The influence of R substituents in triphenylphosphinegold(I) carbonimidothioates, Ph3PAu[SC(OR) = NPh] (R = Me, et and iPr), upon in vitro cytotoxicity against the HT-29 colon cancer cell line and upon apoptotic pathways

Article abstract of DOI:10.1016/j.jinorgbio.2013.05.011

The Ph₃PAu[SC(OR) = NPh], R = Me (1), Et (2) and iPr (3), compounds are significantly cytotoxic to the HT-29 cancer cell line with 1 being the most active. Based on human apoptosis PCR-array analysis, caspase activities, DNA fragmentation, cell

Full text of DOI:10.1016/j.jinorgbio.2013.05.011

Journal of Inorganic Biochemistry 127 (2013) 24–38
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
The influence of R substituents in triphenylphosphinegold(I)
carbonimidothioates, Ph3PAu[SC(OR) = NPh] (R = Me, Et and iPr),
upon in vitro cytotoxicity against the HT-29 colon cancer cell line and
upon apoptotic pathways
b
a
Chien Ing Yeo a, Kah Kooi Ooi b, Abdah Md Akim b, , Kok Pian Ang , Zainal Abidin Fairuz ,
⁎
Siti Nadiah Binti Abdul Halim a, Seik Weng Ng a,c, Hoi-Ling Seng a, Edward R.T. Tiekink
a,⁎⁎
a
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra Malaysia, UPM Serdang, Selangor 43400, Malaysia
c
Chemistry Department, Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
The Ph3PAu[SC(OR) = NPh], R = Me (1), Et (2) and iPr (3), compounds are significantly cytotoxic to the
HT-29 cancer cell line with 1 being the most active. Based on human apoptosis PCR-array analysis, caspase
activities, DNA fragmentation, cell apoptotic assays, intracellular reactive oxygen species (ROS) measure-
ments and human topoisomerase I inhibition, induction of apoptosis is demonstrated and both the extrinsic
and intrinsic pathways of apoptosis have been shown to occur. Compound 1 activates the p73 gene, whereas
each of 2 and 3 activates the p53 gene. An additional apoptotic mechanism is exhibited by 2, that is, via the
JNK/MAP pathway.
Received 7 January 2013
Received in revised form 23 May 2013
Accepted 23 May 2013
Available online 28 May 2013
Keywords:
Phosphinegold(I) compounds
Carbonimidothioate
Thiolate
© 2013 Elsevier Inc. All rights reserved.
Apoptosis
Cancer
Cell cycle
1. Introduction
arthritis and undergoing chrysotherapy are at a lower risk of developing
cancer, and ii) anti-inflammatory drugs such as 6-mercaptopurine also
Chrysotherapy, the use of gold formulations in medicine, primarily
revolves around their use in the treatment of rheumatoid arthritis [1,2].
Gold compounds belong to the disease modifying anti-rheumatic drug
(DMARD) class of anti-arthritic agents and are known to halt the pro-
gression of the disease and to ease clinical manifestations. There are
two classes of gold drugs with the first comprising, typically, polymeric,
charged and water soluble gold(I) thiolates, with Myocrisin® (sodium
gold(I) thiomalate) being a prominent example, Fig. 1(a). By contrast,
the second class of compound is monomeric, neutral and lipophilic,
with Auranofin® (triethylphosphinegold(I) tetraacetylthioglucose)
being the only clinically employed example, Fig. 1(b).
While work on anti-arthritic activity of gold compounds continues
[3], most recent attention in developing molecular gold compounds
for therapeutics revolves around their potential as anti-tumour agents
[1,4–8]. Links between anti-arthritic and anti-cancer activities relate
to two clinical observations [9–11], namely i) patients with rheumatoid
display an anti-tumour activity. Early studies relating to anti-tumour
potential indicated that gold(I) thiolates were generally inactive
but that compounds related to Auranofin® demonstrated potential
and, accordingly, significant early work focussed on developing
phosphinegold(I) thiolates as anti-tumour agents [5]. While inves-
tigations continue with this class of compound [12,13], along with
organometallic gold(I) derivatives, i.e. carbenes [14–17] and alkynyl
species [18,19], it is likely that most recent attention is directed towards
developing gold(III) anti-tumour agents.
Motivated by the isoelectronic relationship between gold(III) and
platinum(II), and the well-known anti-cancer drugs containing the lat-
ter element [20], such as cisplatin (cis-diamminedichloroplatinum),
gold(III) compounds also attracted early attention but the investigated
derivatives were often reduced in vivo. More recently, with tailored li-
gand donor sets, gold(III) compounds, often featuring an Au\C bond,
proved stable in the reducing mammalian environment and demon-
strated exciting potential as anti-tumour drugs [21–26].
In the present contribution, building upon earlier work on
phosphinegold(I) thiolates, e.g. 6-mercaptopurinates [27], 2-
mercaptocarboxylates [28], and dithiocarbamates and xanthates [29], in-
vestigations of the cytotoxicity profiles against the colon cancer HT-29
⁎
Corresponding author.
⁎⁎ Corresponding author. Tel.: +60 3 7967 6775; fax: +60 3 7967 4193.
E-mail addresses: abdah@medic.upm.edu.my (A.M. Akim),
Edward.Tiekink@um.edu.my (E.R.T. Tiekink).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.05.011

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
25
was found to reduce the anti-tumour effects of chemotherapeutic drugs
[45]. Recently, multidrug resistance was found to be induced by adrena-
line in HT-29 cells by the upregulation of ABCB1 gene expression [44]. De-
spite the HT-29 cell line having numerous chromosomal aberrations, it
exhibits significant genomic stability during passage in culture [46]. Final-
ly, recent studies revealed that adrenaline could increase the cell prolifer-
ation and induce the cisplatin-resistance in HT-29 cells by the activation
of the NFκB pathway and subsequent induction of microRNA-55
(miR-155) [47], indicating that screening of novel agents is urgently
needed to combat this disease.
Herein, the determination of selected biological effects in human
HT-29 colon cells, resulting from the treatment of different gold com-
pounds, is reported. The highly transformed HT-29 colon adenocarci-
noma cell line as a model of late cancer stage was employed in the
present study. Parameters of proliferation and cell viability were de-
termined to assess time and concentration dependencies. To gain in-
sight into the mechanism of action of the investigated compounds,
studies of human apoptosis PCR-array analysis, caspase activities, DNA
fragmentation, cell apoptotic assays, intracellular reactive oxygen spe-
cies (ROS) measurements and human topoisomerase I inhibition tests
were performed.
Fig. 1. Chemical structures of gold-based DMARDS: (a) Myocrisin® and (b) Auranofin®.
cancer cell line, a cancer of particular relevance to Asian populations, of a
series of triphenylphosphinegold(I) carbonimidothioates are reported,
see Fig. 2 for chemical structures. A particular motivation for the present
study was the recently reported cytotoxicity, including against HT-29,
exhibited by related platinum(II) carbonimidothioates [30].
Colon carcinoma is the third leading cause of death from cancer
[31] and adding to the therapeutic challenge is the observation that
the occurrence of synchronous colorectal cancers has risen globally
[32]. While traditional chemotherapy has improved outcomes for pa-
tients with advanced colorectal cancer, the efficacy of these chemical
agents is still limited and chemo-resistance remains a major problem.
Thus, the elucidation of the underlying mechanisms associated with
the disease would improve the prognosis [33].
HT-29 cells derived from a human colon adenocarcinoma were
first established in culture by Fogh [34] and have been widely used
as a cultured epithelial cell model for the study of a variety of plasma
membrane receptors including aα-adrenergic receptors [35,36], insu-
lin receptors [37] and vaso-active intestinal peptides [38,39]. HT-29
cells share a common trait with other cancer cells in that glucose me-
tabolism, culminating in lactic acid production, is impressively high
[40]. A metabolic feature of HT-29 cells which may distinguish them
from other cancer cells is that the removal of glucose from the culture
media does not result in the death of all cells. Thus, a subpopulation of
HT-29 cells can be maintained in permanent culture without glucose
[41,42].
2. Experimental
2.1. Instrumentation
1H and 13C{1H} NMR spectra were recorded in CDCl3 solution at
25 °C on a Bruker Avance 400 MHz NMR spectrometer with chemical
shifts relative to tetramethylsilane as internal reference. 31P{1H} NMR
spectra were recorded in CDCl3 solution on the same instrument at
25 °C but the chemical shifts were recorded relative to 85% aqueous
H3PO4 as external reference; abbreviations for NMR assignments: s,
singlet; d, doublet; t, triplet; q, quartet; sept, septet; and m, multiplet.
Fig. 3 shows the numbering scheme adopted for the NMR spectra. IR
spectra were obtained as KBr pellets on a Perkin Elmer RX1 FTIR spec-
trophotometer. Elemental analyses were performed on a Perkin Elmer
PE 2400 CHN Elemental Analyser. Melting points were determined on
a Krüss KSP1N melting point apparatus.
2.2. Synthesis
All reactions were carried out under ambient conditions. All chemicals
and solvents were sourced commercially and used as received.
The human colon is particularly sensitive to stress because of the close
distribution of noradrenergic fibres to its basement lamina [43] and colo-
rectal cancer cells are known to express adrenergic receptors [44]. In re-
cent studies, mouse model experiments showed that psychological stress
2.2.1. Thiocarbamides
The thiocarbamides, ROC(=S)N(H)Ph, for R = Me, Et and iPr,
were prepared as described in the literature whereby the respective
alcohol was reacted with PhN = C = S [48]. Using this procedure
the following compounds were prepared.
2.2.1.1. MeOC(=S)N(H)C6H5. 1H NMR (400 MHz, CDCl3, 25 °C): δ 8.67
(s, br, 1H, NH), 7.37–7.17 (m, br, 5H, aryl-H), 4.14 (s, 3H, OCH3) ppm.
13C{1H} NMR (400 MHz, CDCl3): δ 188.7 (Cq), 137.0 (C1), 129.1 (C3),
125.6 (C2), 121.8 (C4), 58.8 (OCH3) ppm. Anal. Calc. for C8H9NOS: C,
Fig. 2. Chemical structures of the investigated triphenylphosphinegold(I)
carbonimidothioates.
Fig. 3. Numbering scheme employed the NMR spectroscopic study.

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C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
57.46; H, 5.42; N, 8.38%. Found: C, 57.48; H, 5.25; N, 8.33%. IR (KBr
disc, cm−1): 3184 (br) ν(N–H), 1448 (s) ν(C–N), 1207 (s) ν(C = S),
1059 (s) ν(C–O). M.pt: 93–94 °C.
JCP = 8.9 Hz), 129.6 (d, Cα, JCP = 226.4 Hz), 129.0 (d, Cγ, JCP =
45.8 Hz), 128.7 (C3), 122.2 (C4), 121.9 (C2), 70.4 (CH), 22.1
(CH3) ppm. 31P{1H}NMR (400 MHz, CDCl3): δ 37.9 ppm. Anal.
Calc. for C28H27AuNOPS: C, 51.46; H, 4.16; N, 2.14%. Found: C, 51.23;
H, 3.88; N, 2.24%. IR (KBr disc, cm−1): 1436 (s) ν(C = N), 1148 (s)
ν(C–O), 1099 (s) ν(C–S). M.pt: 139–142 °C.
2.2.1.2. EtOC(=S)N(H)C6H5. 1H NMR (400 MHz, CDCl3): δ 8.68 (s, br,
1H, NH), 7.36–7.17 (m, br, 5H, aryl-H), 4.63 (s, br, 2H, OCH2), 1.41
(t, 3H, CH3, J = 7.10 Hz) ppm. 13C{1H} NMR (400 MHz, CDCl3, 25 °C):
δ 188.7 (Cq), 137.2 (C1), 129.0 (C3), 125.4 (C2), 121.6 (C4), 68.8
(OCH2), 14.1 (CH3) ppm. Anal. Calc. for C9H11NOS: C, 59.64; H, 6.12;
N, 7.73%. Found: C, 59.49; H, 6.09; N, 7.73%. IR (KBr disc, cm−1): 3214
(br) ν(N–H), 1446 (s) ν(C–N), 1199 (s) ν(C = S), 1038 (s) ν(C–O).
M.pt: 66–68 °C.
2.3. X-ray crystallography
Crystals of 3 suitable for X-ray diffraction were obtained by slow
evaporation from its chloroform/hexane (2/1) solution held at room
temperature. Crystal data for C28H27AuNOPS: M = 653.50, T =
100(2) K, monoclinic, P21/n, a = 11.0687(3), b = 26.5500(6), c =
17.7763(5) Å, β = 104.368(3)°, V = 5060.6(2) Å3, Z = 8, Dx = 1.715,
F(000) = 2560, μ = 5.981 mm−1, no. of absorption corrected [50]
unique data (Bruker SMART APEX-II CCD [51] using Mo Kα radiation
so that θmax = 27.5°) = 11,595, no. of parameters = 599, R (8755
data with I ≥ 2σ(I)) = 0.039, wR (all data) = 0.078. The structure
was solved by direct-methods (SHELXS97 [52]) and refined (anisotropic
displacement parameters, H atoms in the riding model approxima-
tion and a weighting scheme w = 1 / [σ2(Fo2) + 0.022P] where
P = (F2o + 2F2c) / 3) with SHELXL97 on F2 [52]). The maximum and
minimum residual electron density peaks of 1.95 and 1.00 eÅ−3 were
located 1.00 and 0.60 Å from the Au1 and Au2 atoms, respectively.
CCDC deposition number: 908287. Figs. 4 and 5 were drawn with
ORTEP for Windows [53] and QMol [54], respectively. DIAMOND [55]
was employed for the crystal packing diagrams in the Supplementary
materials.
2.2.1.3. iPrOC(=S)N(H)C6H5. 1H NMR (400 MHz, CDCl3): δ 8.68 (s, br,
1H, NH), 7.41–7.15 (m, br, 5H, aryl-H), 5.66 (sept, 1H, OCH, J =
6.23 Hz), 1.41 (d, 6H, CH3, J = 6.24Hz) ppm. 13C{1H} NMR (400 MHz,
CDCl3, 25 °C): δ 187.9 (Cq), 137.2 (C1), 129.0 (C3), 125.3 (C2), 121.3
(C4), 77.2 (OCH), 21.7 (CH3) ppm. Anal. Calc. for C10H13NOS: C,
61.50; H, 6.71; N, 7.17%. Found: C, 61.89; H, 6.83; N, 7.28%. IR (KBr
disc, cm−1): 3169 (br) ν(N–H), 1450 (s) ν(C–N), 1206 (s) ν(C = S),
1090 (s) ν(C–O). M.pt: 75–77 °C.
2.2.2. Triphenylphosphinegold(I) carbonimidothioates
Ph3PAu[SC(OR) = NC6H5], for R = Me (1), Et (2) and iPr (3), were
obtained from the reaction of the Ph3PAuCl precursor (synthesized by
the reduction of KAuCl4 by sodium sulfite followed by the addition of
Ph3P) with one mole equivalent of ROC(=S)N(H)Ph in the presence
of base following a literature precedent [49].
2.2.2.1. Preparation of Ph3PAu[SC(OiPr) = NC6H5] (3). NaOH (0.5 mmol)
in MeOH (5 mL) was added to a suspension of Ph3PAuCl (0.5 mmol) in
MeOH (20 mL), followed by the addition of iPrOC(=S)NHC6H5 in
MeOH (20 mL). The resulting mixture was stirred for 3 h at 50 °C.
After cooling, an equal volume of dichloromethane was added and
the solution was left for slow evaporation at room temperature, yield-
ing colourless crystals after 2 weeks. Compounds 1 and 2 were pre-
pared and crystallised similarly.
2.4. Cell culture and inhibition of cell growth
Human HT-29 colon carcinoma cell line was obtained from ATCC:
The Global Bioresource Center and maintained in culture as described
by the provider. The cells were routinely grown in RPMI 1640 medi-
um containing 10% foetal calf serum (FCS) and antibiotics at 37 °C
and 6% CO2. For evaluation of growth inhibition tests, the cells were
seeded in 96-well plates (Techno Plastic Products, TPP, Plastik für
die Zellkultur, Switzerland) and grown for 24 h in complete medium.
The stock solutions of metal compounds were prepared by dissolving
the compounds in 1 mL of DMSO to reach a concentration of 10−2 M.
They were then diluted in RPMI medium and added to the wells
(100 μL) to obtain a final concentration ranging between 0 and 80 μM.
DMSO at comparable concentrations did not show any effects on cell
cytotoxicity. Stock solutions of the compounds were diluted directly
in culture medium to the required concentration and added to the cell
culture. After 24 h incubation at 37 °C, 20 μL of a solution of MTT
(3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) in PBS
(2 mg mL−1) was added to each well, and the plates were then incu-
bated for 2 h at 37 °C. The medium was then aspirated and DMSO
(100 μL) was added to dissolve the precipitate. The absorbance of
each well was measured at 580 nm using a 96-well microplate read-
er and compared to the values of control cells incubated without test
compound. The IC50 values for the inhibition of cell growth were
determined by fitting the plot of the percentage of surviving cells
against the drug concentration using a sigmoidal function (Origin
v7.5).
2.2.2.2. Ph3PAu[SC(OMe) = NC6H5] (1). Yield: 0.288 g (91%) colourless
crystals. 1H NMR (400 MHz, CDCl3): δ 7.53–7.42 (m, br, 15H, Ph3P),
7.06–6.69 (m, br, 5H, aryl-H), 3.91 (s, 3H, CH3) ppm. 13C{1H} NMR
(400 MHz, CDCl3): δ 164.6 (Cq), 151.1 (C1), 134.3 (d, Cβ, JCP
=
55.2 Hz), 131.6 (d, Cδ, JCP = 9.1 Hz), 129.5 (d, Cα, JCP = 225.9 Hz),
129.1 (d, Cγ, JCP = 45.8 Hz), 128.8 (C3), 122.5 (C4), 121.9 (C2), 55.3
(CH3) ppm. 31P{1H}NMR (400 MHz, CDCl3): δ 38.0 ppm. Anal. Calc.
for C26H23AuNOPS: C, 49.93; H, 3.71; N, 2.24%. Found: C, 49.66; H,
3.47; N, 2.22%. IR (KBr disc, cm−1): 1435 (s) ν(C = N), 1145 (s)
ν(C–O), 1099 (s) ν(C–S). M.pt: 142–144 °C.
2.2.2.3. Ph3PAu[SC(OEt) = NC6H5] (2). Yield: 0.284 g (88%) colourless
crystals. 1H NMR (400 MHz, CDCl3): δ 7.54–7.44 (m, br, 15H, Ph3P),
7.07–6.71 (m, br, 5H, aryl-H), 4.35 (q, 2H, CH2, J = 7.09 Hz), 1.34 (t, 3H,
CH3, J = 7.10 Hz) ppm. 13C{1H} NMR (400 MHz, CDCl3): δ 164.0
(Cq), 151.2 (C1), 134.3 (d, Cβ, JCP = 55.2 Hz), 131.6 (d, Cδ, JCP
=
9.0 Hz), 129.5 (d, Cα, JCP = 235.9 Hz), 129.1 (d, Cγ, JCP = 45.8 Hz),
128.8 (C3), 122.4 (C4), 121.9 (C2), 63.9 (CH2), 14.6 (CH3) ppm. 31P
{1H}NMR (400 MHz, CDCl3): δ 37.8 ppm. Anal. Calc. for C27H25AuNOPS:
C, 50.71; H, 3.94; N, 2.19%. Found: C, 50.45; H, 3.55; N, 2.19%. IR (KBr
disc, cm−1): 1438 (s) ν(C = N), 1131 (s) ν(C–O), 1100 (s) ν(C–S).
M.pt: 134–136 °C.
2.5. Extraction of RNA, and RT2 Profiler PCR microarray
Total RNA was extracted from cultured HT-29 cells using a high-
purity RNeasy Mini Kit (Qiagen, USA). The real-time PCR for micro-
array assay was performed using the RT2 Profiler PCR array (apoptosis,
human) according to the manufacturer's protocol (http://www.qiagen.
com/Products/Catalog/Assay-Technologies/Real-Time-PCR-and-RT-PCR-
Reagents/RT2-Profiler-PCR-Arrays?catno=PAHS-012Z#productdetails).
Gene expression was compared according to the CT value.
2.2.2.4. Ph3PAu[SC(OiPr) = NC6H5] (3). Yield: 0.297 g (90%) colourless
crystals. 1H NMR (400 MHz, CDCl3): δ 7.54–7.44 (m, br, 15H, Ph3P),
7.07–6.70 (m, br, 5H, aryl-H), 5.29 (sept, 1H, CH, J = 6.19 Hz), 1.33
(d, 6H, CH3, J = 6.20 Hz) ppm. 13C{1H} NMR (400 MHz, CDCl3): δ
163.1 (Cq), 151.4 (C1), 134.3 (d, Cβ, JCP = 55.3Hz), 131.6 (d, Cδ,

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
27
Fig. 4. Molecular structures of the two independent molecules comprising the crystallographic asymmetric unit in Ph3PAu[SC(OiPr) = NPh] (3). Displacement ellipsoids are shown
at the 50% probability level.
2.6. Caspase activity (caspases 3, 7, 8, 9 and 10)
Caspase activity was assayed by measuring the light intensity using a
kit (Caspase Assay, Milipore) and a luminometer (Perkin Elmer HTS
7000, France). Briefly, cells were cultured in 96-well plates in a final
volume of 200 μL following the manufacturer's protocols (caspase 3/7:
http://www.millipore.com/catalogue/item/apt423; 8: …/apt408; 9: …/
apt409; 10: …/apt174). Then 50 μL caspase reaction buffer was added
and incubated at room temperature for 1 h before measurement.
2.7. Membrane permeability study by AOPI staining
HT-29 cells at a concentration of 5 × 103 cells/well in 96 well
plates were treated with the IC50 concentration of each compound
(dissolved in 1% DMSO) and incubated for 24 h. Untreated cells
were included as a negative control. Treated cells were harvested
Fig. 5. Overlay diagram of the two independent molecules in Ph3PAu[SC(OiPr) = NPh]
(3). The black (darker) trace corresponds to the Au1-containing molecule. The images
have been aligned so that the ipso-carbon atoms of the phosphine are overlapped.
from the culture flask. 1× EDTA free-PBS was used to wash the cells

28
C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
twice before transferring to a microcentrifuge tube. The cells were
centrifuged at 1000 g for 10 min. Subsequently, the cells were suspended
in 100 μL 1× PBS. Then, a 5 mg/mL acridine orange (AO) (Sigma) and
propidium iodide (PI) (Sigma) mixture was added to the cells at 1:1
ratio for staining. This was followed by chilling on ice for 10 min. The mix-
ture (20 μL) was aliquoted onto a slide and covered with a cover slip and
viewed under an Olympus BX-51 fluorescence microscope. Images were
captured by an attached Olympus CMAD-2 camera. The mode of cell
death was then determined.
24 h. After that, 3 μL of 6× loading buffer was added to 20 μL of the
given reaction mixture and each electrophoresis was performed at
80 V for 90 min in TAE buffer, pH 8.1, using 1.5% agarose gel. After elec-
trophoresis, the agarose gel was stained with ethidium bromide (EB) so-
lution (0.5 μg/mL).
3. Results and discussion
3.1. Synthesis and spectroscopic characterization
2.8. Human topoisomerase I inhibition assay
The Ph3PAu[SC(OR) = NC6H5], R = Me (1), Et (2) and iPr (3), com-
pounds were prepared in high yields via the metathetical reaction of
Ph3PAuCl with the respective ROC(=S)N(H)Ph thiocarbamide. The
compounds are air- and light-stable crystalline solids. The NMR spectra
exhibited the anticipated characteristics with resonances being assigned
based on literature precedents [56]. It was thought worthwhile to mon-
itor the stability of 1–3 by a time-dependent 1H NMR study. It was ob-
served that the 1H NMR spectra of freshly prepared solutions of each of
1–3 were indistinguishable from the spectra measured after three days
and after one week. However, after one month standing evidence for dis-
sociation of the compounds was found through the appearance of reso-
nances due to the N–H proton of the original thiocarbamide. The IR
spectra of the compounds were noteworthy for the absence of a broad
absorption at ca 3200 cm−1 assigned to ν(N–H) in the thiocarbamide
molecules, and the red shift of ca 100 cm−1 to lower wavenumber of
the absorption due to ν(C = S) in the thiocarbamide consistent with
the reduction of the double bond character of this bond upon complexa-
tion in 1–3. The compounds are soluble in chlorinated solvents, partially
soluble in acetonitrile and DMSO, are sparingly soluble in alcohols, and
insoluble in water.
The human DNA topoisomerase I inhibitory activity was determined
by measuring the relaxation of supercoiled plasmid DNA pBR 322. Each
reaction mixture contained 10 mM Tris–HCl, pH 7.5, 100 mM NaCl,
1 mM phenylmethylsulfonyl fluoride, α-toluenesulfonyl fluoride, PMSF,
and 1 mM 2-mercaptoethanol, 0.25 μg plasmid DNA pBR 322, 1 unit of
human DNA topoisomerase I, and the test compound (dissolved in
dimethylformamide (DMF)) at a specified concentration. The total vol-
ume of each reaction mixture was 20 μL and these mixtures were pre-
pared on ice. Upon enzyme addition, reaction mixtures were incubated
at 37 °C for 30 min. The reactions were terminated by the addition of
2 μL of 10 % SDS, and then followed by 3 μL of dye solution comprising
0.02% bromophenol blue and 50% glycerol. SDS is required to denature
topoisomerase I, preventing further functional enzymatic activity. Each
mixture was applied to 1.2 % agarose gel and underwent electrophoresis
for 5 h at 33 V with running buffer of Tris-acetate EDTA, TAE. The gel was
stained, destained and photographed under UV light using a Syngene Bio
Imaging system and the digital image was viewed with Gene Flash
software.
2.9. Intracellular ROS measurements
3.2. Molecular structure
5-(and-6)-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate
(carboxy-H2DCFDA) (Sigma, USA) was used to detect intracellular
ROS according to the manufacturer's instructions. For ROS quantifica-
tion, cells were seeded in 96-well black plates (Greiner Bio-One, France)
and treated with the trial compound (dissolved in 1% DMSO) at the
indicated IC50 concentrations for 24 h. Afterwards, cells were washed
with PBS (phosphate buffeted saline) and incubated with 10 μM carboxy
H2DCFDA in DPBS (Dulbecco's phosphate buffeted saline) for 1 h. Cells
were then washed and fluorescence was measured by a plate reader
(Perkin Elmer, France) with an excitation wavelength of 485 nm and
an emission wavelength of 535 nm.
As illustrated in Fig. 4, two independent molecules comprise the
crystallographic asymmetric unit in 3 and selected geometric param-
eters for these are collected in Table 1. The gold atom is in an essentially
linear geometry defined by sulphur and phosphorus atoms. The Au\P
bond length is shorter than the Au\S bond length, and the angle
subtended at the gold atom deviates from 180° as discussed below. Com-
paring the S1\C1 and C1\N1 bond lengths of the carbonimidothioate
ligand with those found in the uncoordinated molecule [48], i.e.
1.6679(19) and 1.337(2) Å, respectively, confirms that the anion
is functioning as a thiolate ligand towards gold. There is a systematic
variation of angles around the quaternary C1/C11 atoms which follow
the expected trends with the widest angle involving the larger sulphur
atom and the doubly-bonded nitrogen atom, and the narrowest
angle involving the two singly-bonded atoms. The orientation of
the [−SC(OiPr) = NPh] anion places the O1/O2 atom in close prox-
imity to the Au1/Au2 atom, i.e. 3.106(3) and 2.909(3) Å, respective-
ly, and this is likely responsible for the observed deviations from
linearity.
The overlay diagram shown in Fig. 5 indicates significant differences
in molecular conformation for the independent molecules and some of
these are reflected in the geometric parameters. Most notably, the O1
atom is 0.2 Å further away from the Au1 atom that the corresponding
interaction between O11 and Au2, i.e. 3.106(3) cf. 2.909(3) Å. This fea-
ture explains the small elongation of the Au2\S2 bond, the greater de-
viation of the S2–Au2–P2 angle from linearity, and systematic variations
in the angles around the S2 and C11 atoms compared to the equivalent
parameters in the Au1-containing molecule. Further, there is a signifi-
cant conformational difference between the molecules which is best
quantified in the dihedral angle formed between the least-squares
plane through the three ipso-carbon atoms of the phosphine ligand
and that through the SC(=N)O chromophore. While this dihedral
angle is close to perpendicular in the Au2-containing molecule,
i.e. 87.66(18)°, it is somewhat splayed in the Au1-containing molecule,
2.10. Nucleolytic study
Reagents were used as supplied. The pBR 322, gene ruler 1 kb DNA
ladder, ethidium bromide, loading buffer, Tris, and agarose were
bought from BioSyn Tech (Fermentas). All solutions for DNA experi-
ments were prepared with ultra-pure water from an Elga PURELAB
ULTRA Bioscience water purification system with UV light accessory.
The Tris–NaCl buffer was prepared using ultra-pure water. The Tris–
NaCl buffer contained 5 mM Tris and 50 mM NaCl. The pH of the buffer
solution was adjusted to pH 7.2 by addition of concentrated hydrochloric
acid or sodium hydroxide. All stock solutions of buffers were autoclaved
at 121 °C. Agarose gel electrophoresis experiments were carried out on
supercoiled plasmid DNA pBR322 (4.4 kb) using a horizontal gel system.
For the cleavage studies, each 20 μL sample consisted of the compound
dissolved in a certain amount of 1% DMF, DNA, and the required volume
of additional buffer. All samples were incubated in the dark in an incuba-
tor at a temperature of 37 °C. Each reaction mixture was prepared
by adding: 1 μL of compound (vary stock concentration) to 0.5 μL
of supercoiled plasmid DNA pBR322 (0.5 μg/μL) and Tris–NaCl buffer
pH 7.2. T he resultant solution was made up to 20 μL with an additional
buffer solution. All the reaction mixtures were incubated at 37 °C for

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
29
Table 1
Table 2
Selected geometric parameters (Å, °) for 3.
Cytotoxic activities of 1–3 and cisplatin against HT-29
cell model after 24 h treatment.a
Compound
Parameter
IC50 (μM)
Au1–S1
Au1–P1
C1–S1
C1–N1
2.3136 (12)
2.2643 (12)
1.765 (6)
1.281 (6)
3.106 (3)
176.43 (5)
104.92 (18)
114.0 (3)
125.9 (4)
120.0 (5)
−163.4 (5)
14.6 (4)
2.3200 (13)
2.2632 (13)
1.762 (5)
1.268 (6)
2.909 (3)
173.12 (5)
100.78 (18)
112.8 (3)
126.7 (4)
120.6 (4)
−179.6 (5)
0.5 (4)
1
2
3
11.3
17.0
16.8
25.0
0.3
0.3
0.2
0.2
Au1…O1
Cisplatin
S1–Au1–P1
Au1–S1–C1
S1–C1–O1
S1–C1–N1
O1–C1–N1
Au1–S1–C1–N1
Au1–S1–C1–O1
Au2–S2
Au2–P2
C11–S2
C11–N2
Au2…O2
S2–Au2–P2
Au2–S2–C11
S2–C11–O2
S2–C11–N1
O2–C11–N2
Au2–S2–C11–N2
Au2–S2–C11–O2
a
Mean and standard deviation of at least two inde-
pendent experiments conducted in triplicate.
It has been reported that apoptotic cells exhibit increased plasma
membrane permeability to certain fluorescent dyes [60,61]. Acridine
orange (AO) is a membrane-permeable, monovalent, cationic dye,
which binds to nucleic acids. In cells, a low concentration of AO causes
a green fluorescence, while at high concentrations a red fluorescence
is apparent [62]. When viewed by fluorescence microscopy, cells with
an intact membrane appear to have a bright-green nucleus while apo-
ptotic cells exhibit a bright-green nucleus with condensation of chroma-
tin (as dense-green areas). On the other hand, propidium iodide (PI) is
impermeable to intact plasma membranes, but it easily penetrates the
plasma membrane of dead or dying cells and intercalates with DNA
or RNA producing a bright-red fluorescence [63]. Therefore, cells that
have lost membrane integrity will show red staining throughout the cy-
toplasm and a halo of green staining on the cell surface (plasma mem-
brane). Apoptosis can be characterized by nuclear condensation with
cytoplasm alteration and nuclear fragmentation [64]. The AO/PI staining
showed that majority of HT-29 cells underwent apoptosis after treat-
ment with 1–3. The morphological characteristics include condensation
of nuclear chromatin and cytoplasm, membrane blobbing and the forma-
tion of apoptotic bodies. The apoptotic bodies appear to be round or oval
masses of cytoplasm, and smaller than the size of the cell of origin. Inter-
estingly, some of the 2- and 3-treated cells exhibited enlargement of cell
volume and formation of multinucleated cells. Further, most of the cells
were clumped together in a few colonies (Fig. 7c, d); this phenomenon
was not apparent for the cells treated with 1. The control cells appeared
to be shiny, clear and healthy. A small number of necrotic cells with rup-
tured plasma membrane were observed (Fig. 7e). Finally, the number of
apoptotic cells for the 1–3-treated cells was significantly greater than for
the cisplatin-treated cells.
i.e. 75.78(16)°; similar trends are evident in the torsion angles listed in
Table 1. Minor differences in geometric parameters and conformation
notwithstanding, to a first approximation, the molecular structure de-
scribed here for 3, matches those reported previously for 1 [49] and 2 [57].
The most noteworthy feature of the crystal packing is the presence
of Au…π(arene) interactions occurring between the two molecules
comprising the asymmetric unit. Interactions of this type have been
recognised as being important in the supramolecular chemistry of
gold compounds [58,59] and in the present case, the distance between
the Au2 atom and the ring centroid of the C21–C26 phosphine-bound
phenyl ring is 3.85 Å with the angle of approach being 26.4° for symme-
try operation −1 + x, y, z. These interactions combine with a number
of edge-to-face C–H…π(arene) interactions to form supramolecular
layers in the ac-plane, see Fig. S1 and geometric details in the Supple-
mentary data. Connections between layers also involve edge-to-face
C–H…π(arene) interactions as detailed in Fig. S2.
3.5. Determination of mode of cell death (DNA fragmentation study)
3.3. Inhibition of HT-29 colon cancer cell growth
One of the hallmarks of the terminal stages of apoptosis is the frag-
mentation of chromosomal DNA that proceeds in a two-step manner:
the DNA is initially cleaved into 50–300 kb fragments and eventually
into oligonucleosomal pieces [65,66]. In this study, the confirmation of
apoptosis-induced cell death of HT-29 cells after exposure to 1–3 is
presented in Fig. 8 which shows DNA ladder fragmentation.
Compounds 1–3 were evaluated in vitro by testing for the inhibition of
cell proliferation activity against human colon cancer cell lines (HT-29),
using cisplatin as the positive control. The effects of the compounds on
the growth of the selected cell lines were evaluated after 24 h and the
obtained IC50 values are listed in Table 2. From these data it can be seen
that 1–3 each promoted significant inhibition of cell growth and potency
levels comparable or greater than exhibited by cisplatin. Compound 1 was
the most active among the series, exhibiting potency about twice that of
cisplatin. The substitution of the O-bound methyl group in 1 with ethyl
(2) and isopropyl (3) resulted in a reduction of potency, with 3 being
marginally more potent than 2. The viability of the HT-29 cells after treat-
ment with 1–3 at different concentrations is plotted in Fig. 6. Exposure to
increasing concentrations of 1–3 resulted in a significant dose-dependent
inhibition of cell growth. This cell mortality was triggered by apoptotic
processes characterized by chromatin condensation and DNA fragmenta-
tion, as discussed below.
3.6. Analysis of apoptotic pathway (PCR array study, caspase activity and
measurement of reactive oxygen species (ROS) production)
In general, the apoptotic process can be divided into two catego-
ries, i) the intrinsic mitochondrial-mediated pathway activated by
signals emanating from cellular damage sensors or other types of
severe cell stress, and ii) the extrinsic apoptotic pathway initiated by li-
gand engagement of cell surface receptors such as CD95, APO-1, FAs and
TNF receptors [67,68]. The first intrinsic pathway involves the release of
pro-apoptotic proteins from mitochondrial intermembrane space that
in turn activate caspase proteases. Other than that, proteins of the
BCL-2 family, which comprise pro-apoptotic (e.g. BAX, BAK, BNIP3) as
well as anti-apoptotic (e.g. CL2, BCL-xL) members, are major regulators
of the intrinsic pathway as they form homo- and hetero-oligomers
which act directly at the outer mitochondrial membrane. The ratio of
pro- to anti-apoptotic oligomers has been suggested to play an important
3.4. Membrane permeability study (AO/PI apoptotic cell study)
The evaluation of the membrane integrity of the HT-29 cells after
treatment with the respective IC50 dose of each compound was ac-
complished by staining with AO/PI as shown in Fig. 7.

30
C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
factors. The three different proposed apoptotic pathways induced by
1–3 are shown in Fig. 9.
Many cytotoxic metal complexes target DNA by binding it and in-
ducing cell death through disruption of the cell cycle or by strand
scission [75]. As such, studies on the aqueous chemistry of coordina-
tion compounds, their binding to DNA and their nucleolytic properties
are of fundamental importance. The effect of varying the concentration
of compounds 1–3 was investigated over the range 1–80 μM in
Tris–NaCl buffer (TN buffer), (5 mM Tris; 50 mM NaCl) at pH 7.2
with an incubation time of 24 h and an incubation temperature of
37 °C in the presence of 0.5 μg plasmid DNA, pBR322. Interestingly,
compounds 1–3 were found to induce DNA cleavage activity by
converting most of the supercoiled DNA to nicked and linear DNA
in increasing complex concentration from 1 to 80 μM (Fig. 10a–c, L3–8).
This indicates that each of 1–3 possesses a high tendency to cause DNA
damage.
Generally, all compounds possess a similar apoptotic pathway in
which p53/p73 is activated when DNA repair systems are overburdened
due to excessive DNA damage. The p53/p73 genes also act as an up-
stream inducer of apoptosis by activating the expression of genes
encoding either MMP-inducing proteins of the BCL-2 family, such as
BAX, BID, NOXA and PUMA, or other apoptotic and cell cycle regulators
[76–78]. The up-regulation of the p53/p73 genes indicates that 1–3
could induce DNA damage in HT-29 cells and is consistent with the
DNA fragmentation results discussed above (Fig. 8). Curiously, gene ex-
pression of p53 and its binding protein were found only in cases when
treatment was with 2 and 3. Compound 1 was found to trigger the gene
expression of p73 only, as seen by about a 3500-fold greater increase in
TP73 expression compared to the untreated control cells (Table 3).
The p73 gene is one of the two homologues of the tumour suppressor
p53 and, as a result of differential promoter usage and alternative splic-
ing, is expressed in different isoforms [79]. These isoforms contain a
transactivation domain at their amino terminal ends, collectively termed
TAp73 which, like p53, has long been known to induce programmed cell
death [80]. The induction of pro-apoptotic genes is likely to represent the
underlying mechanism in both intrinsic and extrinsic apoptosis. Funda-
mentally, the expression of TAp73 is activated by E2F-1, transcription
factor 1, and this appears to be required for E2F-1-induced apoptosis at
least in some assay systems [81–84], strongly suggesting that TAp73
can physiologically contribute to programmed cell death. Moreover,
the high expression of the BNIP3L gene, a cell death inducer, in 2- and
3-treatment (79.43- and 126.21-fold, respectively) is supportive evi-
dence for the activation of the p53 gene. Generally, p53 can directly
up-regulate the expression of BNIP3L, which is known to be highly in-
duced in wild-type p53-expressing cells, due, in part, to sequestering of
p53 and CBP (CREB-binding protein) to BNIP3L during hypoxia. In
hypoxia-exposed cells, apoptosis is reduced with functional p53 follow-
ing BNIP3L knockdown. Tumorigenicity of wild-type versus mutant
p53-expressing tumours in vivo is also promoted by BNIP3L knock-
down. Thus, BNIP3L, capable of attenuating tumorigenicity, mediates
p53-dependent apoptosis under hypoxia, which provides a novel un-
derstanding of p53 in tumour suppression [85].
Fig. 6. HT-29 cell viability after treatment of 1–3 at different concentrations (μM).
***P value = 0.005 in case.
role in determining cell death [69–71]. Although it is well established
that many organelles, such as mitochondria, endoplasmic reticulum,
Golgi apparatus, and lysosomes contribute to apoptosis, the majority of
research thus far has focused on the role of mitochondria [72].
Mitochondria play a pivotal role in apoptosis by coordinating
caspase activation through the release of apoptogenic factors such
as the cytochrome c [73]. In metal-induced apoptosis, it is thought
that the mitochondria are most pertinent in mediating apoptosis via
metal-induced reactive oxygen species [74]. Although divergent with
respect to induction, extrinsic and intrinsic apoptotic pathways tend
to converge at later stages.
In this study, investigations into the mechanisms of 1–3-induced
HT-29 cell death in vitro have been conducted. The results clearly indicate
that the gold compounds trigger different levels of anti/pro-apoptotic
gene expression. This work provides evidence that the cytotoxicity
of 1–3 is related to the induction of a p53/p73-dependent activation
of the mitochondrial pathway of apoptosis, demonstrating the anti-
cancer properties of 1–3. Interestingly, the apoptosis PCR array analysis
demonstrated that 1–3-induced apoptosis involved both intrinsic and
extrinsic pathways mediated by different kinds of apoptosis-inducing
The activation of p53/p73 genes leads to high expression of BAX
and BCL-2-associated X protein. In short, mitochondrial membrane
permeability is regulated through a family of proto-oncogenes. The
BCL-2 family of proto-oncogenes comprises anti-apoptotic (BCL-2) or
pro-apoptotic (BAD, BAX) [86]. Once activated, BAX is inserted into the
mitochondrial membrane and increases membrane permeability, there-
by promoting apoptosis [87]. The anti-apoptotic protein, BCL-2, inhibits
the ability of BAX to increase membrane potential [88]. In the treated
HT-29 cells, BCL-2 was down regulated (Table 3). The loss of mitochon-
drial transmembrane potential is likely to be the initial event leading to
apoptosis [89] and results in dissipation of the mitochondrial inner
membrane potential (ΔΨm), disruption of the outer mitochondrial
membrane and subsequent release of proteins such as the cytochrome
c from the intermembrane region [90].

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
31
Fig. 7. Images of AOPI staining of HT-29 cells after being treated with each compound at the IC50 dose: (a) treated with cisplatin, (b) 1, (c) 2, (d) 3 and (e) untreated cells (negative
control). The red colour is produced by the PI stain which penetrates the nuclear matter when the cell membrane integrity is disturbed. Cells with an intact membrane are stained
green. Apoptotic cells are stained green with nuclei stained red, and contain multiple yellow/green dots of condensed nuclei. Necrotic cells were stained bright-red due to the influx
of PI stain. In (a), the green arrow points to a healthy cell, the blue arrow points to an apoptotic cell with a fragmented nucleus and condensed chromatin, and the red arrow points
to a necrotic cell. Magnification = 100×.
Cytochrome c, which binds to apoptotic activating factor-1 (APAF-1),
was also significantly expressed in HT-29 cells treated with 1–3 (Table 3)
and is known to activate caspase-9 (Fig. 11; Tables 3 and 4) leading in
turn to the activation of caspase-3 (Fig. 12; Tables 3 and 4); caspase-3
is the most potent effector caspase with many substrates [91,92]. It
is thought that mitochondrial-induced apoptosis may also involve a
caspase-independent pathway. After an apoptotic stimulus, an increase
in mitochondrial membrane potential allows for the release of
apoptosis-inducing factor (AIF), which activates a DNase leading to
apoptosis [93,94].
An inhibitory effect on BCL-2 expression in HT-29 cells was shown
upon treatment with 1–3 complexes (Table 3). As mentioned above,
the BCL-2 family is crucial in determining cell fate in the apoptotic
pathway as they influence mitochondrial membrane polarization and

32
C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
Fig. 8. Gel images of the DNA fragmentation analysis. The HT-29 cells were cultured for
24 h in DMEM control media, in the presence of 11.3 μM of 1 (L2), 17.0 μM 2 (L4) and
16.8 μM 3 (L6). DNA was extracted from the cultures and DNA fragmentation was eval-
uated using electrophoresis with 2% agarose gel. L1, L3 and L5 are 1 kb DNA ladders.
Compounds 1–3 show the presence of fragmented ladders (indicated by arrows)
which suggest the cell death by apoptosis.
cleavage-mediated caspase activation, which are the ultimate effectors
in apoptosis signalling. Although little is known about the transcription-
al factors that control the expression of BCL-2-related proteins, NF-κB is
a known transcriptional factor involved in their regulation. Moreover,
diminished expression of BCL-2 has been observed in cell types under-
going apoptosis [93–95]. Over and above these considerations, the bal-
ance between BAX and BCL-2 is crucial to the sustenance of apoptosis in
the intrinsic pathway [96].
In the extrinsic pathway, apoptosis is mediated by death receptors.
As an example, FAS interacts with the FAS receptor, leading to an activa-
tion of caspase-8 (Fig. 13; Tables 3 and 4) and caspase-10 [97]. The
strong homology between caspase-8 and caspase-10 may suggest that
both enzymes share similar substrate specificity and biological function
[98]. Furthermore, active caspase-8 and caspase-10 could directly
Fig. 10. Electrophoresis results of incubating pBR322 (0.5 μg/μL) in the presence of (a) 1,
(b) 2, (c) 3 in 5 mM TN buffer (pH 7.2) with increasing concentration (1 μM-80 mM)
(L2, DNA alone; L3, 1 μM compound; L4, 5 μM compound; L5, 10 μM compound; L6,
20 μM compound; L7, 40 μM compound; L8, 80 μM compound; L1, 1 kb DNA ladder)
for 24 h in an oven incubator at 37 °C.
cleave and activate downstream effector proteases, such as caspase-3,
causing apoptosis [99,100]. The present study showed that treatment
of HT-29 cells with 1–3 increased FAS gene expression, Table 3. It is
Fig. 9. Proposed signalling pathway of apoptosis induced by 1, 2 and 3.

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
Table 3 (continued)
33
Table 3
Effects of 1–3 on apoptosis-related gene expression in HT29 cells.a
Up–down regulation
Fold regulation
Up–down regulation
Fold regulation
(compared to control group)
(compared to control group)
1
2
3
1
2
3
CD27
TNFRSF9
TNFSF10
CD70
TNFSF8
TP53
TP53BP2
TP73
TRADD
TRAF2
TRAF3
TRAF4
9.88
1.00
212.50
1.00
1.00
1.00
1.00
3487.55
85.11
1166.52
3.53
6.36
20.05
12.72
−1.00
47.68
20.52
9.33
−1.00
23.13
50.17
2.58
47.68
20.52
9.33
−1.00
6.36
20.05
12.72
−1.00
335.93
81.69
30.18
−1.00
ABL1
AKT1
APAF1
BAD
BAG1
BAG3
BAG4
BAK1
BAX
11.07
1.00
6.04
6.56
1.00
86.30
1.00
1.06
2748.95
1.00
−13.38
−2.72
−10.87
1.00
22.19
−1.00
2.77
3.20
83.59
12.37
−1.00
5.56
7.01
86.14
191.61
2.05
1.03
1583.24
−1.00
−13.40
−2.73
−10.89
−1.00
−1.00
−1.00
11.38
397.64
4.45
−1.03
3225.56
−1.00
−13.40
−2.73
−10.89
27.20
−1.00
−1.00
21.24
BCL10
BCL2
23.34
−1.00
BCL2A1
BCL2L1
BCL2L10
BCL2L11
BCL2L2
BCLAF1
BFAR
BID
BIK
NAIP
Bold-face: up-regulated genes; italic: down-regulated genes.
a
Data represent the mean of samples 1–3-induced fold-change in gene expression
relative to control-treated cells (n = 3), p b 0.05.
12.39
1.00
1.00
known that in response to DNA damage, p53 activates genes such as
BAX and FAS via an extrinsic pathway [101,102], Fig. 9. As mentioned
above p73 is an isoform of p53 and some of their target genes overlap
[103]. In turn, caspase-8, caspase-9, caspase-10 and caspase-3 were ac-
tivated with an increase in their enzymatic activities (Figs. 11–13;
Tables 4 and 5). Surprisingly, 1 and 3 showed a significant enzymatic ac-
tivity and gene expression level on caspase-10 (Tables 3 and 5) but, 2
did not. Caspase-10 is classified as an initiator caspase and it is reported
to play an important role in maintaining homeostasis and in the immune
response, especially through the apoptotic signal from FAS. Caspase-10 is
also involved in the elimination of the T-cells which react to self-antigen.
Therefore, the introduction of mutations in caspase-10 leads to a de-
crease in the sensitivity to FAS- or TRIL-mediated apoptosis, and causes
autoimmune diseases [104]. For example, human immunodeficiency
virus (HIV) appears to down-regulate the apoptosis induction pathway
by the suppression of caspase-10 to propagate themselves in the host
cells [105].
It is possible that caspase-8 and caspase-10 also could cleave the
BCL-2 family member Bid, a BH3 interacting domain death agonist, to
truncated BID (tBID), which binds to proapoptotic protein BAX, resulting
in ΔΨm disruption and the release of cytochrome c [98,106]. BID is
generally considered as a molecular linker bridging death receptor and
mitochondria pathways [107]. The 1–3 compounds also induced an
up-regulating effect on BID expression in HT-29 cells (Table 3), which
provides additional supporting evidence for the proposed apoptotic
pathways.
Only 2 was shown to induce an up-regulating effect on TNF (tumour
necrosis factor) and TNFR (tumour necrosis factor receptor) genes in
HT-29 cells with 40.19- and 83.02-fold enhancement, respectively,
compared with the untreated control cells (Table 3); 1 and 3 caused
down regulation of these genes under the same experimental condi-
tions. In addition to caspase activation, stimulated death receptors
often result in the activation of other signalling pathways that play a
significant role in apoptosis. Moreover, TNFR activation results in the
initiation of the JNK/MAP kinase pathway leading to the phosphoryla-
tion of pro- and anti-apoptotic proteins and regulation of gene expres-
sion. The JNK pathway is implicated in inhibiting the anti-apoptotic
factor BCL-2 and the phosphorylation of an essential transcription
factor, c-JUN [108]. In addition, p38 MAPK was shown to have
pro-apoptotic effects, whereas ERK (Extracellular Regulated Kinase)
was shown to have anti-apoptotic effects [109,110]. Thus, it is proposed
that 2 also induces apoptotic activity through a JNK/MAP kinase path-
way (Fig. 9).
−1.65
17.22
36.96
−23.68
−19.59
1.23
21.34
11.28
−23.72
−19.63
−2845.89
−1.00
−767.84
4.06
1.10
15.46
36.89
−23.54
−19.32
−2847.33
−1.00
−767.32
19.10
−1.00
−1.26
−1.00
126.21
−1.11
189.84
10.82
BIRC2
BIRC3
XIAP
BIRC6
BIRC8
BNIP1
BNIP2
BNIP3
BNIP3L
BRAF
NOD1
CARD6
CARD8
CASP1
CASP10
CASP14
CASP2
CASP3
CASP4
CASP5
CASP6
CASP7
CASP8
CASP9
CD40
CD40LG
CFLAR
CIDEA
CIDEB
CRADD
DAPK1
DFFA
−2840.64
1.00
−766.42
8.16
1.00
−1.26
1.00
−8.73
1.18
1.00
−1.00
−1.26
−1.00
79.43
2.34
369.30
6.04
6.05
284.31
97.32
284.31
12.59
97.32
27.96
1.00
1.00
1.00
1.00
112.34
112.57
−87.14
1.00
93.79
−1.45
1.00
149.91
1.00
−6.57
330.38
93.79
1.80
5.60
1.42
1.84
1.00
1.00
1.28
−1.44
1.00
71.41
−18.79
−6.34
−2.74
−1.20
1.00
1397.53
−1.00
−1.00
369.30
283.78
76.00
1.00
−1.00
27.96
27.46
283.79
100.36
−87.31
−1.00
47.46
1.20
−2.10
329.77
149.64
−6.58
414.53
47.46
3.14
−1.00
−1.80
−1.14
−1.00
1.89
582.18
−1.00
97.14
6.04
−1.00
112.56
−1.00
−1.00
1397.53
−1.00
212.33
221.76
−4.39
−1.00
1.89
−1.46
−1.30
19.86
71.28
−6.58
19.86
FADD
FAS
71.28
FASLG
GADD45A
HRK
IGF1R
LTA
LTBR
MCL1
NOL3
PYCARD
RIPK2
TNF
TNFRSF10A
TNFRSF10B
TNFRSF11B
TNFRSF1A
TNFRSF21
TNFRSF25
755.87
−1.00
−1.80
1.00
−1.00
47.68
20.52
6.48
10.52
335.93
−18.83
−6.36
−2.75
−1.21
−1.00
−1.36
−1.00
1.78
3.50
19.86
71.28
40.19
1.66
122.39
67.74
83.02
−1.36
−1.00
It was thought of interest to also determine whether the observed
cytotoxicity of 1–3 was correlated with the generation of ROS. The
production of cellular ROS (mainly H2O2) was analysed using the
−1.36
48.89
(continued on next page)

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C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
Fig. 11. In situ caspase-9 detection. The HT-29 cells were cultured for 24 h in (a) control media, (b) in the presence of 11.3 μM 1, (c) 17.0 μM 2 and (d) 16.8 μM 3. Caspase-9 (green)
was detected using the fluorescent caspase-9 substrate (FAM-LEHD-FMK). Observations: (a) little detection of caspase-8 is seen in control HT-29 cells, whereas (b), (c) and (d) show
robust caspase-9 detection.
fluorogenic freely permeable tracer DCFH-DA which is deacylated by
intracellular esterases to the non-fluorescent compound, DCFH, and
oxidized to the fluorescent compound, DCF, through the action of
peroxides in the presence of ROS such as hydroxyl radical and, espe-
cially, hydrogen peroxide [111,112]. In the presence of 1–3, cellular
ROS generation exceeded about 5-fold that of the negative control
(untreated cells), indicating that the trial compounds do induce sig-
nificant H2O2 generation (Fig. 14). Many reports have showed that
ROS generation may be either a cause or a consequence of the PTP
opening and mitochondrial alterations in apoptosis [113,114]. The
mitochondria are a source of ROS and, simultaneously, a target of exces-
sive ROS generation. Besides, excess ROS increases the mitochondrial
Table 4
Quantification of caspase-3/7, 8 and 9 expression in HT-29 cells cultured in the pres-
ence of 1–3.a
Absorbance
Caspase
Control
1
2
3
Caspase 3/7
Caspase 8
Caspase 9
76
75
71
0.3
0.2
0.2
19257
20558
19516
0.2
0.3
0.2
45059
56697
23390
0.3
0.2
0.4
17939
38479
42793
0.3
0.3
0.2
a
The green fluorescent signal apparent in Figs. 10–12 is a direct measure of the amount
of active caspases present in the cells at the time the reagent was added. Means and stan-
dard deviations are calculated based on at least two independent experiments conducted
in triplicate. Excitation wavelength = 490 nm and emission wavelength = 520 nm.

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
35
Fig. 12. In situ caspase-3/7 detection. The HT-29 cells were cultured for 24 h in (a) control media, (b) in the presence of 11.3 μM 1, (c) 17.0 μM 2 and (d) 16.8 μM 3. Caspase-3/7
(green) was detected using the fluorescent caspase-3/7 substrate (FAM-DEVD-FMK). Observations: (a) little detection of caspase-3/7 is seen in control HT-29 cells, whereas (b), (c) and
(d) show robust caspase-3/7 detection.
membrane permeability and damages the respiratory chain resulting in
increased ROS production [74]. The disruption in the mitochondrial
membrane causes the release of cytochrome c from the mitochondria,
which initiates events leading to apoptosis. Although the exact mecha-
nism is undefined, ROS are thought to play a role in TNFR and FAS
receptor-mediated apoptosis [115].
and chromosome segregation [116]. DNA topoisomerases are important
enzymes in the nucleus that modify the topological state of DNA through
the introduction of transient breaks in the phosphodiester backbone of
the DNA [117]. These enzymes have been identified as important targets
in cancer chemotherapy and microbial infections [118]. Topoisomerase I
inhibitors are proven to possess a wide range of anti-tumour activities
and are among the most widely used anti-cancer drugs clinically
[119–122]. Accordingly, it was thought worthwhile to evaluate the
potential of 1–3 to inhibit topo I.
3.7. Human topoisomerase I inhibition study
The investigation of small molecules with topoisomerase I (topo I)
has gained recent prominence due to its potential in the development
of anti-cancer agents. DNA topoisomerases are enzymes involved in
the supercoiling of DNA and play important roles in many aspects of
DNA processing including replication, transcription, recombination
In the DNA relaxation assay employed herein, one unit of human topo
I can completely convert all the supercoiled plasmid pBR322 (4.4 kb) to
fully relaxed topoisomer, which is the completely unwound covalently
bonded closed circular DNA (Fig. 15a–c, lane 4). This is found in the
slowest moving DNA band (labelled Form II) which contains the fully

36
C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
Fig. 13. In situ caspase-8 detection. The HT-29 cells were cultured for 24 h in (a) control media, (b) in the presence of 11.3 μM 1, (c) 17.0 μM 2 and (d) 16.8 μM 3. Caspase-8 (green)
was detected using the fluorescent caspase-8 substrate (FAM-LETD-FMK). Observations: (a) little detection of caspase-8 is seen in control HT-29 cells, whereas (b), (c) and (d) show
robust caspase-8 detection.
relaxed closed circular pBR322 and the originally present, small amount
of nicked DNA [122]; the difference in plasmid size may account for the
inability to separate the fully relaxed pBR322 from the nicked pBR322.
Incubating pBR322 with the highest concentrations of 1–3 (0.125 to
4.0 μM) resulted in no cleavage or unwinding of the DNA as the banding
pattern is the same as the control (Fig. 15a–c, lane 3). Compounds 2 and
3 exhibited a full inhibitory activity on topo I at the lowest concentration,
2 μM, and both compounds started to show topoisomers at 0.25 μM. By
contrast, 1 showed the lowest inhibitory capability on topo I at double
the concentration of other two compounds in achieving full inhibition
on topo I activity (Fig. 15a–c, lanes 6–11).
Table 5
Quantification of AFC generation in HT-29 cells cultured in the presence of 1–3 upon
cleavage of the AEVD-AFC substrate by caspase-10.a
Absorbance
Caspase
Control
84 0.3
1
2
3
Caspase 10
18,399
0.3
831
0.4
6471
0.2
a
AEVD-AFC emits blue light (λmax = 400 nm) upon cleavage of the substrate by
caspase-10, and free AFC emits a yellow-green fluorescence (λmax = 505 nm). Means
and standard deviations are calculated on the basis of at least two independent experi-
ments conducted in triplicate.
Fig. 14. Interaction of 1–3 with HT-29 cells and production of ROS after treatment at doses
corresponding to the IC50 value of 1–3 (11.3, 17.0 and 16.8 μM, respectively) for 16 h and
labelled with carboxy-H2DCFDA for 1 h, when the fluorescence was measured.

C.I. Yeo et al. / Journal of Inorganic Biochemistry 127 (2013) 24–38
37
Fig. 15. Human topoisomerase I inhibition assay by gel electrophoresis. Electrophoresis results of incubating human topoisomerase I (1 unit/21 μL) with pBR322 (0.25 μg) in the
absence or presence of 0.125–4.0 μM of 1 (a), 2 (b) and 3 (c). Lanes 1 & 5, gene ruler 1 Kb DNA ladder; lane 2, DNA alone; lane 3, DNA + 4.0 μM trial compound (control); lane 4,
DNA + 1 unit Human Topoisomerase I (control); lanes 6–11, DNA + X μM trial compound + 1 unit Human Topoisomerase I where X = 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 μM,
respectively.
Fig. 16. Effect of sequence of mixing in the human topoisomerase I inhibition assay. Electrophoresis results of incubating human topoisomerase I (1 unit/21 μL) with pBR322
(0.5 μg/μL) and 3 μM (a) 1, (b) 2 and (c) 3. Lane 1: 1 kb DNA ladder; lane 2: DNA alone; lane 3: DNA + compound alone; lane 4: DNA + 1 unit topo I; lane 5: simultaneous mixing
DNA + topo I + compound; lane 6: incubated topo I + compound for 30 min, then addition of DNA with further incubation for another 30 min; lane 7: incubated DNA + compound for
30 min, then addition of topo I and further incubation for another 30 min.
No inhibitory activity of topo I was observed in the presence of 1–3
alone as the DNA bands are the same as those observed for DNA incu-
bated only with topo I (data not shown). However, when pBR322 was
incubated with human topo I, increasing concentrations of 1–3 lead to
i) the reduction of the nicked band (containing nicked and fully relaxed
DNA), and ii) to the formation of various faster moving bands of
topoisomers with different degrees of relaxation. At 0.5 μM of 1–
3, unchanged supercoiled DNA band starts to appear (Fig. 15a–c,
lane 8). The appearance of slower moving bands of less relaxed
topoisomers was observed with increasing concentrations of 1–3
(Fig. 15a–c, lanes 6–11). These results showed that 1–3 are able to in-
hibit the function of topo I by relaxing the supercoiled pBR322, and
also show that the degree of inhibition is dependent on concentra-
tion. The above also indicates that 1–3 are topo I inhibitors and not
topo I poisons, which prevent the nicked DNA from relegation.
As a preliminary investigation into the mechanism of topo I inhibi-
tion, three variations of mixing the DNA, topo I and the gold com-
pound were explored. When incubating the topo I with each of 1–3
prior to the addition of DNA, there is extensive inhibition as a fastest
wide band appears and this comprises supercoiled DNA and poorly
relaxed DNA (Fig. 16a–c, lane 6). The light bands of topoisomers
with different degrees of relaxation can be observed in Fig. 16a–c.
When the DNA was first incubated with the gold compound for
30 min before the addition of topo I or when the three components
are mixed simultaneously, the fastest moving band is wider, thereby
appearing to be nearer to the slowest moving band (Form II). These
observed differences in inhibition of topo I suggest initial binding of
the respective gold compound to either the topo I or the DNA to
give rise to differences in the mode of action. Further investigations
in mechanisms of topo I inhibition by 1–3 are underway.
4. Conclusions
Three Ph3PAu[SC(OR) = NPh], R = Me (1), Et (2) and iPr (3) ex-
hibit significant cytotoxicity to the HT-29 cancer cell line with 1 being
the most active of the series. The compounds uniformly induce apo-
ptosis and the possible mechanisms have been elucidated: both ex-
trinsic and intrinsic pathways have been demonstrated. Compound
1 activates the p73 gene, whereas each of 2 and 3 activates p53. Com-
pound 2 causes apoptosis by an additional mechanism, namely the
JNK/MAP pathway.
Acknowledgements
The authors thank the Ministry of Higher Education (Malaysia) for
funding studies in metal-based drugs through the High-Impact Re-
search scheme (UM.C/HIR-MOHE/SC/12). Further support from the
University of Malaya (RG125/10AFR) is also gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2013.05.011.
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