that migrated into the wound area was counted manually, and the
calculated migration rate is reported in Fig. 5. The results, shown in
Fig. 5A and 5B, indicate that, in comparison to the control, cell
migration was significantly inhibited (30-40% inhibition) after 24 h
treatment with the metal complexes, and the effect is even higher
(40-60% inhibition) after 48 h.
Fig. 5 The effect of the gold complexes 1, 5, 6 and 10 on the
migration of HeLa cells. (A) Wounds were created in the confluent
monolayers and photos were taken at 0, 24 and 48 h after
treatment with the gold complexes (4× magnification). (B) Graphical
representation of the percentage of migration of HeLa cells after
treatment with IC50 concentrations of the gold complexes. Data are
the mean of three independent experiments ± the standard
deviation (**p < 0.01, ***p < 0.001).
Effect of the gold complexes on actin assembly
The polymerization of actin filaments is considered an essential step
for the formation of cell membrane protrusions, which can help in
cell migration as well as stress fibre assembly.36 As shown in Fig. 5,
the gold complexes inhibited the migration of HeLa cancer cells, so
we next examined the effect the complexes had on actin
polymerization. In this assay, the cytoskeleton of the HeLa cells
treated with the gold complexes (1, 5, 6 and 10) was visualized
using rhodamine phalloidin, a red fluorescent dye which specifically
stains F-actin proteins. It was observed that treatment with the gold
complexes causes a decrease in F-actin extensions at the periphery
and inhibited the formation of stress fibre assemblies. The control
cells displayed elongated stress fibres with broad lamellipodia
whereas after treatment with the gold complexes, the staining was
rather granulated and condensed (Fig. 6) and the tumor cells were
Fig. 6 Disruption of F-actin assemblies due to the gold complexes 1,
5, 6, and 10. HeLa cells were treated with IC50 concentrations of the
gold complexes and stained with rhodamine-phalloidin (red
fluorescent dye: actin filament) and Hoechst 33242 (nucleus: blue).
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TrxR inhibition
ARTICLE
population in the G0/G1 phase was observed after treatment with
the complexes. Moreover, complex treatment leads to an increase
in the S phase population without altering the G2/M phase. These
results indicate the metal complexes inhibit the cell cycle
progression in the S phase and also induce apoptosis through sub
G1 arrest.
The thioredoxin (Trx) system consists of NADPH, thioredoxin
reductase (TrxR), and Trx, and plays a central role in several cellular
homeostasis processes, including the regulation of transcription
factors and the removal of reactive oxygen species which
overcomes oxidative stress.37 Therefore, the inhibition of TrxR has
recently emerged as an attractive strategy in cancer treatment
since the overexpression of TrxR in various aggressive tumors is
considered to be responsible for the development of drug
resistance.38 It has been shown that many of the reported gold(I)
compounds exhibit antiproliferative activity through strong TrxR
inhibition.39-40 To investigate whether the newly synthesized gold
complexes could inhibit TrxR in HeLa cells, the lipoate reduction
assay was carried out.41 In this assay, the ability of live HeLa cells to
reduce the cell permeable cofactor (lipoate) was monitored
colorimetrically over a period of 180 min after 6 h treatment with
the gold complexes (1, 5, 6 and 10). Auranofin was used as a
positive control in this experiment. As shown in Fig. 7, treatment
with the neutral gold complexes 1 and 6 lead to strong inhibition of
TrxR (70-80%) in HeLa cells in comparison to auranofin. Moreover, a
significant inhibition of TrxR (50-60%) was observed for the cationic
complexes 5 and 10 relative to the vehicle treated control cells. The
higher TrxR inhibition of the neutral gold(I) complexes may be due
to participation in ligand exchange reactions with the
selenocysteine residue of TrxR. Overall, these results indicate the
newly synthesized gold complexes are comparable to auranofin in
their ability to inhibit TrxR in HeLa cells.
(A)
(B)
Reactive oxygen species (ROS)
It is well-known that TrxR enzyme inhibition causes an increase in
hydrogen peroxide and reactive oxygen species levels which leads
to an imbalance in cell redox conditions, thus resulting in
mitochondrial membrane permeabilization and cell apoptosis.42 To
determine if the TrxR inhibitory properties of the gold complexes
would lead to increased ROS levels in HeLa cells, we carried out a
ROS assay using 6-carboxy-2',7'-dichlorodihydrofluorescein
diacetate (Carboxy-DCFDA),
a non-fluorescent dye which, on
hydrolysis by ROS, produces green fluorescent DCF. The intensity of
the DCF fluorescence directly correlates with the intracellular ROS
levels and is measured by flow cytometry. As shown in Fig. 8, a 2-4
fold increase in DCF fluorescence was observed due to H2DCF
oxidation after treatment with the complexes 1, 5, 6 and 10 in
comparison to the control cells, indicating the gold complex
treatment leads to an increase in ROS levels. Based on these results,
we propose that the antiproliferative activity of the gold complexes
was due to TrxR based ROS mediation.
Fig. 7 (A) TrxR inhibitory activity of the gold complexes 1, 5, 6 and
10 in HeLa cells after treatment with 1.25 µM concentration over a
period of 180 min. (B) Concentration-dependent inhibition of TrxR
activity in HeLa cells treated with 0.625–10.0 µM gold complexes
for 6 h and monitoring the lipoate reduction colorimetrically after
180 min incubation.
Cell cycle distribution
To investigate the influence of the gold complexes on the cell cycle
distribution of HeLa cells, propidium iodide staining was carried out.
The cells were treated with complexes 1, 5, 6 and 10 and after 48 h
analysed by flow cytometry (Fig. 9). The results from the histograms
indicate that the gold complexes induce apoptosis in HeLa cells with
a profound increase of sub G1 cell population. Quantitative analysis
revealed that, compared to the control cells (2.6%), the percentage
of sub G1 phase cells increased significantly to 9.6, 16.3, 11.1 and
18.5% after treatment with complexes 1, 5, 6 and 10, respectively
(Fig. 9B). Simultaneously,
a significant decrease in the cell
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for 48 h. Propidium iodide stained cells were analysed by flow
cytometry. Data from 10,000 cells were collected for each data file.
Histograms are representatives of three independent experiments.
(B) The percentage of cells in each phase (G0/G1, S, and G2/M) was
quantified using BD Accuri C6 software.
Caspase-3 activity assay
Previous reports have suggested that gold(I) complexes can induce
apoptosis in cancer cells through the activation of caspases.43-44
Among the caspases, caspase-3 is the main downstream effector
caspases, and the activation of this enzyme leads to the cleavage of
a variety of critical cellular proteins.45 Therefore, we investigated
the ability of the gold complexes 1, 5, 6 and 10 to activate caspase-3
in HeLa cells after 24 h treatment with IC50 concentrations. As
shown in Fig. 10, treatment with the gold complexes markedly
stimulated caspase-3 activity in comparison to the control. The
increment of caspase-3 concentration was significant for all
complexes relative to the control with a 2-3 fold increase in
caspase-3 activity. Taken together, these results indicate that HeLa
cells treated with the gold complexes showed several hallmarks of
apoptotic cell death, including strong induction of caspase-3
activity.
Fig. 8 Effect of the gold complexes 1, 5, 6 and 10 on the production
of ROS in HeLa cells. ROS levels were determined by monitoring the
florescence of DCF via flow cytometric analysis in live HeLa cells
treated with the complexes. The fold increase in ROS was quantified
using BD Accuri C6 software. Error bars represent one standard
deviation. Data are the mean of three independent experiments ±
the standard deviation (*p < 0.05, **p < 0.01).
(B)
Fig. 10 Caspase-3 activity in HeLa cells after treatment with
complexes 1, 5, 6, and 10 for 24 h. Caspase-3 activation was
determined by mixing the cell lysates with DEVD-pNA (N-acetyl-Asp-
Glu-Val-Asp p-nitroanilide) and monitoring the colorimetric
substrate hydrolysis at 405 nm using an absorbance plate reader.
The values are shown as a fold-increase compared to the control.
Data are the mean of three independent experiments ± the
standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001).
Hoechst staining
It was considered of interest to examine the effect of the gold
complexes on morphological changes, such as DNA fragmentation
associated with caspase-3 and increased ROS, which are known to
cause apoptosis.46 HeLa cells treated with IC50 concentrations of the
gold complexes 1, 5, 6 and 10 for 48 h were stained with the
nuclear staining dye Hoechst 33342. The results, shown in Fig. 11,
indicated that untreated control cells showed no obvious
morphological changes (all the cells exhibited uniform rounded cell
morphology), whereas treatment with the gold complexes lead to
Fig. 9 (A) Effect of gold complexes 1, 5, 6 and 10 on the cell cycle
distribution of HeLa cells after treatment with IC50 concentrations
6 | Dalton Trans., 2018, 00, 1-3
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fragmentation of nuclei (indicated by red arrows), which is a
hallmark of apoptosis. As expected, the cationic complexes 5 and 10
were potent in inducing nuclear fragmentation in HeLa cells.
Conclusions
In conclusion, we have synthesized a series of gold complexes
containing methoxy-substituted aryl phosphine ligands and
investigated their anticancer properties towards a panel of five
different human tumor cell lines. Most of the synthesized
complexes displayed promising in vitro anticancer activities with
IC50 values in the range of low micromolar and even nanomolar
concentrations on various cancer cell lines. The structure activity
relationships of the gold complexes reveal that methoxy
substituents on the phosphine ligand are crucial for the enhanced
anticancer activity and selectivity towards cancer cells. Among the
series, the lipophilic cationic complexes 5 and 10 displayed potent
cytotoxicity towards all the tested cell lines with IC50 values in the
range of 0.19-0.77 µM. In contrast, the neutral hydrophilic gold(III)
complexes 4 and 9, and their gold(I) counterparts 1 and 6, displayed
similar antiproliferative effects with moderate cytotoxicity. It was
also observed that there was no significant correlation between the
halide bound to gold (Cl, Br, I) and their cytotoxicity. The more
physiologically relevant 3D tumor spheroid assay demonstrates the
superior potency of these cationic complexes. It is also evident that
the observed significant cytotoxicity of the gold complexes was
through TrxR inhibition, increased ROS and apoptosis induction with
sub G1 cell cycle arrest and caspase-3 activation. Taken together,
these findings clearly demonstrate the potential of the gold(I)
complexes in anticancer drug development.
Fig. 11 Nuclear morphological changes in HeLa cells after treatment
with IC50 concentrations of the gold complexes 1, 5, 6 and 10 for 48
h (red arrows indicate fragmented nuclei-apoptotic cells).
Annexin V-FITC/propidium iodide dual staining assay
Since the metal complexes induce apoptosis in HeLa cells through
sub G1 cell cycle arrest and caspase-3 stimulation, the Annexin V-
FITC/propidium iodide double staining assay was carried out to
quantify the gold complex-induced apoptosis. As shown in Fig. 12,
the populations of early apoptotic (LL) and late apoptotic (LR) cells
were dramatically increased to 22.6-42.3% after treatment with the
gold complexes in comparison to the vehicle treated cells (10.9%).
Experimental
Chemistry
The cationic complexes
apoptotsis, with more than 40% of the cells being apoptotic
whereas the neutral gold complexes and displayed
approximately 25% of the apoptotic cells. These results further
confirm the apoptosis-inducing ability of the gold complexes in
HeLa cells.
5 and 10 were potent in inducing
Tris(4-methoxyphenyl)phosphine
and
tris(2,6-
dimethoxyphenyl)phosphine were purchased from Sigma. [PhICl2],47
[AuCl(tht)]48 and [AuCl(PPh3)]49 were prepared following literature
methods. [AuX{P(C6H4-4-OMe)3}] (X = Cl, Br, I) were prepared by a
modified literature method, the details of which are given below. 1H
(300 MHz) and 31P (121 MHz) NMR spectra were measured as
CDCl3 solutions 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 constant (J)
are given in Hz. Mass spectra were acquired on a Bruker Autoflex
Speed (MALDI) or Perkin Elmer Axion 2 TOF (ESI) spectrometer. In
general, the mass spectra for the gold(I) halide complexes failed to
show peaks assignable to the [M]+ or [M-halide]+ fragments, but
intense peaks due to the [AuL2]+ ion; similar behaviour has been
reported previously.50
1
5
X-ray crystallography
Crystals of complexes 4-10 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
SMART51 and SAINT52 were used for data collection in φ- and ω-
scan modes and data processing, respectively, and absorption
corrections using SADABS.53 The structures were solved using direct
methods and refined with full-matrix least-squares methods
Fig. 12 Flow cytometric analysis of the apoptotic and necrotic cells
induced by the gold complexes 1, 5, 6 and 10. HeLa cells were
stained with Annexin V-FITC and PI after 48 hours of incubation
with the IC50 concentrations of the metal complexes (LL: live; LR:
early apoptotic; UR: late apoptotic; UL: necrotic).
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on F2 using the SHELX-TL package.54-55 The CCDC numbers for To a solution of [AuCl(tht)] (0.716 g, 2.23 mmol) in CH2Cl2 (30 mL),
complexes 4-10 are 1811850-1811856.
cooled to -20°C, was added P(C6H3-2,6-{OMe}2) (1.000 g, 2.24
mmol). The clear, colorless solution was stirred for 15 min, during
which time a white precipitate formed. After warming to room
temperature, the solid dissolved and the solution was filtered
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 (1.45 g, 96%). 1H
NMR: δ 7.29 (t, J = 8.3 Hz, 3H), 6.50 (dd, JHH = 8.3 Hz, JPH = 4.5 Hz,
6H), 3.56 (s, 18H). 31P NMR: δ -32.5 (s). MALDI-MS (m/z): 1081.38
[AuL2]+, 1313.34 [Au2L2Cl]+.
Preparation of [AuCl{P(C6H4-4-OMe)3}] (1)
To a solution of [AuCl(tht)] (0.91 g, 2.83 mmol) in CH2Cl2 (30 mL),
cooled to 0°C, was added P(C6H4-4-OMe)3 (1.00 g, 2.83 mmol). The
clear colorless solution was stirred for 30 min, filtered through
Celite and MeOH added to the filtrate. The volume of the solution
was reduced in vacuo, precipitating a white solid, which was filtered
off, washed with MeOH and dried in vacuo. 1H NMR: δ 7.41-7.50 (m,
6H), 6.95-7.00 (m, 6H), 3.87 (s, 9H). 31P NMR: δ 29.2 (s). MALDI-MS
(m/z): 901.28 [AuL2]+, 1133.26 [Au2L2Cl]+.
Preparation of [AuBr{P(C6H3-2,6-{OMe}2)3}] (7)
Preparation of [AuBr{P(C6H4-4-OMe)3}] (2)
To a solution of [AuCl{P(C6H3-2,6-{OMe}2)3}] (150 mg, 0.22 mmol) in
CH2Cl2 (30 mL) was added a solution of NaBr (35 mg, 0.34 mmol) in
MeOH (15 mL). After stirring the solution for 10 min, the solvent
was removed in vacuo and the residue was dissolved in CH2Cl2. The
mixture was filtered through Celite and MeOH was added to the
filtrate. The volume of the solution was reduced in vacuo,
precipitating a white solid which was filtered off, washed with
To a solution of [AuCl{P(C6H4-4-OMe)3}] (150 mg, 0.26 mmol) in
CH2Cl2 (10 mL) was added a solution of LiBr (35 mg, 0.40 mmol) in
MeOH (5 mL). After stirring the solution for 15 min, the solvent was
removed in vacuo and the residue was dissolved in CH2Cl2. Filtration
through Celite gave a clear solution, to which MeOH was added.
The volume of the solution was reduced in vacuo, precipitating a
white solid which was filtered off, washed with MeOH and dried in
vacuo (118 mg, 73%). 1H NMR: δ 7.42-7.49 (m, 6H), 6.96-6.99 (m,
6H), 3.86 (s, 9H). 31P NMR: δ 31.6 (s). MALDI-MS (m/z): 901.34
[AuL2]+, 1177.28 [Au2L2Br]+.
1
MeOH and dried in vacuo (153 mg, 96%). H NMR: δ 7.29 (t, J = 8.3
Hz, 3H), 6.51 (dd, JHH = 8.3 Hz, JPH = 4.5 Hz, 6H), 3.56 (s, 18H). 31P
NMR: δ -28.5 (s). MALDI-MS (m/z): 636.06 [M-Br]+, 1081.28 [AuL2]+,
1357.21 [Au2L2Br]+.
Preparation of [AuI{P(C6H3-2,6-{OMe}2)3}] (8)
Preparation of [AuI{P(C6H4-4-OMe)3}] (3)
Compound 8 was prepared analogously to the bromo complex
above from [AuCl{P(C6H3-2,6-{OMe}2)3}] (150 mg, 0.22 mmol) and
NaI (50 mg, 0.33 mmol) to give the product as a white solid (165
mg, 97%).1H NMR: δ 7.30 (t, J = 8.2 Hz, 1H), 6.51 (dd, JHH = 8.3 Hz,
JPH = 4.5 Hz, 2H), 3.56 (s, 6H). 31P NMR: δ -20.9 (s). MALDI-MS (m/z):
1081.34 [AuL2]+.
Compound 3 was prepared analogously to the bromo complex
above from [AuCl{P(C6H4-4-OMe)3}] (150 mg, 0.26 mmol) and NaI
(60 mg, 0.40 mmol) to give the product as a white solid (166 mg,
1
96%). H NMR: δ 7.43-7.50 (m, 6H), 6.97-7.00 (m, 6H), 3.86 (s, 9H).
31P NMR: δ 35.9 (s). MALDI-MS (m/z): 901.32 [AuL2]+, 1225.23
[Au2L2I]+.
Preparation of [AuCl3{P(C6H3-2,6-{OMe}2)3}] (9)
Preparation of [AuCl3{P(C6H4-4-OMe)3}] (4)
To a solution of [AuCl{P(C6H3-2,6-{OMe}2)3}] (400 mg, 0.6 mmol) in
CH2Cl2 (20 mL), cooled to -78°C, was added a solution of PhICl2 (165
mg, 0.6 mmol) in CH2Cl2 (10 mL). The colorless solution turned
yellow, the cold bath was removed, and the solution was left to stir
for 10 min. The solution was filtered through Celite and hexane was
added to the filtrate. The volume of the solution was reduced in
vacuo, precipitating an orange solid, which was filtered off, washed
with hexane and dried in vacuo (430 mg, 97%). 1H NMR: δ 7.39 (t, J
= 8.5 Hz, 3H), 6.4-6.6 (m, 6H), 3.94 (s, 6H), 3.62 (s, 6H), 3.49 (s, 6H).
31P NMR: δ 2.5. (s). ESI-MS (m/z): 709.0577 [M-Cl]+. Calcd for
C24H27AuCl2O6P: 709.0588
To a solution of [AuCl{P(C6H4-4-OMe)3}] (150 mg, 0.26 mmol) in
CH2Cl2 (10 mL) was added a solution of PhICl2 (74 mg, 0.27 mmol) in
CH2Cl2 (10 mL). The colorless solution turned yellow and was left to
stir for 10 min. The solvent was removed in vacuo and the residue
dissolved in CH2Cl2. After filtration through Celite, hexane was
added and the volume of the solution was reduced in vacuo,
precipitating a bright yellow solid, which was filtered off, washed
with hexane and dried in vacuo (162 mg, 96%). 1H NMR: δ 7.60-7.67
(m, 6H), 7.07-7.03 (m, 6H), 3.92 (s, 9H). 31P NMR: δ 41.3 (s).
Preparation of [Au{P(C6H4-4-OMe)3}2]PF6 (5)
Preparation of [Au{P(C6H3-2,6-{OMe}2)3}2]PF6 (10)
To a solution of [AuCl{P(C6H4-4-OMe)3}] (151 mg, 0.26 mmol) in
CH2Cl2 (10 mL) was added P(C6H4-4-OMe)3 (91 mg, 0.26 mmol) and
TlPF6 (95 mg, 0.27 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. The white
solid that precipitated was filtered off, washed with hexane and
To a solution of [AuCl{P(C6H3-2,6-{OMe}2)3}] (120 mg, 0.18 mmol) in
CH2Cl2 (20 mL) was added P(C6H3-2,6-{OMe}2) (79 mg, 0.18 mmol)
and TlPF6 (65 mg, 0.19 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. The
white solid that precipitated was filtered off, washed with hexane
and dried in vacuo (216 mg, 99%). 1H NMR: δ 7.33 (t, J = 8.3 Hz, 6H),
6.51 (dt, JHH = 8.3 Hz, JPH = 2.2 Hz, 12H), 3.28 (s, 36H). 31P NMR: δ -
22.0 (s), -144.3 (sept, JPF = 712 Hz). MALDI-MS (m/z): 1081.32 [M-
PF6]+.
1
dried in vacuo (265 mg, 98%). H NMR: δ 7.39-7.46 (m, 12H), 7.09-
7.12 (m, 12H), 3.90 (s, 18H). 31P NMR: δ 41.9 (s), -144.3 (sept, JPF
712 Hz). MALDI-MS (m/z): 901.33 [M-PF6]+.
=
Preparation of [AuCl{P(C6H3-2,6-{OMe}2)3}] (6)
8 | Dalton Trans., 2018, 00, 1-3
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Cell culture
ARTICLE
495 nm, em 515 nm). Propidium iodide would enter cells with
damaged membranes and undergo a 40-fold enhancement of
fluorescence after binding with nucleic acids, producing a bright red
fluorescence in dead cells (ex 535 nm, em 617 nm).
Ovarian (SKOV-3), prostate (PC-3), fibrosarcoma (HT1080),
glioblastoma (U87MG) and normal human embryonic kidney (HeK-
293T) cells were obtained from the American Type Culture
Collection (Rockville, MD, USA). Cervical (HeLa) cancer cells were Wound healing assay
generously provided by Professor Joseph Trapani (Peter MacCullum
HeLa cells (2×105/mL) were seeded in a 6 well plate in 2 mL
Cancer Centre, Melbourne). PC-3 and HeLa cells were grown in
Roswell Park Memorial Institute (RPMI-1640, GIBCO) media
whereas SKOV-3, HT1080, U87MG and Hek-293T were cultured in
Dulbecco's Modified Eagle's Medium (DMEM, GIBCO) media; in
both cases the media were supplemented with 1% penicillin-
streptomycin and 10% FBS (Foetal Bovine Serum). All cells were
maintained in an incubator humidified atmosphere containing 95%
air and 5% CO2 at 37 °C. 0.05% Trypsin-EDTA
(ethylenediaminetetraacetic acid) was used for harvesting cells for
subculture. For all the assays, stock solutions of the gold complexes
were prepared in DMSO (10 mM) and the final treatment
concentrations (0.01-100 µM) were made in complete growth
medium.
complete medium and incubated overnight. Wounds were created
across the well using a 200 µL pipette tip then washed with PBS
(phosphate buffered saline) to remove the cell debris. The cells
were incubated for a further 48 h either with or without the gold
phosphine complexes (IC50 concentrations) in complete growth
medium. The cells were observed under an inverted microscope at
4× magnification immediately (0 h) and after 24 and 48
h
treatment. The number of cells migrated into the wound area were
counted manually. The data reported in Fig. 5B are the mean ±
standard deviation from three independent experiments.
Effect on F-actin
HeLa cells (2×105/well) were seeded on cover slips in a 6 well plate
and allowed to adhere overnight. After 24 h treatment with the
gold complexes, the cells were washed with PBS (phosphate
buffered saline) and fixed with 4% paraformaldehyde. After fixation,
the cells were permeabilized with 0.01% Triton-X100 and incubated
with rhodamine phalloidin (Thermo Fisher) for 1 h at room
temperature. The excess dye was removed by washing with PBS
and the cells were further incubated with Hoechst 33242 (2 µg/mL)
for 10 min at room temperature to counterstain the nucleus. The
coverslips were then mounted with ProLong Gold anti-fade reagent
(Molecular probes), and the cells were observed under a confocal
microscope (Nikon). Images were captured using 20× objective lens.
MTT assay
The MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide} assay is based on the reduction of a yellow coloured MTT
solution to purple formazan crystals by the active mitochondria of
viable cells. Thus, the amount of produced formazan crystals is
proportional to the number of viable cells. In this assay, SKOV-3, PC-
3, HT1080, U87MG, HeLa cancer cells and normal HeK-293T cells
were seeded in a 96 well plate at a density depending on their
doubling times, and cells were incubated overnight at 37 °C. After
incubation, the cells were exposed to different concentrations (100,
10, 1, 0.1 and 0.01 µM) of the metal complexes 1-8 and 10 for 72 h.
1% DMSO in complete medium was used as a vehicle control. At the
end of the incubation, the medium containing metal complexes was
removed, and 100 µL of MTT solution (0.5 mg/mL in serum-free
medium) was added to each well and further incubated for 4 h in
the dark at 37 °C. The excess unreacted MTT was removed and the
formazan crystals were dissolved in 100 µL of DMSO. The
absorbances of the solutions were recorded at 570 nm using a
micro plate reader (SpectraMax, Molecular Device). Cell viability
was calculated as the percentage ratio of sample absorbance to the
vehicle treated control absorbance. The IC50 values were calculated
using Probit software [29]. The results are presented as means of
three independent experiments.
TrxR inhibition assay (Lipoate reduction assay)
This assay involves addition of DTNB [5,5′-dithiobis(2-nitrobenzoic
acid)] to the cells followed by lipoate (cell permeable cofactor)
addition and measuring the formation of newly reduced thiol. HeLa
cells (10000 cells/well) in a 96 well plate were seeded and allowed
to grow overnight. The cells were incubated with different
concentrations of the gold complexes (0.625, 1.25, 2.5, 5 and 10
µM) in complete growth medium for 6 h. Then, the medium
containing the gold complexes was removed from the each well and
replaced with 100 µL of HBSS (Hanks balanced salt solution)
containing 20 mM lipoate and 1 mM DTNB. The plates were
monitored immediately and every 30 min for
a change in
Spheroid inhibition assay
absorbance due to the reduction of DTNB at 405 nm over a period
of 180 min using a micro plate reader (SpectraMax, Molecular
Device).
HeLa cells (5000 cells/well) were seeded in 96 well ULA (ultra-low
attachment) round-bottom plates and allowed to grow spheroids
for 2 days. The spheroids of approximately 100 µm were treated
with IC50 concentrations of the gold complexes or cisplatin for a
period of 96 h. The culture media from the wells were refreshed
every two days without altering the drug concentration. The growth
and surface area of the spheroids were monitored using phase
contrast microscopy after 48 and 96 h treatment. To visualize the
live/dead cells after 72 h treatment with the complexes, 3D
spheroids were incubated with Calcein AM (2 µM) and propidium
iodide (4 µM) for 30 minutes and imaged directly using an inverted
fluorescence microscope. Live cells were distinguished by the
presence of ubiquitous intracellular esterase activity, as determined
by the enzymatic conversion of the virtually non-fluorescent cell-
permeant calcein AM to the intensely green fluorescent calcein (ex
Reactive Oxygen Species
HeLa cells (2×105/well) in a 6 well plate were cultivated under
standard conditions and were incubated with the gold complexes
for 48 h. After incubation, cells were harvested and resuspended in
1 mL of PBS buffer containing 10 µM CM-DCFDA (6-carboxy-2',7'-
dichlorodihydrofluorescein diacetate) at room temperature in the
dark for 15 min. The excess dye was removed by washing with PBS,
and the cells were immediately analyzed for green fluorescence
using a BD Accuri C6 flow cytometer.
Cell cycle
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HeLa cells were seeded into a 6 well plate at a density of 2×105 cells
per well and allowed to adhere overnight. The cells were incubated
with IC50 concentrations of the gold complexes in complete culture
medium for 48 h. After incubation, the cells were harvested using
0.25% trypsin-EDTA, washed with cold PBS and fixed with 70% cold
ethanol. The ethanol-fixed cells were further washed with PBS and
stained with propidium iodide staining buffer. Then, 10000 cells
from each sample were analysed for propidium iodide fluorescence
using a BD Accuri C6 flow cytometer.
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incubated with IC50 concentration of the complexes for 24 h were
harvested with 0.05% trypsin-EDTA and washed with PBS. The
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There are no conflicts to declare.
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10 | Dalton Trans., 2018, 00, 1-3
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DOI: 10.1039/C8DT01724G
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DOI: 10.1039/C8DT01724G
Gold(I) and Gold(III) Phosphine Complexes: Synthesis, Anticancer
Activities Towards 2D and 3D Cancer Models, and Apoptosis Inducing
Properties
T. Srinivasa Reddy, Steven H. Privér, Vijay V. Rao, Nedaossadat Mirzadeh,* Suresh K.
Bhargava*
Here in we report the synthesis of gold(I), gold(III) of tris(4-methoxyphenyl)phosphine and
tris(2,6-dimethoxyphenyl)phosphine and their anticancer activity towards 2D and 3D cancer
models.
Actin polymerization
Cell cycle arrest
inhibition
O2
Au1
P2
C8
P1
C15
C1
O1
O3
Nuclear fragmentation
Apoptosis