Antiproliferative activity exerted by tricyclohexylphosphanegold(I) n-mercaptobenzoate against MCF-7 and A2780 cell lines: the role of p53 signaling pathways

Article abstract of DOI:10.1007/s10534-020-00269-7

Based on the recent studies depicting the potential of heterometallic gold complexes as potent antiproliferative agents, herein we first reported the preliminary mechanistic data on the in-vitro antiproliferative activity of tricyclohexylphosphanegold(I)

Full text of DOI:10.1007/s10534-020-00269-7

Biometals
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Antiproliferative activity exerted
by tricyclohexylphosphanegold(I) n-mercaptobenzoate
against MCF-7 and A2780 cell lines: the role of p53 signaling
pathways
.
Kok Pian Ang Pit Foong Chan Roslida Abd Hamid
.
Received: 9 August 2020 / Accepted: 4 November 2020
Ó Springer Nature B.V. 2020
Abstract Based on the recent studies depicting the
potential of heterometallic gold complexes as potent
antiproliferative agents, herein we first reported the
preliminary mechanistic data on the in-vitro antipro-
liferative activity of tricyclohexylphosphanegold(I) n-
mercaptobenzoate, Cy3PAu(n-MBA) where n = 2 (1),
3 (2) and 4 (3), and MBA = mercaptobenzoic acid,
treated using MCF-7 breast cancer and A2780 ovarian
cancer cells, respectively. 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
was used to assess the cytotoxicity of both cancer cells
treated with 1–3, respectively. The IC50 of 1–3 were
applied to the subsequent assays including cell inva-
sion and thioredoxin reductase (TrxR) as well as
ubiquitin activities specifically on Lys48 and Lys63-
linked polyubiquitin chains via flowcytometric anal-
ysis. The mechanistic effect of 1–3 towards both cells
were evaluated on human p53 signaling gene expres-
sions via RT2 profiler Polymerase Chain Reductase
(PCR) array. 1–3 were found to be highly cytotoxic
towards both MCF-7 and A2780 cancer cell lines with
the compounds were more sensitive towards the latter
cells. 1–3 also suppressed TrxR and cell invasion
activities by modulating p53 related genes related with
proliferation, invasion and TrxR activities i.e. CCNB1,
TP53, CDK4 etc. 1–3 also regulated Lys48 and Lys63-
linked polyubiquitination by reactivation of p53,
suggesting the ability of this gene in regulating
inhibition of cytoskeletal reorganization via epithe-
lial–mesenchymal transition (EMT), required for
tumor progression. Taken together, the overall find-
ings denoted that 1–3 exerted potent antiproliferative
activity in MCF-7 and A2780 cells via activation of
the p53 signaling pathway.
Keywords p53 Á Gold (1) complexes Á Thioredoxin
reductase Á Cell invasion Á Antiproliferative Á MCF-7
Introduction
Concurrent global modernization leads to changes in
several habits such as dietary intake, sedentary
lifestyles and occupational exposure to heavy metals
caused several health problems. Amongst them,
cancer is one of the most major public health issues
and ranked the top three leading causes of death after
infectious diseases and cardiovascular diseases. Up to
date, there was a total of 14.1 million new cancer cases
have been reported and the mortality rate of cancer is
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10534-020-00269-7) con-
tains supplementary material, which is available to authorized
users.
K. P. Ang Á P. F. Chan Á R. A. Hamid (&)
Department of Biomedical Science, Faculty of Medicine
and Health Sciences, Universiti Putra Malaysia,
43400 Serdang, Selangor, Malaysia
e-mail: roslida@upm.edu.my
123

Biometals
about 9.1 million by the year 2012 (Ferlay et al. 2015).
Breast cancer ranked the first reported cancer cases
and cancer killer among women of all ethnic back-
grounds across the world. In Malaysia, breast cancer
affected status is about 1 in every 19 Malaysian
women who are diagnosed by the age of 85. Approx-
imately, 4000 women are diagnosed each year and it
commonly affects the women aged between 35 to 60
and 40% of the incident rate aged below 50 years old.
The risk factors of breast cancer including early
menarche or late menopause, late-stage at first full-
term pregnancy, high body mass index after meno-
pause and exposure to ionizing radiation (Almutlaq
et al. 2017). Simultaneously, ovarian cancer is the 5th
most common cancer amongst Malaysian women.
Statistically, about 500 women were diagnosed with
ovarian cancer every year in Malaysia. The known risk
factors of ovarian cancer including nulliparity, late
menopause, early menarche, use of infertility drugs e.
g. contraceptive pills and family history of breast or
ovarian cancer.
development efforts to (re-)activate p53 in established
tumors. (Stegh 2012). It is a tumor suppressor protein
that regulates the expression of a variety of genes,
including apoptosis, growth inhibition, cell cycle
progression inhibition, differentiation and accelera-
tion of DNA repair, genotoxicity, and aging after
cellular stress. The p53 pathway is composed of a
network of genes and their products that are targeted to
respond to a variety of intrinsic and extrinsic stress
signals that impact upon cellular homeostatic mech-
anisms that monitor DNA replication, chromosome
segregation and cell division (Vogelstein et al. 2000).
Activation of p53 can be induced by a number of stress
signals, including oxidative stress, activated oncoge-
nes and DNA damage (Harris and Levine 2005).
Whilst, the p53 protein is employed as a transcrip-
tional activator for p53-regulated genes, resulted in
cell cycle arrest, cell senescence and apoptosis (Jin
and Levine 2001).
Auranofin,
triethylphosphinegold(I)
tetraac-
etathioglucose, a gold-based anti-inflammatory com-
pound was serendipitously discovered for its potential
inhibitory activity on malignancies. The auranofin
discovery enabled researchers to explore and develop
more anti-cancer potential drugs utilizing gold com-
plexes. A hypothesis was made from the presence of
phosphine ligand on gold complexes which demon-
strated significant cytotoxicity on cancer cells (Trudu
et al. 2015; Tiekink 2002). Herein, we investigated
newly synthesized auranofin analogues, tricyclo-
hexylphosphanegold(I) n-mercaptobenzoate, Cy3-
PAu(n-MBA) where n = 2 (1), 3 (2) and 4 (3);
MBA = mercaptobenzoic acid, to assess their antipro-
liferative efficacy by determining their ability to
regulate cellular signaling cascades via p53-mediated
signaling pathway which includes gene expression
events, reorganization of cytoskeletal networks and
inhibition of metastasis by preventing cancer cells
invasion (Sui et al. 2015) through applying human
breast adenocarcinoma cell line (MCF-7) and human
ovarian carcinoma cell lines (A2780) as representative
in vitro cancer cells models. This is the first report on
the antiproliferative potential and its underlying
mechanisms of 1–3 against cisplatin resistant cancer
cells, MCF-7 and A2780 cell lines, respectively.
Several treatment options for cancer are usually
adopted and it includes radiotherapy, surgical
removal, and chemotherapy, with several promising
outcomes associated with various side effects (Sharma
et al. 2001). However, chemotherapy that usually
adopts antineoplastic drugs in killing rapid dividing
cancer cells and further suppresses their proliferation
is greatly utilized to increase the life expectancy of
cancer patients. Most of the antineoplastic-based
chemotherapy drugs are metal-based complexes and
some are probably made of platinum complex
employed for their anti-cancer properties. Cisplatin,
the conventional antineoplastic drug of this family is
one of the most widely used and the most effective
cytotoxic agent in the treatment of solid tumors such as
breast and ovarian cancers with the cure rate of
70–80% (Brezden et al. 2000; Donzelli et al. 2004;
Taguchi et al. 2005). However, chemotherapy using
cisplatin is highly limited in cancer patients due to the
development of resistance by tumor cells (Boulikas
and Vougiouka 2003).
It is ubiquitously known that p53 represents an
attractive target for the development of anticancer
therapies. It has been found most associated with
human tumors of all genes, motivated drug
123

Biometals
Materials and methods
Synthesis and characterisation
20 mL acetonitrile and the resulting mixture was
stirred for 3 h at 50 °C. The solution mixture was
extracted with dichloromethane and added with
equivolume of acetonitrile, which was left for slow
evaporation at room temperature, yielding crystals
after 2 weeks. Using the same procedures, compounds
2 and 3 were prepared and crystallised similarly.
All chemicals and solvents were used as purchased
without purification. All reactions were carried out
under ambient conditions. Elemental analyses were
performed on a Perkin Elmer PE 2400 CHN Elemental
Analyser (USA). Melting points were determined on a
Kru¨ss KSP1N melting point meter (Germany). 1H and
13C NMR spectra were recorded in CDCl3 solutions on
a Bruker Avance 400 MHz NMR spectrometer (USA)
with chemical shifts relative to tetramethylsilane.
31P{1H} NMR spectra were recorded in CDCl3
solution on the same instrument but with the chemical
shifts recorded relative to 85% aqueous H3PO4 as the
external reference; abbreviations for NMR assign-
ments: s, singlet; d, doublet; t, triplet; m, multiplet; br,
broad. IR spectra were obtained on a Perkin Elmer
Spectrum 400 FT Mid-IR/Far-IR spectrophotometer
(USA) from 4000 to 400 cm-1; abbreviations: s,
strong; br, broad. Powder X-ray diffraction (PXRD)
data were recorded with a PANalytical Empyrean
XRD system (UK) with Cu-Ja1 radiation
1
Yield: 0.281 g (89%) colourless crystal. H NMR
(400 MHz, CDCl3, 25 °C): d 13.47 (s, 1H, COOH),
8.31 (dd, 1H, H3, JHH = 7.90, 1.66 Hz), 7.64 (dd, 1H,
H6, JHH = 7.78, 1.22 Hz), 7.26 (td, 1H, H5, JHH-
= 7.49, 1.81 Hz), 7.17 (td, 1H, H4, JHH = 7.56,
1.24 Hz), 2.02–1.18 (m, br, 33H, Cy3P) ppm.
13C{1H} NMR (400 MHz, CDCl3, 25 °C): d 168.2
(COOH), 139.4 (C1), 137.3 (C6), 133.0 (C3), 131.2
(C5), 131.1 (C2), 125.3 (C4), 33.2 (d, i-PC6H11,
JCP = 28.20 Hz), 30.8 (s, o-PC6H11), 27.0 (d, m-
PC6H11, JCP = 12.01 Hz), 25.8 (s, p-PC6H11) ppm.
31P{1H}NMR (400 MHz, CDCl3, 25 °C): d 57.8 ppm.
Anal. Calc. for C25H38AuO2PS: C, 47.62; H, 6.07.
Found: C, 47.72; H, 6.22. IR: 2925 (br) m(O–H), 1716
(s) m(C=O). M.pt 174.0–175.0 °C.
Cy3PAu(3-mba) (2)
˚
(k = 1.54056 A) in the 2h range 5°–50°. The com-
1
Yield: 0.287 g (91%) pale yellow crystal. H NMR
parison between experimental and calculated (from
CIF’s) PXRD patterns were performed with X’Pert
HighScore Plus. Stability of 1–3 in DMSO were tested
using NMR spectroscopic method; 1–3 are stable up to
3 weeks with no visible changes observed from the
spectra.
(400 MHz, CDCl3, 25 °C): d unobserved (1H,
COOH), 8.37 (s, 1H, H2), 7.71 (d, br,1H, H4,
JHH = 7.80 Hz), 7.67 (d, br,1H, H6, JHH = 7.72 Hz),
7.17 (t, 1H, H5, JHH = 7.74 Hz), 2.11–1.24 (m, br,
33H, Cy3P) ppm. 13C{1H} NMR (400 MHz, CDCl3,
25 °C): d 171.7 (COOH), 144.0 (C1), 137.2 (C6),
133.7 (C2), 128.6 (C3), 128.0 (C5), 124.8 (C4), 33.4
(d, i-PC6H11, JCP = 27.66 Hz), 30.8 (s, o-PC6H11),
27.1 (d, m-PC6H11, JCP = 11.73 Hz), 25.9 (s, p-
PC6H11) ppm. 31P{1H}NMR (400 MHz, CDCl3,
25 °C): d 57.1 ppm. Anal. Calc. for C25H38AuO2PS:
C, 47.62; H, 6.07. Found: C, 47.23; H, 6.28. IR: 2925
(br) m(O–H), 1681 (s) m(C=O). M.pt 248.5–251.0 °C.
Synthesis of gold complexes
The preparation of 1–3 was similar, so that the
preparation of 1 is described in detail as a represen-
tative example. The Cy3PAuCl precursor employed in
the synthesis was prepared following standard proce-
dures by reducing KAuCl4 (Sigma-Aldrich, USA)
using one mole excess sodium sulfite (Merck, Ger-
many), followed by addition of one mole equivalent of
Cy3P (Merck, Germany).
Cy3PAu(4-mba) (3)
Yield: 0.293 g (93%) colourless crystal. 1H NMR
(400 MHz, CDCl3, 25 °C): d unobserved (1H,
COOH), 7.78 (d, br, 2H, H3 and H5, JHH = 8.48 Hz),
7.61 (d, br, 2H, H2 and H6, JHH = 8.48 Hz), 2.11–1.24
(m, br, 33H, Cy3P) ppm. 13C{1H} NMR (400 MHz,
CDCl3, 25 °C): d 171.0 (COOH), 153.3 (C4), 131.9
(C3), 129.6 (C2), 123.4 (C1), 33.4 (d, i-PC6H11,
Preparation of Cy3PAu(2-mba) (1)
NaOH (0.5 mmol, 0.020 g) in water (5 mL) was
added to a suspension of Cy3PAuCl (0.5 mmol,
0.256 g) in acetonitrile (20 mL), followed by addition
of 2-mercaptobenzoic acid (0.5 mmol, 0.077 g) in
123

Biometals
Cell lines
JCP = 27.64 Hz), 30.8 (s, o-PC6H11), 27.1 (d, m-
PC6H11, JCP = 11.85 Hz), 25.9 (s, p-PC6H11) ppm.
31P{1H}NMR (400 MHz, CDCl3, 25 °C): d 57.3 ppm.
Anal. Calc. for C25H38AuO2PS: C, 47.62; H, 6.07.
Found: C, 47.65; H, 6.26. IR: 2924 (br) m(O–H), 1670
(s) m(C=O). M.pt 283.5–285.0 °C (Fig. 1).
Breast adenocarcinoma cell line, (MCF-7) was pur-
chased from the American Type Culture Collection
(ATCC), USA [ATCC catalog No.: HTB-22]. Human
ovarian cancer cell line (A2780) was purchased from
the European Collection of Cell Cultures (ECACC),
UK [ECACC catalog No.: 93112519].
Materials
Cell culture and treatment
Roswell park memorial institute medium (RPMI-
1640) with high glucose, antibiotics with 10,000 U/mL
penicillin and 10,000 lg/mL streptomycin, fetal
bovine serum (FBS) were purchased from ScienCell
Research Laboratories, USA. 0.5% Trypsin–EDTA
(10 9) was obtained from Gibco, Life Technology,
UK. MatrigelTM Invasion Chamber is purchased from
BD Biosciences, USA. For the solvent, ethanol (95%
and absolute) was purchased from HmbG chemicals,
USA and dimethyl sulfoxide (DMSO) was purchased
from MERCK, Germany. All the other chemicals and
reagents used in this study were of analytical grade
unless otherwise stated.
The MCF-7 breast cancer cells and A2780 ovarian
cancer cells were cultured and maintained under
standard culture conditions: 37 °C in 5% CO2 incu-
bator. Both cell lines were cultured in RPMI-1640
media supplemented with 10% FBS and 1% antibi-
otics. In this study, the trial compounds, namely
Cy3PAu(2-mba) (1), Cy3PAu(3-mba) (2) and Cy3-
PAu(4-mba) (3) respectively, associated with appli-
cation of cisplatin as standard (positive) control. All
compounds were dissolved in dimethyl sulfoxide
(DMSO); the stock solution was prepared by dissolv-
ing the compounds in 1 mL of DMSO to obtain the
stock concentration of 80 mM (80 mM).
Fig. 1 Chemical structures of 1–3
123

Biometals
Cell proliferation assay
both types of cultured cells were treated with the
respective IC50 concentrations of compounds 1–3 for
24 h at 37 °C in the 5% CO2 incubator. The cells were
then harvested and collected, rinsed twice with PBS,
followed by addition of 1 mL of lysis buffer consists
of 10 mM Tris–HCL, 0.1 M EDTA, 5 g/mL SDS, pH
8.0 to obtain the cell lysate. The experiment was
conducted according to the instruction of manufac-
turer protocol (Mitochondrial thioredoxin reductase
assay kit, Sigma-Aldrich, USA) and lastly the activity
of the enzyme was measured at 412 nm by using
spectrophotometer (Thermo Scientific, USA).
Both MCF-7 and A2780 cancer cells were seeded at
the density of 1 9 105 cells/well in 96-well plate.
Compounds 1–3 were further diluted with RPMI-1640
media and added to the well (100 lL) to obtain the
final concentration of 0, 1, 5, 10, 20, 40, and 80 lM
with 0 lM served as negative control; followed by
incubation for 24 h at 37 °C in 5% CO2 incubator. The
cytotoxicity effects of 1–3 towards proliferation of
MCF-7 and A2780 cells were evaluated through the
MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenylte-
trazolium bromide) assay by referring to established
methods (Mosmann 1983). The MTT powder was
prepared by dissolving into 1 9 PBS and filtered
using 0.22 lm filter to make a concentration of 2 mg/
mL. The prepared MTT reagents were stored at 4 °C
and covered with aluminum foil to prevent from light
exposure.
Flow cytometry analysis on ubiquitin activities
(Lys48-linked and Lys63-linked
polyubiquitination)
The MCF-7 cells and A2780 cells were cultured in
6-well plate with the density of 1 9 106 cells/well,
followed by overnight-attachment, and application of
respective IC50 concentrations of compounds 1–3,
with negative control received no treatment. The
samples were then incubated for 24 h at 37 °C in 5%
CO2 incubator. After harvest, the cells were further
washed twice with ice cold PBS to remove remnants
and debris. The ubiquitin activities (Lys48-linked
polyubiquitination and Lys63-linked polyubiquitina-
tion) were analyzed via flow cytometer (FACSCal-
ibur, BD Biosciences, USA); activity of anti-ubiquitin
Lys48-FITC was measured at 495 nm meanwhile
activity of anti-ubiquitin Lys63-Alexa Fluor was
measured at 647 nm, by following the guideline
provided in manufacture’s protocols (Merck-Milli-
pore, USA).
After the incubation period, 20 lL of 2 mg/mL
MTT solution was added into each of the well by using
multi-channel pipette. The plate was incubated again
in CO2 incubator for 2 h in dark condition. After that,
the solution in the well plate was discarded by
aspiration, and thereafter 100 lL of DMSO was added
into each well to dissolve the purple formazan crystal
that formed at the well plate. The plate was shaken for
25 s to ensure formazan crystal had fully dissolved
and homogenized. The plate was analyzed by micro-
plate reader (BioTek EL808) at the wavelength of 570/
630 nm. By using the MTT assay, the number of
surviving cells after treatment of compounds 1–3 at
every time interval was expressed in percentage by
using the formula as below:
Percentage of cell viability ð%Þ
Cell invasion assay
Absorbance of treated cells
¼
 100
Absorbance of untreated cells
Cell invasion assay was carried out to investigate the
inhibitory activity of compounds 1–3 towards invasion
and metastasis of cancer cells, by using MatrigelTM
Invasion Chamber (BD Biosciences, USA) as the in-
vitro invasion model. The experiment was conducted
according to the attached manufacturer’s instruction.
Briefly, the inserts of the invasion chamber were pre-
coated with 8-lm pore size polycarbonate membrane
which give the close morphology to the endothelial
cells of blood vessels, hence it was a repre-
sentable model to study the inhibitory effect of
compounds towards the invasion and metastasis of
Graphs of various concentrations against cell
viability were plotted from the calculation obtained
both MCF-7 and A2780 cells, respectively. From the
curves illustrated, IC50 for the compounds against
MCF-7 and A2780 cells were respectively obtained.
Thioredoxin reductase (TrxR) assay
MCF-7 and A2780 cells were respectively cultured at
the cell density of 1 9 108 cells/well in 6-well plate
and allowed for attachment overnight. Afterwards,
123

Biometals
Results
cancer cells. The MCF-7 and A2780 cancer cells were
seeded at the density of 1 9 105 cells in 0.5 mL of
media per inserts, followed by addition of 0.5 mL of
media in respective well. The cells were then treated
with respective IC50 concentrations of 1–3 for 24 h.
After the incubation period, the cells were fixed (with
methanol) and stained with haematoxylin and eosin
(H&E) according to the guideline and the inserts was
observed under light microscope at the magnification
of 9 100.
Characterization of compounds 1–3 elucidated
by NMR
The compounds, Cy3PAu(n-MBA) with n = 2 (1), 3
(2) and 4 (3) were prepared in high yield following the
precedented procedures (de Vos et al. 1999). The
crystals obtained are well soluble in chlorinated
solvents and are stable against air, moisture and light.
The 1H NMR spectra of 1–3 measured in CDCl3
showed the expected resonances attributed to n-mer-
captobenzoic acid (MBA) and tricyclohexylphos-
phine, which confirmed the successful coordination
of the thiolate and phosphine ligands. The signals
correlated to aromatic protons of –SC6H4COOH
moiety appeared at downfield in the range of
8.37–7.17 ppm, opposed to the deshielded protons of
–P(C6H11)3 that were observed at 2.11–1.18 ppm. For
1 and 2, four characteristic resonances were observed
for aromatic protons while 3 gave rise to two pairs of
broad doublets with poorly resolved coupling pattern.
In the 1H spectrum of 1, a resonance corresponded to –
COOH was identified at 13.47 ppm but the respective
resonance was not observed in the case of 2 and 3. The
resonances ascribed to the respective carbon nuclei of
–SC6H4COOH and P(C6H11)3 were unambiguously
identified in the 13C{1H} spectra of 1–3; anticipated
splitting patterns were observed for carbon coupled
with phosphorous. In the 31P NMR, a single resonance
was detected at * 57.0 ppm, confirmed the unifor-
mity of the compounds prepared. To evaluate the
samples integrity of 1–3 in DMSO, time-dependence
1H NMR measurements were conducted in d6-DMSO
and the result confirmed that 1–3 stayed intact up to at
Human p53 signaling pathway via RT2 profiler
PCR array
The MCF-7 breast cancer cells and A2780 ovarian
cancer cells were seeded at the density of 1 9 108
cells per 25 cm2 culture flask and allowed for
attachment for overnight at 37 °C in 5% CO2 incuba-
tor. On the next day, the cells were treated with
respective IC50 concentration of compounds 1–3 and
further incubated for 24 h in standard culture condi-
tion. Prior to RNA extraction, the cells were harvested
and washed twice with ice cold PBS, the samples
firstly undergo DNA elimination followed by RNA
extraction according to the manufacturer’s instruction
(DNA elimination: RNase-Free DNase Set; RNA
extraction: RNeasyÒ Mini Kit; both from Qiagen,
USA). The RNA of samples was further converted into
cDNA by using the RT2 first strand kit (Qiagen, USA)
through the guideline provided accordingly. Lastly,
the mechanistic studies on gene regulation was
conducted via the RT2 Profiler PCR array (Human
p53 signalling pathway, PAHS-027Z). The gene
expression regulated by 1–3 towards MCF-7 and
A2780 cells were compared between treatment groups
to negative control according to threshold level (Ct
values).
1
least 3 weeks with no changes observed in the H
spectra. The infrared spectra of 1–3 displayed the
characteristic bands of m(O–H) and m(C=O) at approx-
imate 2900 and 1700 cm-1 respectively. Upon vary-
ing the position of –COOH at the benzene ring, a red
shift to lower wavenumber was observed, i.e.
1716 cm-1 to 1681 cm-1 and 1670 cm-1 when
n = 2, 3 and 4 respectively. ESI (Suppl 1) showed
that the powder X-ray patterns of 1–3 measured on
bulk materials are in close agreement with the patterns
calculated from the CIF files of single crystals.
Statistical analysis
Experimental data were expressed as mean stan-
dard deviation (SD). Group of data were compared
with one-way ANOVA with post-hoc Tukey multiple
comparison test for comparison. Statistical signifi-
cance was defined at p \ 0.05.
123

Biometals
Table 1 Half maximal (50%) inhibitory concentration of 1–3
towards two selected in-vitro cancer cell models: MCF-7 breast
cancer cell lines, and A2780 ovarian cancer cell lines, com-
pared with cisplatin
Compounds/cancer MCF-7 breast A2780 ovarian
cell lines cancer cells (lM) cancer cells (lM)
1
8.14
7.26
1.19
2.28
0.785
2
3
9.03
Cisplatin
30.53
26.8
The cytotoxicity activity of 1–3 towards both cancer cell lines
were measured in terms of inhibition of proliferation through
the MTT viability assay. The cells were incubated for 24 h in
5% CO2 incubator at 37 °C, and the respective IC50 was
calculated based on corresponding
x
and
y
values
(x = concentration applied) and (y = percentage of cell
viability) from the graph and data calculation software,
GraphPad Prism 5.0
Cytotoxicity of compounds 1–3
towards proliferation inhibition of MCF-7 cancer
cells and A2780 cancer cells
The two selected in-vitro cancer cell lines, MCF-7 and
A2780 were used as representative cancers in current
studies. The cytotoxicity of compounds 1–3 towards
both cancer cell lines were evaluated via the MTT
viability assay and the corresponding IC50 values were
found to be 8.14, 7.26 and 9.03 lM against MCF-7
breast cancer cell lines, and 1.19, 2.28 and 0.78 lM
against A2780 ovarian cancer cell lines, respectively
(Table 1).
Fig. 2 The inhibitory effects of 1–3 towards mitochondrial
thioredoxin reductase (TrxR) enzymatic activity of a MCF-7
breast cancer cell lines and b A2780 ovarian cancer cell lines for
24 h, respectively. Cisplatin was used as positive control.
Results with different superscript letters were statistically
different (p \ 0.005)
Compounds 1–3 inhibited mitochondrial
thioredoxin reductase activity in MCF-7
and A2780 cancer cells
compared to untreated cells (negative control) by
assuming full enzymatic activity of TrxR. Similarly,
when treated with cisplatin (standard, or positive
control), TrxR activity was also suppressed to 67.63%.
There was not much difference in terms of its
suppression rate amongst the tested compounds, with
a range from 37.3 to 40.3%. Whilst cisplatin exerted
slightly lower suppression rate (32.7%) than 1–3. In
contrast, the treatment with compounds 1–3 on A2780
cancer cells also exerted similar reducing pattern in
TrxR activity to 9.62%, 68.59% and 61.54%, respec-
tively (Fig. 2b). Whilst, cisplatin that served as
positive control suppressed the activity of TrxR to
42.31%. Compound 1 showed highest inhibitory rate
(90.38%) on A2780 cells and showed significant
In this experiment, activity of mammalian mitochon-
drial thioredoxin reductase (TrxR) was analyzed by
measuring the amount of 5,50dithiobis (nitrobenzoic
acid) (DTNB) reduced by TrxR enzyme; DTNB was
used as an alternative substrate of TrxR other than the
NADPH. In this study, all gold compounds 1–3
exhibited inhibitory effect on thioredoxin reductase
(TrxR) enzymatic activity in both MCF-7 (Fig. 2a)
and A2780 (Fig. 2b) cancer cell lines. As shown in
Fig. 2a, the percentage (%) of TrxR activity in MCF-7
cells treated with 1–3 was significantly attenuated to
62.70%, 52.14% and 59.70% respectively, as
123

Biometals
Compounds 1–3 suppressed cell invasion of MCF-
7 and A2780 cells
difference as compared to 2 and 3, including the
positive control cisplatin respectively.
In this study, compounds 1–3 inhibited cell invasions
on MCF-7 and A2780 cancer cells, respectively. As
illustrated in Fig. 4a–h, the invasion rate of MCF-7
cells treated by compounds 1–3 (Fig. 4c–e) through
matrigel membrane was significantly decreased to
9.29% 0.20, 7.41% 0.28 and 8.45% 0.24,
respectively, compared with negative control (nor-
malized to 100%) (Fig. 4a), whereas the positive
control (cisplatin), significantly decreased cell inva-
sion at the rate of 33.41% 1.28, shown in Fig. 4b.
There were no significant differences amongst the
compounds, yet the suppression by all compounds
were significantly higher than cisplatin.
Compounds 1–3 modulated both Lys48-
and Lys63-linked polyubiquitination
The ubiquitin activities (Lys48 and Lys63) were
measured via flow cytometer at 495 nm laser illumi-
nations. Addition of anti-ubiquitin antibodies (anti-
ubiquitin Lys48 and anti-ubiquitin Lys63) in the assay
was also analyzed for anti-ubiquitin Lys48-FITC and
anti-ubiquitin Lys63-Alexa Fluor at 647 nm. Prior to
the analysis, the forward scatter and side scatter were
established to exclude out cell aggregates and cell
debris. The activities were represented as percentage
of ubiquitin expression. Control was adjusted and
calibrated to the area within 101 to 102 of the
histograph, whereas untreated and treatment groups,
tagged with antibodies were expressed in area M2, if
they are greater than 102.
Likewise, similar inhibitory pattern on A2780 was
also observed when cells were treated with the gold
series 1–3, respectively as shown in Fig. 4f–j. For the
treatment groups, the invasion rate of A2780 cancer
cells
significantly
decreased
by
1–3
to
Promotion of Lys48-linked polyubiquitin and sup-
pression of Lys63-linked polyubiquitin by compounds
1–3 towards MCF-7 cancer cells were summarized in
Fig. 3A(a–e). Meanwhile, results of A2780 ovarian
cancer cells were summarized in Fig. 3B(a–e). As
illustrated in Fig. 2A(a–e), application of IC50 of
compounds 1–3 towards MCF-7 cancer cell lines for
24 h possessed higher Lys48-linked polyubiquitin
activities and relatively lower Lys63-linked polyubiq-
uitin activities. Exposure of MCF-7 cancer cells
towards treatment resulted in increased population of
detected Lys48-linked polyubiquitin (Fig. 3A(c–e))
compared to negative control (Fig. 3A(a)). Meanwhile
cisplatin showed less detection of Lys48-linked
polyubiquitin (Fig. 3A(b)) in contrast with treated
groups. In addition, application of treatments
(Fig. 3A(h–j)) including cisplatin-treated (Fig. 3(g))
exhibited lower detection of Lys63-linked
polyubiquitin.
36.21% 0.68, 48.52% 2.75 and 47.56% 1.99
(Fig. 4h–j) respectively. The positive control (cis-
platin) exhibited the cell invasion rate at
42.15% 2.13, (Fig. 4g) which gave almost similar
efficacy as 2 and 3, but not 1. All treatment groups in
both cancer cells, MCF-7 and A2780 cancer cells
significantly suppressed the cell invasion rate as
compared to negative control (normalized to 100%,
respectively (p \ 0.005). The graphical results of
cancer cells invasion studies were summarized in
Fig. 4k (for MCF-7 breast cancer cells) and Fig. 4l
(for A2780 ovarian cancer cells).
Compounds 1–3 regulated p53 related gene
expression in MCF-7 and A2780 cells
In order to analyze the ability of compounds 1–3 to
regulate the most common cellular signaling cascade
in both selected cancer cells, the human p53 signaling
pathway, consists of the key components of down-
stream targeted genes involved in cell proliferation
and cell cycle, apoptosis, organization of cytoskeletal
networks as well as cellular migration and metastasis.
However, in this study, we just focused on the related
genes which involved in the activities studied which
are cell proliferation, TRxR and ubiquitin activities as
well as cell invasion. Herein, treatment of compounds
1–3 towards MCF-7 cells (Table 2) induced
Whilst, when tested on A2780 ovarian cancer, the
candidate gold compounds 1–3 exhibited similar
activity as breast cancer cells, whereby higher
Lys48-linked polyubiquitin activities were detected
compared to Lys63-linked polyubiquitin (Fig. 3B(a–
e)).
123

Biometals
Fig. 3 A Histograms
depicting MCF-7 cells
stained using the anti-
ubiquitin Lys-48 specific,
FITC conjugate and
antiubiquitin Lys-63
specific, Alexa FluorÒ 647
conjugate upon treatment
with (a) negative control
(untreated cells),
corresponding IC50 values
of (b) cisplatin; (c) 1, (d) 2,
and (e) 3 for 24 h.
B Histograms depicting
A2780 cells stained using
the anti-ubiquitin Lys-48
specific, FITC conjugate and
antiubiquitin Lys-63
specific, Alexa FluorÒ 647
conjugate upon treatment
with (a) negative control
(untreated cells),
corresponding IC50 values
of (b) cisplatin; (c) 1, (d) 2,
and (e) 3 for 24 h
123

Biometals
Fig. 3 continued
123

Biometals
Fig. 4 Qualitative findings of cell invasion assay illustrated by
1–3 towards MCF-7 breast cancer cells, with a negative control,
b cisplatin, c 1, d 2, and e 3; followed by A2780 ovarian cancer
cells, with f negative control, g cisplatin, h 1, i 2, and j 3; The
graphical results of cell invasion were further amended in
k MCF-7 breast cancer cells and l A2780 ovarian cancer cells.
Results were mean of three independent experiment (n = 3);
each microscopic image is representative from ten random
microscopic field from the same Matrigel invasion chamber, and
the number of cells were calculated by using the cell counter.
[Magnification: 9 100]. Data with different superscript letters
were significantly different, (p \ 0.005)
expression of extrinsic apoptosis pathway genes
together with its upstream regulator, up-regulation of
TNF gene and down-regulation of FAS gene families.
Furthermore, many other important genes associated
with extrinsic apoptosis and p53 signal transduction
pathway was also found to be modulated (up-regulated
and down-regulated) upon treatment with compounds
1–3.
As 1–3 have shown high antiproliferative activities
towards MCF-7 cells, related tumor suppressor genes
123

Biometals
Table 2 p53 related gene
expression level upon
treatment with 1–3 towards
MCF-7 breast cancer cells,
compared to the negative
control group
Gene
Up/Down Regulation
Fold Regulation
(compared to control group)
2
1
8.39
20594.90
306.55
1.52
50.21
38.31
-11.63
-6.63
404.50
-83.28
2.62
3
APAF1
ATM
5.37
13.92
-11.23
-51.62
-127.11
10.70
-1.70
578.52
-1.00
ATR
BAI1
-1.05
BAX
86.59
BBC3
1.05
BCL2
-23.16
-1.67
-32.89
-1.70
BCL2A1
BID
2346.42
-3.34
46.52
-6.63
BIRC5
BRCA1
BRCA2
BTG2
-1.04
4.72
26.35
-1.07
9.57
2.05
3.29
1.04
-4.78
CASP2
CASP9
CCNB1
CCNE1
CCNG1
CCNH
CDC25A
CDC25C
CDK1
CDK4
CDKN1A
CDKN2A
CHEK1
CHEK2
CRADD
DNMT1
E2F1
3.47
69.07
326.28
-2.97
4.28
57.13
-82.14
30.91
-9.78
-1.23
-22.78
-3.41
-15.03
-46.53
2.28
-84.67
1717.67
-78.45
-5.67
2896.31
-2.16
-3.76
-7.38
-22.01
-12.73
-79.34
-259.57
187.40
8841.04
-1.06
-3.66
-2.92
-2.27
1.76
5.54
5428.13
-4.70
1.53
1.69
-1.03
13.42
-21.46
7.71
-1.57
-12.59
2.86
-16.38
-6.74
-126.57
-52.48
-2.16
-2.91
39.40
-3.29
2.41
23.75
-32.00
7.41
E2F3
10.56
-7.11
6.63
-1.13
EGFR
EGR1
-1.72
1.12
EI24
ESR1
-2.13
-1.45
-19.43
-15.03
-1.43
-1.22
-2.22
347.29
-1.06
2.62
-27.10
-16.68
-30.06
-11.24
-7.62
FADD
FAS
FASLG
FOXO3
GADD45A
GML
-9.74
-4.90
-30.27
-7.01
-61.82
-5.70
-2.33
-7.46
-97.01
-76.64
-5.58
-17.43
-4.70
HDAC1
HK2
-2.12
1GF1R
IL6
1.39
-4.36
-2.89
-3.07
-4.72
3.34
-1.67
JUN
-1.76
KAT2B
KRAS
-9.81
109.61
-5.22
724.08
-10.56
-23.10
-106.15
-40.79
-10.48
-4.50
MCL1
MDM2
MDM4
MLH1
MSH2
MYC
-1.82
-2.27
1.18
-2.46
-1.21
1.17
60.97
-1.24
-64.45
-10.78
-5.17
45.57
2.07
652.58
-3.48
-2.19
-1.80
-1.56
33.82
1.43
891.44
2.49
1.98
-4.30
-61.55
-3.56
-6.21
-2.82
MYOD1
NF1
NFKB1
PCNA
PIDD
13.61
-1.58
-1.56
-16.11
-73.52
-714.11
-177.29
-14.72
-5.90
-585.60
-43.83
-14.66
-1.14
PPM1D
PRC1
PRKCA
PTEN
PTTG1
RB1
RELA
PRPM
SESN2
SIAH1
SIRT1
STAT1
TADA3
TNF
-1.15
-2.96
4.92
-22.84
-2.27
-15.56
-98.36
-19.84
-18.51
321.79
-4.02
-5.18
-6.52
-6.65
-2.17
-16.26
1.26
24.25
-2.58
-1.31
-2.91
-1.65
3.65
-5.79
-14.82
5.93
8.49
Data represent means of
samples 1–3 induced fold-
change in gene expression,
relative to control treated
cells. (n = 3) p \ 0.05.
Red, up-regulated genes;
blue, down-regulated genes
and plain text, no
TNFRSF10B
TNFRSF10D
TP53
6.72
1.78
1.31
2.51
-1.27
-1.39
8.00
2.33
6.45
TP53AIP1
TP53BP2
TP63
349.70
103.25
-1.48
274.37
248.99
12.04
-132901.60
-1.29
3.40
765.36
2.01
12.61
-2.31
-3.34
1351.17
5.73
2.37
-2336.28
-10.41
TP73
223.82
3.96
TRAF2
TSC1
-3.43
-14342168.63
-6.20
WT1
XRCC5
detectable change
123

Biometals
directly/indirectly involved in MCF-7 cell prolifera-
tion were shown to be upregulated by 1–3 which are
p21 encoded genes, CDKN1A (28-, 1874.2-fold),
CDKN2A (5.54-, 5428.13 and 8841.04- fold). In
addition 1 and 2 also significantly upregulated ATM
by 20,594.90- and 578.52-fold, respectively. Whilst,
1–3 also downregulated CDK4 (- 46.63, - 2.27
and - 259.57 fold), CDK1 (- 15.03, - 2.92 and -
79.34 fold), CCNB1 (- 82.14, - 84.07 and - 2.97
fold), CCNG1 (- 9.78, - 78.45 and - 2.16 fold)
CDC25A (- 22.78, - 7.38 and - 22.01 fold), PCNA
(- 10.78, - 43.83 and - 714.11 fold), CDC25C
hand, BAX (50.21-, 86.59- and 10.70-fold), BID (404.50-,
2346.42- and 46.52-fold), CCNE1 (30.91-, 1717.67-,
2896.31- fold), E2F1 (2.41-, 7.71- and 7.41-fold), APAF-
1 (8.39-, 5.37- and 13.92-fold), KRAS (3.34-, 109.61-,
724.08-fold), TP53 (8-, 2.33 and 6.45-fold), TP53AIP1
(349.70-, 3.40- and 765.36-fold), TP53BP2 (103.25-,
12.61- and 2.01-fold), TP73 (274.37-, 223.83- and
1351.17-fold), TNF (3.65-, 8.49- and 5.93-fold), TRAF-
2 (249-, 3.96- and 5.74-fold) were significantly upregu-
lated by 1–3, and TSC1 by 1 and 3 (12.04- and 2.37-fold),
respectively.
Table 3 shows the p53 mediated gene expression
data on A2780 ovarian cells treated with 1–3, respec-
tively. 1–3 also exhibited high antiproliferative, TrxR
inhibition and anti-invasive activities which are con-
firmed by the downregulation of several related
oncogenes as follows; BCL2 (- 57.68, - 3.78 and -
10.41-fold), BCL2A1 (- 11.19, - 2.22 and - 9.00-
fold), BIRC5 (- 4.99, - 34.49 and - 10.27-fold),
BRCA1 (- 4.72, - 2.62 and - 18.10-fold), BRCA2
(- 2.85, - 4.11 and - 2.04-fold), CCNB1 (- 22.63,
- 10.56 and - 7.11-fold), CCNH (- 3.48, - 15.65
(- 3.41, - 3.66
(- 132,901.60, - 14,342,168.63
fold), respectively. Whilst, MYC, IGF1R and ESR1
were only downregulated by and by
and - 12.73
fold),
WT1
and - 2336.28
2
3
(- 2.82, - 4.50 fold), (- 4.36, - 7.46 fold) and
(- 6.74, - 16.68 fold), respectively.
On the other hand, it has been reported that TrxR
inhibitory directly modulated the cell cycle arrests as
well as apoptosis. Hence, there is a direct correlation
between TrxR activity with the modulation of cell
cycle and apoptotic-related genes (Selenius et al.
2012). This activity was significantly suppressed by 1–
3 and this was confirmed by modulation of several
genes directly/indirectly related with the TrxR sup-
pression/inhibition i.e. BCL2 (- 11.63, - 23.16
and - 32.89-fold), BID (404.50-, 2346.42- and
46.52-fold), CCNB1 (- 82.14, - 84.67 and - 2.97-
fold), CDK4 (- 46.53, - 2.27 and - 259.57-fold),
MDM2 (- 2.27, - 4.30, and - 23.10-fold) respec-
tively. Meanwhile, ESR1 was only modulated by 2 and
3 by - 6.74 and - 16.68-fold; PRC1 by 1 only
(- 5.90-fold).
and - 6.41-fold),
CDC25A
(- 17.98, - 13.70
and - 17.49-fold), CDC25C (- 67.65, - 40.12 and
- 50.21-fold), CHEK1 (- 69.07, - 5.86 and -
37.27-fold), CHEK2 (- 5.78, - 2.95 and - 5.86-
fold), EGFR (- 60.89, - 19.11 and - 22.29-fold),
GML (- 18.20, - 17.39 and - 8.46-fold), HK2
(- 25.96, - 3.86 and - 89.26-fold), NFKB1 (-
23.10, - 4.44 and - 15.67-fold), MDM4 (- 5.66,
- 5.13
and - 5.17-fold),
RELA
(- 10.63,
- 3.56 and - 6.50-fold) and TP63 (- 11.46,
- 11.54 and - 8.62-fold), respectively. Whilst, com-
pounds 1 and 3 were shown to down-regulate CDK1
(- 2.14 and - 4.23-fold), CDK4 (- 3.92 and -
6.28-fold) and MDM2 (- 2.51 and - 3.07-fold),
respectively.
Notwithstanding, all compounds were also shown to
suppress MCF-7 cancer cells invasion and this has been
confirmed with the downregulation of several genes
related with invasion i.e. BIRC5 (- 83.28, - 3.34
and - 6.63-fold), CDK4 (- 46.53, - 2.27 and -
259.57 fold), PCNA (- 10.78, - 43.83, - 714.11-
fold), NFKB1 (- 64.45, - 585.60 and - 73.52-fold),
respectively. Whilst, CCNH (- 5.67, - 3.76-fold),
MYC (- 2.82, - 4.50-fold), RELA (- 6.52 and -
18.51-fold) and TP63 (- 2.31 and - 3.34-fold) were
significantly downregulated by 2 and 3; JUN by 1 and 3
(- 3.07 and - 76.64-fold); EGFR by 1 and 2
(- 7.11, - 12.59 fold); PRKCA by 1 (- 2.96-fold);
STAT1 by 2 (- 2.91 fold), respectively. On the other
On the contrary, 1–3 significantly upregulated
APAF1 (3.73-, 62.25- and 35.26-fold), CASP9
(28.44-, 5.94- and 53.82-fold), CDKN1A (14.20-,
2.00- and 13.91-fold), FAS (29.45-, 17.10- and
18.00-fold), FASLG (66.72-,6.65- and 2.16-fold),
JUN (3.29-, 8.75- and 4.69-fold), KRAS (8.49-, 2.69-
and 25.81-fold), RB1 (41.36-, 2.03- and 55.72-fold),
TP53 (4.32-, 57.68- and 11.79-fold),TP53AIP1 (3.78-,
6.09- and 5.84-fold), TP53BP2 (5.89-, 2.17- and 6.58-
fold), TP73 (3.20-, 8.39- and 6.96-fold) and TSC1
(5.46-,4.74- and 8.57-fold), respectively. In addition,
123

Biometals
Table 3 p53 dependent
gene expression level upon
treatment with 1–3 towards
A2780 ovarian cancer cells,
compared to the negative
control group
Gene
Up/Down Regulation
Fold Regulation
(compared to untreated control)
2
1
3
APAF1
ATM
3.73
62.25
-1.05
-3.71
-1.51
1.72
22.94
-3.78
-2.22
1.78
35.26
33.36
58.49
-35.75
28.25
-1.51
-10.41
-9.00
29.45
-10.27
-18.10
-2.04
-2.97
2.35
20.39
5.21
ATR
BAI1
-65.80
10.34
-1.45
-57.68
-11.19
41.36
-4.99
-4.72
-2.85
-7.57
1.56
BAX
BBC3
BCL2
BCL2A1
BID
BIRC5
BRCA1
BRCA2
BTG2
-34.49
-2.62
-4.11
12.82
2.13
CASP2
CASP9
CCNB1
CCNE1
CCNG1
CCNH
CDC25A
CDC25C
CDK1
CDK4
CDKN1A
CDKN2A
CHEK1
CHEK2
CRADD
DNMT1
E2F1
28.44
-22.63
-5.39
-1.73
-3.48
-17.98
-67.65
-2.14
-3.92
14.20
-1.01
-69.07
-5.78
-23.26
14.03
9.32
14.83
-60.89
-3.29
-2.23
-3.05
13.74
29.45
66.72
12.55
-4.66
-18.20
16.45
-25.96
4.17
5.94
53.82
-7.11
-3.01
-1.07
-6.41
-17.49
-50.21
-1.75
-1.30
13.91
-1.21
-37.27
-5.86
-6.36
25.28
8.11
-10.56
-1.04
-27.86
-15.65
-13.70
-40.12
-4.23
-6.28
2.00
-1.62
-5.86
-2.95
-7.26
11.54
45.76
2.53
-19.11
2.87
-5.96
2.98
8.75
E2F3
2.89
EGFR
EGR1
-22.29
-2.64
-1.41
-1.14
10.06
18.00
2.16
EI24
ESR1
FADD
FAS
17.10
6.65
FASLG
FOXO3
GADD45A
GML
-12.38
-1.27
-17.39
58.89
-3.86
1.10
-17.01
8.75
5.28
2.69
14.93
-1.92
-8.46
29.04
-89.26
4.03
-3.33
4.69
4.69
25.81
-1.80
-1.38
-5.17
-6.13
1.17
HDAC1
HK2
1GF1R
IL6
-4.13
3.29
JUN
KAT2B
KRAS
1.77
8.49
MCL1
MDM2
MDM4
MLH1
MSH2
MYC
3.16
-23.10
-3.07
-5.13
-4.12
53.45
-4.16
16.56
22.94
-4.44
-14.52
-38.48
3.16
17.63
-68.12
16.34
-9.88
2.03
-3.56
-7.15
9.99
-12.19
-47.50
-2.48
-5.39
-16.00
2.60
-2.51
-5.66
-4.25
-1.32
1.79
1.61
1.05
MYOD1
NF1
NFKB1
PCNA
PIDD
8.10
-2.60
-23.10
-1.74
-13.07
-5.43
-1.22
-54.57
1.66
-1.06
-15.67
-1.54
-71.01
-3.01
1.62
PPM1D
PRC1
PRKCA
PTEN
PTTG1
RB1
RELA
PRPM
SESN2
SIAH1
SIRT1
STAT1
TADA3
TNF
-26.72
5.58
-2.25
41.36
-10.63
-8.49
-4.89
-45.89
-2.68
-2.30
3.29
-72.00
-2.46
-85.04
4.32
3.78
5.89
-11.46
3.20
29.45
5.46
-3.27
55.72
-6.50
-6.63
-5.28
-4.59
-1.93
-1.03
4.43
-17.36
-3.51
-55.33
11.79
5.84
6.58
-8.62
6.96
43.11
8.57
-9.96
2.04
Data represent means of
samples 1–3 induced fold-
change in gene expression,
relative to control treated
cells. (n = 3) p \ 0.05.
Red, up-regulated genes;
blue, down-regulated genes
and plain text, no
TNFRSF10B
TNFRSF10D
TP53
-9.38
57.68
6.09
2.17
-11.54
8.39
-1.62
4.74
3.71
1.78
TP53AIP1
TP53BP2
TP63
TP73
TRAF2
TSC1
WT1
-11.60
1.95
XRCC5
detectable changes
123

Biometals
BAX (10.34-, and 28.25-fold), BID (41.36- and 29.45-
fold), FOXO3 (12.55- and 14.93- fold) and TRAF2
(29.45- and 43.11- fold) were only up-regulated by 1
and 3 by and respectively. Whilst, PTEN (16.34- and
5.58-fold) and CASP2 (2.13- and 2.35-fold) were
significantly upregulated by 2 and 3; SESN2 (9.99-
fold) by 2 only.
triaza-5-phosphabicyclo[3.3.1]nonane
(Darensbourg et al. 2004).
(DAPTA)
It has been reported that phosphinegold(1) thiolates
were more cytotoxic than gold(I) thiolates, and that
phosphinegold(I) thiolates had more promising cyto-
toxicity profiles than their chloride analogues, indi-
cating the importance of both the phosphine and the
thiolate ligand for cytotoxic activity (Gandin et al.
2010). Therefore, here in we reported the cytotoxic
potential of phosphanegold(1) thiolate derivatives;
tricyclohexylphosphanegold(I) n-mercaptobenzoate,
Cy3PAu(n-mba) where n = 2 (1), 3 (2) and 4 (3),
against MCF-7 breast cancer cells and A2780 ovarian
cancer cells in order to understand their potential as
antiproliferative agent. Compounds 1–3 have been
shown to suppress the cell proliferation of cisplatin
sensitive cells lines, MCF-7 and A2780 cell lines,
respectively with low IC50 values in low lM range. In
a similar study by deVos et al. (2002), the similar
phosphanegold (1) complexes have also been previ-
ously tested for their IC50 using Sulfurhodamine B
(SRB) assay towards MCF-7 cells, which reported
lower range of ID50 values (411–973 ng/mL) and
comparable to cisplatin compared to the our current
findings. In addition, triethylphosphanegold(1)-n-
MBA and triphenylphosphanegold(1)-n-MBA were
also tested by the same authors and were shown to
exert higher ID50 compared to the former complexes
(de Vos et al. 2002). In another study by de Vos et al.
(2004), the cytotoxicity of tricyclohexyphosphane-
gold (1) with dithhiocarbamates and xanthates as
ligands were determined which yielded lower ID50 of
dithiocarbamates compared to xanthates, respectively.
Nevertheless, the hypothesis that either dithiocarba-
mates or xanthates can influence the cytotoxicity of
phosphanegold(1) was opposed when Jamaluddin
et al. (2013) reported that the dithiocarbamates in salt
form exhibited IC50 above 80 lM compared to the
ditthiocarbamates as ligand bound to triorganophos-
phanegold(1) which exerted IC50 ranging from 4.4 to
13.6 lM. Therefore, this pattern can also be implied to
our current complexes of 1–3, which can be concluded
that their higher cytotoxicities towards MCF-7 and
A2780 cell lines were mostly contributed to the
tricyclohexylphosphane ligand linearly bound to gold
(1). Furthermore, this is supported by Berner-Price
et al. (1990) who reported that the variation of anionic
ligand was shown to present little effect in cytotoxicity
and this is further supported by Gunatilleke and
Discussion
The synthesized gold(1) complex with bis-ligand i.e.
tricyclohexyl and n-mercaptobenzoic acid (n-MBA)
i.e. 2-MBA, 3-MBA and 4-MBA were successfully
synthesized. Initially, the phosphanegold(1) com-
plexes have been characterized by Tiekink and
workers in the mid-90 s and early 2000s (de Vos
et al. 1999,2002) and their compounds’ crystallization
have also been characterized previously (de Vos et al.
2002; Cookson and Tiekink 1992; Smyth et al. 2001).
In this current study, confirmation of the chemical
structures of 1–3 was characterized by 1H, 13C and 31
P
NMR spectra, respectively via both CDCl3 and d6-
DMSO solution as exhibited in the previous study by
Cookson and Tiekink (Cookson and Tiekink 1992).
They have reported elucidation of the compounds’
structures via 1H and 13C NMR obtained in d6-DMSO
solution were similar to those obtained in chlorinated
solvents suggesting no interaction between the com-
plex and the solvent. Ultimately, 31P NMR spectra of
the compound was shown to possess similar chemical
shifts in both CDCl3 and d6-DMSO, in which these
resonances are in the range expected for complexes of
this type (Cookson and Tiekink 1992), which justified
the implication of 31P NMR to characterize 1–3 in this
current study.
Additionally, DMSO was used to dissolve the
compounds in the biochemical/molecular studies.
Besides, DMF was also been used a stock solution
for gold(1) complexes of water soluble diphos-type
ligand in TrxR activities (Wetzel et al. 2011). Elie
et al. (2009) reported the use of PBS (NaCl 0.9%) to
dissolve the water soluble phosphanegold(1) com-
plexes to determine their cytotoxicities against Jurkat
cells. Other than that, PBS can also be used to dissolve
water-soluble gold(1) phosphine complexes such as
sulfonated arylphosphines (abbreviated TPPMS,
TPPDS, TPPTS), 1,3,5- triaza-7-phosphaadamantane
(TPA) (Daigle et al. 1974) or 3,7-diacetyl-1,3,7-
123

Biometals
Barrios (2006) whom stated that changes of thiolate
ligand only has little effect towards the activities
compared to variation of its phosphine ligand.
inhibition towards A2780 cells compared to other
compounds of 2 and 3. This interesting finding
somehow led us to question the role of mercaptoben-
zoic acid ligand in this experiment. Priorly, Smyth
et al. (2001) reported the polymorphism in 1, which
consists of four distinct forms; three with rod motif
and one with ‘ball’ motif, during the crystallization.
Tiekink (2003) also hypothesized that 2-mercapto-
benzoate series bound to triorganophosphinegold(1)
exhibited interesting profiles due to its selectivity in
the cytotoxic profiles of 2-MBA series Structural wise,
we suspected that 2-MBA might probably play a
partial role in its TrxR inhibition, compared to its other
isomeric compound. According to Varghuese et al.
(2011), activity of drug might change abruptly from
one polymorph to another, which may cause problem
in pharmaceutical industries. In addition, an ideal
approach in understanding the structure–property
relationship in molecular solids could be provided by
the comparison between different polymorphs (Me-
undaeng et al. 2016). Nevertheless, this can only be
confirmed once we conducted the same experiment on
the compounds by adding the isomeric mercaptoben-
zoic ligands alone as controls.
It is well known that cancer constitutes a cell
autonomous genetic or epigenetic disease and hence
the therapeutic efficacy of any antineoplastic agent
will highly rely on their ability to influence the tumor-
host interaction leading to a balanced mounting of
immune response specific for the malignant cells and
its microniche (Zitvogel et al. 2013). Hence, under-
standing the activation status of key signaling path-
ways that underlies components that links immune
responses specific to tumor cells is highly required.
Recently, p53 tumor suppressor as a master regulatory
transcription factor (in cell cycle arrest, apoptosis,
senescence, and metabolism) has gained much focus
due to its vital role in tumor immunology and
homeostatic regulation of immune responses (Menen-
dez et al. 2013; Mun˜oz-Fontela et al. 2016). Here in
our study, we have investigated the ability of com-
pounds 1–3 to activate p53 signaling pathway and
status of p53 mediated genes that will influence tumor
regulation to exert anticancer activity in in vitro MCF-
7 breast cancer and A2780 cells ovarian cancer model
systems.
Phosphanegold(1) compounds can inhibit Trx by
binding to the active site cysteines and through the
oxidation of Trx(SH)2 to Trx(SS) (Lima and Rodri-
guez 2011). TrxR is endowed with a flexible C-termi-
nal extension containing a cysteine/selenocysteine
redox center that can easily interact with different and
chemically unrelated substrates and inhibitors (Nishi-
naka et al. 2001). In a study reported by Fricker
(2010), the ability of the gold(1) compound to inhibit
TrxR activity is due to its thiol-containing molecules,
which may suggest that the role of thiolate ligand in
this experiment. Thiol-containing molecular targets
include the redox enzymes thioredoxin reductase and
glutathione reductase, transcription factors, and cys-
teine proteases such as caspases and cathepsins
(Fricker 2010). Thiolates are known to act as ‘‘soft’
ligands forming covalent bonds with the ‘soft’ gold (1)
ion; corresponding selenolates are ‘‘softer’’ donor
ligands compared to thiolates and, consequently,
behave as better substrates for the gold(I) ion
To investigate the first possibility on the activation
status of p53, we have analyzed the status of thiore-
doxin reductase (TrxR) activity on MCF-7 cells and
A2780 cells upon treatment with 1–3. Tumor sup-
pressor p53 is a redox active transcription factor
whose activation is also mediated by oxidative status
of thioredoxin (Trx) (Liu et al. 2008) Thioredoxin
(Trx) is a dithiol-reducing enzyme that is induced by
ROS and/or oxidative Trx modulates/regulates the
activity of DNA-binding proteins, including p53, Jun/
Fos and NFjB. Our results showed that in both MCF-7
and A2780 cells, the activity of TrxR was highly
suppressed to about 40% compared to untreated
control cells, denoting the compound 1–3 treatment
favors accumulation of oxidized thioredoxin.
Among TrxR inhibitors, gold compounds are very
effective, acting at nanomolar levels, probably due to
the high affinity of gold towards chalcogenides (group
16 donors including S, Se and Te) which renders the
nucleophilic selenolate of reduced TrxR the prime
target site of modification by this metal (Gandin et al.
2010). In the current findings, comparable TrxR
inhibition was seen on MCF-7 cells treated with 1–3,
respectively. Nevertheless, 1 has shown greater TrxR
`
(Chaudiere and Tappel 1984). Therefore, apart from
the MBA and posphine ligands, the presence of
thiolate in our synthesized compounds should has also
been taken into account for its effect in inhibiting/sup-
pressing TrxR.
123

Biometals
In a recent study by Walther et al. (2020) on
carbene-gold(1) thiolate compounds, bound to dithio-
carbamate and D-glucopyranosy, NHC*-
On the other hand, in an ovarian cancer in vitro
model, A2780 cells upon treatment of 1–3, we
observed upregulation of TP53, TP53AIP1, TP53BP2,
TP73, E2F1, APAF1, CASP9 that favor apoptosis
respectively, whilst HK2, BCl-2, CDK-1, and CDK-4
which regulate anti-cancer activity and PCNA prolif-
erative marker were down-regulated and notably, RB1
(senescence associated) gene was up-regulated. These
may indicate that the gold compounds caused signif-
icant oxidative damage by favoring cell death via
apoptosis partially and caused induction of senescence
associated phenotype in partial (Qian and Chen 2010;
Rufini et al. 2013). These are evident from the
downregulation of anticancer genes in A2780 cells
which is distinct from MCF-7 cells. Our results
signified that all compounds exerted antiproliferative
activities in both MCF-7 and A2780 cells which may
be differed in terms of their mechanisms between both
cancer cells.
Polyubiquitination usually occurs at Lys48 and
Lys63 of ubiquitin. It has been well recognized that
while Lys48-linked polyubiquitination serves as a
universal recognition signal that targets proteins for
proteasomal degradation, Lys63-linked polyubiquiti-
nation acts primarily as regulatory rather than prote-
olytic signal (Nathan et al. 2013; Swatek and
Komander 2016). The role of p53 as a transcription
factor is highly achieved by regulating its protein
synthesis and degradation to maintain constant nuclear
concentration (Jain and Barton 2010). At the fulcrum
of this balance, an array of proteins ligates ubiquitin to
lysine residues of target proteins in order to achieve
post translational modification (PTM) and to regulate
its stability. Hammond-Martel et al. (2012) reported
that p53 interacts with array of E3s that targets p53 for
self-destruction; however, p53 also interacts with
distinct set of E3s that mediates its nuclear localization
and stability. For instance, E4F1, an atypical E3
promotes p53-dependent growth arrest probably via
activating cell cycle arrest genes, via ubiquitin lysine
48 chain linkages (Cam et al. 2006). In addition,
another class of ubiquitin lysine E3 favors monomeric
p53 degradation, thereby preventing its multimeriza-
tion required for its transcription activity (Laine et al.
2006). Thus, we determined the context of p53
functioning upon treatment with 1–3 in the present
study. We analysed the status of ubiquitin activities by
measuring the levels of Lys48-linked polyubiquitin
and Lys63-linked polyubiquitin in MCF-7 and A2780
AuSCSNMe2 and NHC*Au–S-GLUC exhibited sig-
nificant TrxR IC50 of 1.2 0.2 and 7.4 0.4 lM,
respectively. This indicated that the same GLUC
ligand in this compound, which also consists in
auranofin did not contribute much to the TrxR
inhibition activities. Marzo et al. (2020) supported
the findings by comparing the TrxR inhibition activity
of Auranofin, Et3PAuCl and Et3PAuI, respectively, to
yield with comparable IC50 of Auranofin and Et3-
PAuCl, but lower IC50 of Et3PAuCl, yet all the IC50
values were still fell in the nanomolar range.
In addition, the cellular TrxR is in mitochondria
plays role in transcription factors for proliferation and
apoptosis inhibition and is found to be over expressed
in breast, ovarian and colorectal tumors favoring drug
resistance (Sasada et al. 1996; Yokomizo et al. 1995;
Bhatia et al. 2016). In our study, reduced TrxR activity
upon treatment with 1–3 denotes that they regulate
cellular proliferation probably by modulating p53
activation and these compounds might directly inter-
act with mitochondria leading to ROS generation
(Muniyappa and Das 2008) and thereby inhibiting
TrxR activity which is also reported for similar gold
nanoparticles (Karatas¸ et al. 2009) and other gold(1)
analogues (Walther et al. 2020; Marzo et al. 2020).
Activation of p53 mediates transcription of array of
genes that regulates cell cycle arrest, apoptosis, or
DNA repair contextually depending upon cell type and
stress. Here in our study we performed qPCR analysis
in MCF-7 cells and A2780 cells upon treatment with
1–3 compounds to understand the role of p53 activa-
tion and their underlying mechanisms of action in
exerting antiproliferative action. In MCF-7 cells, we
observed up-regulation of TP53, TP53AIP1,
TP53BP2, TP73, BAX, APAF1, and TNF respectively,
whilst MDM2, BCL2, NFKB1, CDC25A and CDC25C
that regulate cell cycle activation, PCNA proliferative
marker and RB1 (senescence associated) were down-
regulated. Cumulatively, this denotes the test com-
pounds probably cause significant genome damage
favoring cell death via apoptosis and with concurrent
down-regulation of cell cycle regulators and activates
genes promoting cell death, notably TP53, TP53AIP1,
TP53BP2 and immune regulation BRCA1, BRCA2
etc., (Mun˜oz-Fontela et al. 2016).
123

Biometals
Conclusion
cancer cells respectively. Our results showed levels of
Lys48-linked polyubiquitin was about 92.73%,
89.60% and 90.26%, whilst the activities of Lys63-
linked polyubiquitination was found to be about
6.20%, 20.96% and 30.42%. Similarly, in the A2780
cells, the levels of Lys48-linked polyubiquitin was
96.72%, 98.98%, 98.74%, while Lys-63 activities was
about 31.10%, 40.60%, 9.74% respectively. Our
results depicted that the test compounds increased
the activities of ubiquitin Lys-48 whilst simultane-
ously suppressed Lys-63 in promoting p53 to interact
with chromatin, which led to transcriptional initiation
of cell cycle arrest genes thereby augmented growth
arrest. This postulates that 1–3 exerted potent antipro-
liferative activities probably via activating and main-
taining p53 function as well as preventing its nuclear
export and destabilization (Topisirovic et al. 2009;
Brooks and Gu 2011; Thiery 2002).
Taken all the findings together, candidate gold com-
pounds 1–3 could activate p53 by modulating p53
target genes expressions, which in turn inhibited cell
cycle progression, cytoskeletal reorganization and
tumor invasion on the respected MCF-7 and A2780
cells. The observed antiproliferative efficacy of the
compounds and their ability to induce p53 activation
can be directly associated to their ability to induce
oxidative stress via ROS generation, evidently shown
from reduced TrxR activity in both in vitro models.
Hence, these gold derived drug complexes 1–3 may
have potential to further develop as a better therapeu-
tic agent for cancer therapies.
Acknowledgements This research was funded by Ministry of
Higher Education, Malaysia, Grant Numbers UM.C/HIR-
MOHE/SC/03 and UM.C/HIR-MOHE/SC/12. We thanked
Universiti Malaya for providing the grants under High Impact
Research Schemes and collaborated with us by providing the
compounds and the funding. We also thanked Prof Dr Noor
Saadah Abd Rahman, the Deputy Vice Chancellor (Research
and Innovation) at the University of Malaya for giving us the
consent to publish the data
In support to our findings that the compounds 1–3
mediated cell cycle arrest and inhibited tumor growth
by activating p53, we performed cell invasion assay on
MCF-7 and A2780 cells, respectively. Herein, we
observed that both MCF-7 and A2780 cells exhibited
significant reduction of cell migration which was more
pronounced on MCF-7 cells (9.29% 0.2,
7.41% 0.28 and 8.45% 0.24), as compared to
A2780 cells (41.5% 1.4, 33.86% 1.24 and
30.78% 7.67). The observed difference in reduced
inhibitory cell invasion in A2780 cells could be due to
the increased expression of senescent associated genes
manifested by the compounds, accompanied by the
reduced expression of genes that regulate antiprolif-
erative activity, as priorly discussed. These com-
pounds can alter/regulate the processes of epithelial
mesenchymal transition (EMT) on MCF-7 and A2780
cells, which is crucial for cell migration and tumor
progression (Schaeffer et al. 2014). Furthermore, the
activation of p53 in our study may also favor inhibition
of EMT probably by inhibiting NFjB activation,
evidently manifested from reduced activity of ubiqui-
tin Lys63 in our study which is required for NFjB
activation. Notably, these data were also supported by
Sui et al. (2015) who evidently documented the
inhibition of cancer cell invasion by wild type 53 via
suppression of NFjB mediated activation of fascin, a
cytoskeletal protein, whilst p53 deletion augmented
cell invasion.
Author contributions Conceptualization, RAH; methodology,
RAH; validation, AKP, CPF and RAH; formal analysis, AKP
and CPF; investigation, AKP and CPF; data curation, AKP;
writing—original draft preparation, AKP; writing—review and
editing, RAH; visualization, AKP and CPF; supervision, RAH;
project administration, RAH. All authors have read and agreed
to the published version of the manuscript.
Compliance with ethical standards
Conflict of interest The authors declared no conflict of
interest.
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