A bismuth diethyldithiocarbamate compound promotes apoptosis in HepG2 carcinoma, cell cycle arrest and inhibits cell invasion through modulation of the NF-κB activation pathway

Article abstract of DOI:10.1016/j.jinorgbio.2013.09.018

The compound with R = CH₂CH₃ in Bi(S ₂CNR₂)₃ (1) is highly cytotoxic against a range of human carcinoma, whereas that with R = CH₂CH₂OH (2) is considerably less so. Both 1 and 2 induce apoptosis in HepG2 cells with some evidence for necrosis induced by 2. Based on DNA fragmentation, caspase activities and human apoptosis PCR-array analysis, both the extrinsic and intrinsic pathways of apoptosis have been shown to occur. While both compounds activate mitochondrial and FAS apoptotic pathways, compound 1 was also found to induce another death receptor-dependent pathway by induction of CD40, CD40L and TNF-R1 (p55). Further, 1 highly expressed DAPK1, a tumour suppressor, with concomitant down-regulation of XIAP and NF-κB. Cell cycle arrest at the S and G₂/M phases correlates with the inhibition of the growth of HepG2 cells. The cell invasion rate of 2 is 10-fold higher than that of 1, a finding correlated with the down-regulation of survivin and XIAP expression by 1. Compounds 1 and 2 interact with DNA through different binding motifs with 1 interacting with AT- or TA-specific sites followed by inhibition of restriction enzyme digestion; 2 did not interfere with any of the studied restriction enzymes.

Full text of DOI:10.1016/j.jinorgbio.2013.09.018

Journal of Inorganic Biochemistry 130 (2014) 38–51
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
A bismuth diethyldithiocarbamate compound promotes apoptosis in
HepG2 carcinoma, cell cycle arrest and inhibits cell invasion through
modulation of the NF-κB activation pathway
a
b
b
b,
b
Dayang Hazwani Abang Ishak , Kah Kooi Ooi , Kok-Pian Ang , Abdah Md Akim ⁎, Yoke-Kqueen Cheah ,
c
a
a
a,
Norshariza Nordin , Siti Nadiah Binti Abdul Halim , Hoi-Ling Seng , Edward R.T. Tiekink ⁎⁎
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2013
Received in revised form 26 September 2013
Accepted 30 September 2013
Available online 10 October 2013
The compound with R = CH CH3 in Bi(S CNR ) (1) is highly cytotoxic against a range of human carcinoma,
2
2
2 3
2 2
whereas that with R = CH CH OH (2) is considerably less so. Both 1 and 2 induce apoptosis in HepG2 cells
with some evidence for necrosis induced by 2. Based on DNA fragmentation, caspase activities and human
apoptosis PCR-array analysis, both the extrinsic and intrinsic pathways of apoptosis have been shown to occur.
While both compounds activate mitochondrial and FAS apoptotic pathways, compound 1 was also found to
induce another death receptor-dependent pathway by induction of CD40, CD40L and TNF-R1 (p55). Further, 1
highly expressed DAPK1, a tumour suppressor, with concomitant down-regulation of XIAP and NF-κB. Cell
Keywords:
Bismuth
Apoptosis
2
cycle arrest at the S and G /M phases correlates with the inhibition of the growth of HepG2 cells. The cell invasion
Metallopharmaceuticals
Cell cycle
NF-κB
rate of 2 is 10-fold higher than that of 1, a finding correlated with the down-regulation of survivin and XIAP
expression by 1. Compounds 1 and 2 interact with DNA through different binding motifs with 1 interacting
with AT- or TA-specific sites followed by inhibition of restriction enzyme digestion; 2 did not interfere with
any of the studied restriction enzymes.
©
2013 Elsevier Inc. All rights reserved.
1
. Introduction
[9], for activity against the Leishmaniasis protozoa [10] and for the
targeting of H. pylori [11].
Various bismuth compounds, e.g. bismuth subgallate and bismuth
Despite the widespread use of bismuth compounds in medicine and
the efforts devoted to developing new bismuth compounds for the treat-
ment of a variety of diseases, the biochemistry of bismuth is poorly under-
stood [12,13]. Work has commenced to delineate the biocoordination
chemistry of bismuth involving proteins, enzymes and cell membranes
in order to redress the deficiency in knowledge concerning possible
mechanisms of actions. It seems that the major target sites of bismuth
in proteins and enzymes are both iron(III) sites in transferrin (having ox-
ygen and nitrogen donors) [14,15] and zinc(II) sites in metallothionein
(with thiolate donors) [16]. It is also well established that bismuth
forms very stable complexes with glutathione, which may be involved
in the transport of bismuth in cells and bacteria [17,18].
subnitrate, have been used in the past to treat hypertension, syphilis,
gastrointestinal disorders and skin conditions [1–3]. In particular,
since the 1970s, two bismuth compounds, i.e. bismuth subsalicylate
(
BSS, Pepto-Bismol®; the Procter & Gamble Company, Cincinnati,
Ohio, USA), for the prevention and treatment of dyspepsia and diar-
rhoea, and colloidal bismuth subcitrate (CBS, De-Nol®; Gist Brocades,
Delft, The Netherlands), for the treatment of peptic ulcers, have gained
widespread use as OTC (over the counter) drugs emphasising
the non-toxic nature of bismuth, at least in medicinal doses [4–6].
These bismuth-containing drugs as well as ranitidine bismuth citrate
(
Tritec® and Pylorid®) are being used worldwide in combination
with antibiotics to eradicate infection due to Helicobacter pylori [4–6].
Complimenting clinical applications, new molecular bismuth com-
pounds are under development as anti-cancer agents [7], of direct
relevance to the present study, anti-viral agents [8], anti-microbials
In connection with the anti-cancer potential of bismuth compounds,
bismuth can be used as a protective agent in that pre-administration
with bismuth is thought to protect patients from some of the toxic
side-effects induced by the widely used anti-cancer drug, cisplatin,
without affecting anti-tumour activity [19]. In terms of developing
new drug candidates based on bismuth, interest is increasing with
bismuth thiolates at the fore [20]. For example, studies on bismuth xan-
⁎
Corresponding author.
⁎⁎ Corresponding author. Tel.: +60 3 7967 6775; fax: +60 3 7967 4193.
2 3
thate compounds of the general formula Bi(S COR) , for R=Et, i-Pr and
E-mail addresses: abdah@medic.upm.edu.my (A.M. Akim),
Edward.Tiekink@um.edu.my, Edward.Tiekink@gmail.com (E.R.T. Tiekink).
cyclohexyl, were shown to exert cytotoxic activities against cisplatin-
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.09.018

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
39
sensitive Calu-6 (human lung adenocarcinoma cells) and cisplatin-
insensitive MCF-7 (human breast carcinoma cells) [21]. Subsequently,
related species derived from the reaction of an amine with carbon
disulphide with subsequent reaction with bismuth salts, i.e. bismuth
M.P. = 195 °C. Elemental analysis: Found C, 27.57; H, 4.72; N, 6.41.
−
1
C
15
H
30BiN
3 6
S requires: C, 27.56; H, 4.63; N, 6.43. IR (KBr disk, cm ):
1
1489 m ν(C–N); 1065 s and 906 s ν(C–S). H NMR: δ 3.83 (q, CH
2
, J =
, J = 7.1 Hz). 13C{ H} NMR: δ 202.0 (CS
); 48.5
3 2
1
7.2 Hz); 1.33 (t, CH
(CH ); 12.0 (CH ).
The potassium salt of the di-hydroxylethyldithiocarbamate anion,
CN(CH CH OH) ], was synthesized by the reaction of diethanolamine
dithiocarbamates with the general formula Bi(S
onstrated to exhibit potent in vitro cytotoxicity against a panel of seven
human cancer cell lines [22]. One species in particular, i.e. Bi(S CNEt
1, Fig. 1), proved to be very potent with activity comparable to that ex-
2
CNR
2
)
3
, were also dem-
2
3
2
)
2 3
K[S
2
2
2
2
(
(0.06 mol, 4.82 ml; Sigma-Aldrich), carbon disulfide (0.06 mol, 3.02 ml;
Merck) and potassium hydroxide (0.06 mol; Riendemann Schmidt) in
distilled water (40 ml). The salt (10.00 mmol, 2.19 g) was added to a
hibited by taxol®. Therefore, this was chosen for in vivo anti-tumour
screening against ovarian (OVCAR-3) and colon carcinoma (HT-29) cell
lines in a murine model and proved to exhibit some anti-tumour activity
suspension of BiCl
stirring, the product was left overnight after which yellow-brown crystals
of Bi[S CN(CH CH OH) (2) deposited. Percentage yield: 61%. M.P. =
157–158 °C. Elemental analysis: Found C, 23.79; H, 3.45; N, 5.51. C15
3
(3.39 mmol, 1.069 g) in ethanol (25 ml). After 2 h of
[
22]. Compound 1 and hydroxyl derivative, Bi[S
2 2 2 2 3
CN(CH CH OH) ] (2,
Fig. 1), form the focus of the present investigation.
2
2
2
2 3
]
Herein, in vitro cytotoxicity of 1 and 2 has been evaluated against a
series of six human carcinoma cells, namely HepG2, MCF-7R, A2780,
HT-29, A549 and 8505C. Having exhibited particular potency against
HepG2, an investigation whether 1 inhibited the cell growth of HepG2
cells by regulating the cell cycle was conducted; non-potent 2 was
also investigated in this regard. In addition, the underlying molecular
mechanisms of the different growth-inhibitory effects of 1 and 2 in
HepG2 cells were conducted in order to better understand their effects
on the expression of several genes significantly involved in liver cancer
development, and especially in the human cell cycle and apoptosis. The
expression of these genes was quantified by polymerase chain reaction
H
30-
−
1
BiN
br ν(O–H); 1472 s ν(C–N); 1205 s ν(C–O); 967 m ν(C–S). H NMR: δ
3 3 6
O S requires: C, 24.03; H, 4.03; N, 5.61. IR (KBr disk, cm ): 3251
1
13
4.79 (t, OH, J = 5.4 Hz); 3.90 (t, NCH
2
, J = 6.0 Hz); 3.73 (m, CH
2
O).
C
1
{ H} NMR: δ 203.0 (CS
2
2
); 59.0 (NCH ); 57.0 (OCH ).
2
2.3. Cell viability assay
The HepG2 (human hepatocellular carcinoma), MCF-7R (human
breast carcinoma cells, multidrug resistant strain, Michigan Cancer
Foundation-7), A2780 (human ovarian carcinoma), HT-29 (human
colon adenocarcinoma), A549 (human lung adrnocarcinoma) and
8505C (human thyroid carcinoma) cell lines were obtained from ATCC:
The Global Bioresource Center and maintained in culture as described
by the provider. The cells were routinely grown in an RPMI 1640 medi-
um containing 10% foetal calf serum (FCS) and anti-biotics at 37 °C and
(PCR) microarray analysis. This study indicates the potential utility of
bismuth compounds as a potent class of experimental therapeutics for
liver cancer and lays the foundation for the rational design of new
drugs based on bismuth.
2
. Materials and methods
2
6% CO . 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 a complete medium. The stock solu-
tions of 1 and 2 were prepared by dissolving the compounds in 1 mL of
DMSO to reach a concentration of 10 M. They were then diluted in
an RPMI medium and added to the wells (100 μL) to obtain a final con-
centration ranging between 0 and 80 μM. DMSO at comparable concen-
trations, i.e. b1%, did not show any effects on cell cytotoxicity. Stock
solutions of the compounds were diluted directly in a culture medium
to the required concentration and added to the cell culture. After 24 h
2
.1. Instrumentation
1H and 13C{1H} NMR spectra were recorded in a CDCl
5 °C on a Bruker Avance 400 spectrometer; abbreviations for NMR
3
solution at
−
2
2
assignments: t, triplet, q, quartet and m, multiplet. IR spectra were
obtained as KBr pellets on a Perkin Elmer RX1 FTIR spectrophotometer;
abbreviations for IR assignments: s, strong, m, medium and br, broad.
Elemental analyses were performed on a Perkin Elmer PE 2400 CHN
Elemental Analyser. Melting points were determined on a Krüss
KSP1N melting point apparatus. Powder X-ray diffraction (PXRD) data
were recorded with a PANalytical Empyrean XRD system with Cu-Κα1
radiation (λ = 1.54056 Å) in the 2θ range 5 to 40° with a step size of
of incubation at 37 °C, 20 μL of
a solution of MTT (3-[4,5-
dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) in PBS (phos-
−
1
phate buffer saline, 2 mg mL ) was added to each well, and the plates
were then incubated for 2 h at 37 °C. The medium was then aspirated
and DMSO (100 μL) added to dissolve the precipitate. The absorbance
of each well was measured at 580nm using a 96-well microplate reader
and compared to the values of control cells incubated without a test
compound. The IC50 values for the inhibition of cell growth were deter-
mined by fitting the plot of the percentage of surviving cells against the
drug concentration using a sigmoidal function (Origin v7.5™).
0
.026°. Comparison between experimental and calculated (from CIF's)
PXRD patterns was performed with X'Pert HighScore Plus [23].
2
.2. Reagents and synthesis
2 2
The sodium salt of diethyldithiocarbamate, Na[S CNEt ] (10.00mmol,
2
.25g; Sigma-Aldrich) was dissolved in distilled water (40ml) and added
(3.39 mmol, 1.069 g; R&M Chemicals) in
ethanol (25 ml). After 2 h of stirring, a bright-yellow precipitate
was obtained. This was then recrystallised from a CH CN/CHCl (1:3)
solution yielding a bright-yellow crystalline material, Bi(S CNEt (1),
after the solution was left to stand overnight. Percentage yield: 93%.
slowly to a suspension of BiCl
3
2.4. Membrane permeability study by AO/PI staining
3
3
3
2
2 3
)
HepG2 cells at a concentration of 5 × 10 cells/well in 96 well plates
were treated with the IC50 concentration of each compound and incubat-
ed for 24 h. Untreated cells were included as a negative control. Treated
cells were harvested from the culture flask. 1× EDTA free-PBS was used
to wash the cells 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 AO (acridine orange,
Sigma) and PI (propidium iodide, Sigma) mixture was added to the cells
in a 1:1 ratio for staining. This was followed by chilling on ice for 10 min.
The mixture (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.
2 2 3 2 2 2 2 3
Fig. 1. The structures of Bi(S CNEt ) (1) and Bi[S CN(CH CH OH) ] (2).

40
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
2
.5. DNA fragmentation analysis
7000, France). Briefly, cells were cultured in 96-well plates in a final
volume of 200 mL. Then 50 mL caspase reaction buffer was added and
incubated at room temperature for 1 h before measurement.
6
For the DNA fragmentation assay, 2×10 HepG2 cells were treat-
ed with 0.53 μM (1) and 55.9 μM (2) in an RPMI medium for 24 h at
3
3
7 °C and 5% CO
00 ×g/15 min/10 °C and washed with PBS. The cells were then re-
2
. The cells were harvested and centrifuged at
2.9. Intracellular reactive oxygen species (ROS) measurements
6
−1
suspended at a concentration of 1 × 10 cells mL
buffer (10 mM TriseHCl, 0.1 M EDTA, 5 g mL
with 20 mg L
in an extraction
SDS) and treated
RNase A at 37 °C for 60 min, followed by incubation
5-(And-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate
(Carboxy-H2DCFDA, Sigma, USA) was used to detect intracellular ROS ac-
cording to the manufacturer's instructions. For ROS quantification, cells
were seeded in 96-well black plates (Greiner Bio-One, France) and treat-
ed with 1 or 2 at the indicated IC50 concentrations for 24h. Afterwards, the
cells were washed with PBS and incubated with 10μM carboxy-H2DCFDA
in DPBS for 1h. The cells were then washed and fluorescence measured by
a plate reader (Perkin Elmer, France) with an excitation wavelength of
485 nm and an emission wavelength of 535 nm.
−
1
−
1
−
1
with proteinase K (100 mgL ) at 37 °C for 60min. An equal volume
of saline solution (NaCl 6 M) was added to the cells followed by
centrifuging at 13,000 ×g for 10 min. The supernatant was collected
and two volume equivalents of ethanol (−20 °C) were added. The
samples were centrifuged at 13,000×g for 30min at 4 °C. The superna-
tant was then discarded and the pellets dissolved in TE buffer (1×). The
DNA concentration and quality were analyzed by electrophoresis and
UV/vis on a BioPhotometer Spectrophotometer UV/VIS (Eppendorf,
Germany). The samples were applied to a 1.5% agarose gel and
subjected to electrophoresis for 2 h at 80 V with running buffer of 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™.
2.10. Flow cytometry analysis (fluorescence-activated cell-sorting)
6
2
The HepG2 cells were plated at 1 × 10 cells per T-75 cm flask. The
cells were maintained in a medium supplemented with 0.53 μM (1)
and 55.9μM (2) in DMSO for the treated cells. A control was performed
with DMSO alone. The cells were collected after 6, 12 and 24h. The cells
were harvested, fixed in 70% ethanol and stored at −20 °C. Cells were
then washed twice with ice-cold PBS and incubated with RNase and
the DNA intercalating dye PI. Cell-cycle phase analysis was performed
using a Becton Dickinson Facstar flow cytometer equipped with Becton
Dickinson cell-fit software™.
2
.6. Restriction enzyme inhibition assay
The restriction enzyme inhibitory activity was determined by ob-
serving the resultant band of lambda (λ) DNA. Each reaction mixture
contained 0.25μg of λ DNA, 2μl of 10× restriction enzyme reaction buff-
er, 50 μM of 1 or 2, 5 units of restriction enzyme and sterile deionized
water. The total volume of each reaction was 20 μl. Firstly, λ DNA was
incubated with the bismuth compound at 37 °C for 60 min followed by
addition of the restriction enzyme. The reaction mixture was incubated
for a further 2h at the same temperature. The reactions were terminated
by the addition of 2 μl of 10% SDS, followed by 3 μl of a dye solution
comprising 0.02% bromophenol blue and 50% glycerol. SDS is required
to denature the restriction enzyme, preventing further functional enzy-
matic activity. The mixtures were applied to a 2.0% agarose gel and
subjected to electrophoresis for 2 h at 80 V with running buffer of 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™.
2.11. Cytochrome c detection
6
2
The HepG2 cells were plated at 1 × 10 cells per T-75 cm flask.
The cells were maintained in a medium supplemented with 0.53 μM
(1) and 55.9 μM (2) in 1% DMSO for the treated cells. A control was
performed with 1% DMSO alone. Cytochrome c was detected by
FlowCellect™ Cytochrome C Kit (Millipore, Germany) according to
the manufacturer's protocol (http://www.millipore.com/catalogue/
item/fcch100110#).
2.12. Cell invasion assay
The cell invasion study was conducted employing a BioCoat™
Matrigel™ Invasion Chamber (BD Biosciences, San Jose, CA). Briefly,
0.75 ml of complete medium was added to the bottom of the well. At
2
2
.7. Extraction of RNA, and RT profiler PCR microarray (apoptosis and cell
4
cycle analysis)
the same time, 5 × 10 cells, re-suspended in 0.5 ml complete medium
with or without compound 1 and 2 (0.53 μM and 55.9 μM, respectively),
were added to the insert of the well. The final concentration of DMSO
was identical in all the inserts (0.01%). The cells were then incubated at
6
The HepG2 cells were plated at a density of 3 × 10 cells per T-
7
5 cm2 flask. Total RNA was extracted from cultured HepG2 cells
using a high-purity RNeasy Mini Kit (Qiagen, USA) according to the
manufacturer's protocols. Total RNA samples were dissolved in
diethylpyrocarbonate-treated water and their concentration and
quality were analyzed by electrophoresis and by UV/visible (UV/
vis) spectroscopy on a BioPhotometer Spectrophotometer UV/VIS
2
37°C under 5% CO for 22h. Non-invaded cells in the interior of the insert
were removed with cotton tips. Invaded cells were fixed with methanol
for 2min, followed by staining with 1% Toluidine Blue O (Sigma-Aldrich)
and washing with water. The membrane was finally observed under an
inverted microscope at 200× magnification. Ten fields were randomly
chosen and the numbers of invaded cells were counted. The data are
shown mean ± SEM from three independent experiments.
(
Eppendorf, Germany).
The real-time PCR microarray assay was performed using the RT2
Profiler PCR microarray (Apoptosis (PHAS-012); cell cycle (PHAS-
20)) according to the manufacturer's protocols (Qiagen, Germany).
0
3. Results
2
The human apoptosis RT profiler PCR array profiles the expression of
4 key genes involved in programmed cell death. This array contains
8
3.1. Chemistry
genes that both positively and negatively regulate the cell cycle, the
transitions between each of the phases, DNA replication, checkpoints
and arrest. Gene expression was compared according to the CT value.
Compounds 1 and 2, Fig. 1, exhibited the expected spectroscopic
characteristics (see Section 2.2) confirming their formation. Crystal
structures are known for each of 1 [24] and 2 [25] and feature
anisobidentate coordination of the dithiocarbamate ligands and heavily
distorted geometries owing to the presence of a stereochemically active
lone pair of electrons coupled with secondary Bi…S interactions. PXRD
measured on the samples prepared herein matched those calculated
2
.8. 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

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
41
from the respective single crystal studies; see Fig. S1. This indicates that
the bulk material has the same structure as determined by single crystal
X-ray crystallography. Time-dependent 1H NMR measurements in
DMSO-d6 on each of 1 and 2 showed no change after 36 h, indicating
their stability beyond the time-frame of the biological studies described
below.
3
.2. Inhibition of cancer cell proliferation
Compounds 1 and 2 were evaluated in vitro for their ability to inhibit
cell proliferation against six human cancer lines, i.e. HepG2, MCF-7R,
A2780, HT-29, A549 and thyroid 8505C, with cisplatin, doxorubicin
and paclitaxel as the positive controls. The effects of the compounds
on the growth and proliferation of the selected cell lines were evaluated
after 24h and the obtained IC50 values are listed in Table 1. Generally, 2
was non-potent, only exhibiting limited potency against HepG2. By
contrast, 1 was more cytotoxic than any of the standard drugs in all
the investigated cell lines. The viability of the HepG2 cells after treat-
ment with 1 and 2 at different concentrations is plotted in Fig. 2 which
shows that 1 and 2 inhibited cell proliferation in a concentration-
dependent manner. HepG2 was selected for the subsequent studies
into mechanisms of cell death.
Fig. 2. (a) 1 and (b) 2. Cell viability of HepG2 cells after treatment with 1 and 2 at different
concentrations at 24 h.
3
.3. Membrane permeability study (AO/PI apoptotic cell study)
The assessment of the membrane integrity of the HepG2 cells
most of the 2-treated apoptotic cells with intact plasma membrane,
Fig. 3c, indicating that 2 might also trigger cell necrosis at the same time.
after treatment with the respective IC50 dose of each compound
was accomplished by staining with AO/PI as shown in Fig. 3. Apopto-
tic cells exhibit increased plasma membrane permeability to certain
fluorescent dyes [26]. In this assay, cells with an intact membrane
appear to have a bright-green nucleus while apoptotic cells exhibit
a dark-green nucleus with condensation of chromatin (as dense-
green areas) when viewed by fluorescence microscopy after being
stained with AO [27]. Necrotic cells on the other hand have lost
membrane integrity and show bright-red fluorescence with PI as
this is impermeable to intact plasma membranes but can easily pen-
etrate the plasma membrane of dead or dying cells to intercalate
with DNA or RNA thereby producing a bright-red fluorescence [28].
The AO/PI staining showed that majority of HepG2 cells underwent
apoptosis after treatment with 1 and 2. Some morphological charac-
teristics of apoptotic cells was observed on the treated HepG2 cells,
such as condensation of nuclear chromatin and cytoplasm, mem-
brane blobbing and the formation of apoptotic bodies. Some of the
3.4. DNA fragmentation analysis
Chromosomal DNA fragmentation is one of the hallmarks of the
lethal stages of apoptosis that proceeds in a two-step manner. At first,
the DNA is initially cleaved into 50–300 kb fragments and eventually
into oligonucleosomal pieces [29,30]. The release of apoptotic signals
during the apoptosis process such as the expression of the p53 and
DFFA genes leads to DNA damage. As shown in Fig. 4 (L2, L4), 1 and 2
causes intensified DNA fragmentation in HepG2 cells noticeable by the
generation of ladder fragments in individual cells resulting from DNA
breaks (shown by arrows).
3.5. Restriction enzyme digestion and analysis
The sequence binding selectivity of 1 and 2 to DNA was studied
1
-treated cells exhibited enlargement of cell volume and formation
in detail by measuring the inhibition of twelve different restriction
enzymes which differ in their target sequences. The list of restriction
enzymes and their target sequences (red arrows) are as follows:
of multinucleated cells. After treatment with 1 and 2, the majority
of cells were clumped in few unorganised colonies (Fig. 3b,c),
while this phenomenon was not shown for the untreated cells
(Fig. 3d). Furthermore, a small amount of red fluorescence appears in
1
2
3
.
.
.
Tsp 509I
Hae III
Sal I
5′-↓ A A T T-3′
′-T T A A ↑-5′
5′-G C ↓G C-3′
′-C G ↑C G-5′
5′-G ↓T C G A C-3′
′-C A G C T ↑G-5′
5′-C T G C A ↓G-3′
′-G↑ A C G T C-5′
5′-C A G ↓C T G-3′
′-G T C ↑ G A C-5′
5′-A G T ↓A C T-3′
3′-T C A ↑ T G A-5′
5′-A A ↓T A T T-3′
3′-T T A T ↑A A-5′
5′-A T ↓T A A T-3′
3′-T A A T ↑T A-5′
5′-C↓ A A T T G-3′
3
3
Table 1
3
Cytotoxic activity of 1, 2 and standard drugs against human carcinoma cells after 24 h
treatment.
a
4.
5.
Pst I
3
Cisplatin
Doxorubicin
Paclitaxel
1
2
Pvu II
Sca I
3
Cell line
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
6.
HepG2
MCF-7R
A2780
HT-29
A549
152.0 ± 0.3
19.6 ± 0.2
28.8 ± 0.4
25.0 ± 0.3
35.4 ± 0.4
120.0 ± 0.2
4.6 ± 0.2
5.2 ± 0.4
4.4 ± 0.2
4.8 ± 0.3
N80
N80
0.5 ± 0.4
0.9 ± 0.2
0.5 ± 0.2
3.5 ± 0.3
2.3 ± 0.4
0.2 ± 0.4
55.9 ± 0.3
N80
N80
N80
N80
15.9 ± 0.3
14.2 ± 0.3
N80
N80
N80
7.
8.
9.
Ssp I
Ase I
8505C
N80
N80
Mun I
3′-G T T A A ↑C-5′
a
Means and standard deviations from at least two independent experiments conducted
in triplicate, except for values N80.
(continued on next page)

42
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
(
continued)
(
Figs. 5a, 6a). Conversely, 2 failed to protect the restriction sites in any
10.
11.
12.
EcoR I
5′-G↓ A A T T C-3′
′-C T T A A ↑G-5′
5′-C A↓ T A T G-3′
′-G T A T ↑A C-5′
5′-G T A ↓ T A C-3′
′-C A T ↑ A T G-5′
of the twelve tested restriction enzymes as discerned from the bands
appearing in the gel which correspond to the restriction sites of the en-
zyme (Figs. 5B, 6B).
3
Nde I
3
Bst 11071
3
3
.6. Intracellular signalling cascades
Apoptosis is a highly controlled process of programmed cell death
The twelve restriction enzymes were used to assess the binding se-
for eliminating unwanted cells from the body during organ develop-
ment, immune responses and tissue remodelling. The resistance of
tumours to chemotherapy has been closely related to the inherent
weaknesses in apoptotic pathways in the development of cancer.
Hence, numerous anti-cancer drugs are aimed specifically at the signal-
ling components of cell death and survival pathways [32–34]. In gener-
al, apoptosis is characterised by an individual set of biochemical stages
and morphological changes comprising chromatin condensation,
activation of caspases and chromosomal DNA fragmentation [35].
Cell death pathways affected by 1 and 2 were evaluated by a combi-
lectivity of 1 and 2 towards λ DNA as anti-cancer activity can be manip-
ulated when specific binding occurs with DNA, either by intercalation or
via groove binding. Gel electrophoresis following the digestion with the
twelve restriction enzymes of λ DNA pre-treated with 1 and 2 is illus-
trated in Figs. 5 and 6; precise and reproducible protection patterns
were observed for each DNA interaction study. Based on the results,
the ‘AT’ sequence in Ssp I “AATATT”, Nde I “CATATG” and Bst 11071
“
GTATAC” was sheltered from DNase digestion by 1, whereas the GC,
short AT, AAT and random sequences were not. These conclusions are
based on the fact that digestion with Ssp I, Nde I and Bst 11071 did
not induce DNA scission on the λ DNA suggesting that 1 was bound to
specific DNA sites and protects these from restriction enzymes digestion
2
nation of apoptosis RT profiler PCR microarray analysis, caspase activity
study, DNA fragmentation and ROS production measurements. The pro-
posed signalling pathways are summarised in Fig. 7. Over-expression of
pro-apoptotic genes accompanied by suppression of anti-apoptotic
[
31] while digested DNA bands were shown in the negative control
Fig. 3. AOPI staining of HepG2 cells after being treated with the IC50 value of each compound. (a) doxorubicin; (b) 1; (c) 2; (d) untreated cells (negative control). The red colour was
produced by the PI stain which penetrated the nuclear matter when the cell membrane integrity was disturbed. Cells with intact membrane are stained green. Apoptotic cells are stained
dark-green and contain multiple yellow/green dots of condensed nuclei. Necrotic cells were stained bright-red due to the influx of PI stain. The green arrow in (a) shows a healthy cell. The
blue arrow in (a) shows an apoptotic cell with fragmented nucleus and condensed chromatin. Magnification = 100×.

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
43
up-regulation of the p53/p73 genes shows that the compounds could
induce DNA damage in HepG2 cells, which is consistent with the DNA
fragmentation results discussed above (see Section 3.4 and Fig. 4). At
the same time, the p21 encoded gene, CDKN1A, that functions as a
major inhibitor of p53-dependent apoptosis [36], was up-regulated by
1
and 2, 12- and 22-fold (after 24 h), respectively (Fig. 8). Another up-
stream protein that regulates p53 is the ataxia telangiectasia mutated
(
(
ATM) gene that was up-regulated by 1 and 2 about 98- and 21-fold
after 24 h), respectively (Fig. 8). The ATM gene initiates a mechanism
for controlling cell cycle and apoptotic responses by selectively stimu-
lating the expression of p53 [37], so the up-regulation of CDKN1A
gene and the up-regulation of the ATM gene work against each other
in the enhancement of the p53 pathway.
It is noted that the level of gene expression of ATM by 1 is significant-
ly greater than promoted by 2 which may be one of the reasons why 1
has greater cytotoxicity than 2. The enhancement of p73-induced
apoptosis after treatment with 1 and 2 is due to the presence of c-Abl
protein which also leads to the inception of apoptosis [38] (Table 2;
ABL1 gene). The high expression of the BNIP3L protein, a cell death in-
ducer, in 1- and 2-treated cells (55- and 23-fold) by p53 is considered
as supportive evidence for the activation of the p53 gene as this can
directly up-regulate the expression of the BNIP3L gene which is
known to be highly induced in wild-type p53-expressing cells [39].
The BCL-2-associated X protein (BAX) initiates a second mechanism
of p53/p73-induced apoptosis, which in turn promotes Bax mitochon-
drial translocation and cytochrome c release. In general, mitochondrial
membrane permeability is controlled through a family of proto-
oncogenes, such as anti-apoptotic (BCL-2) and pro-apoptotic (BAD,
BAX) [40]. BAX is introduced into the mitochondrial membrane and
increases membrane permeability, thereby promoting apoptosis once
activated [41,42]. In the treated HepG2 cells, BCL-2 was deactivated by
1 and 2 (Table 2), hence, BAX is able to increase mitochondrial mem-
brane potential without obstruction in the absence of anti-apoptotic
protein, BCL-2.
Fig. 4. DNA fragmentation analysis. The HepG2 cells were cultured for 24h in RPMI 1640 in
the presence of 0.53 μM 1 (L2) and 55.9 μM 2 (L4). DNA was extracted from the cultures
and DNA fragmentation was detected using an electrophoresis system with a 2% agarose
gel. L1, L3 and L5 are 1 kb DNA ladder; L6 is negative control (untreated cells). Formation
of ladders on the gel to indicate DNA fragmentation occurs following treatment with 1 and
2
(indicated by arrows) which suggests that cell death is by apoptosis.
genes was observed following the treatment of HepG2 with 1 and 2.
This work provides evidence that the cytotoxicity of 1 and 2 is related
to the induction of a p53/p73-dependent activation of the mitochondri-
al pathway of apoptosis, demonstrating the anti-cancer properties of
the compounds. Interestingly, the PCR microarray analysis revealed
that 1 induced apoptosis involving both intrinsic and extrinsic pathways
mediated by various types of apoptosis-inducing factors. Both p53 and
p73 were expressed at the same time. Compounds 1 and 2 were
found to trigger the gene expression of p53 (TP53 gene) as seen by
about a 6- and 10-fold increase over TP73 expression (Table 2). The
Generally, the primary event leading to apoptosis is the loss of
mitochondrial trans-membrane potential [43] which results in the com-
promise of the ΔΨm, followed by outer mitochondrial membrane disrup-
tion and release of proteins such as the cytochrome c from the inter-
Fig. 5. Electrophoresis results after incubating λ DNA (0.5 μg/μL) with 5 units of restriction enzyme in the presence or absence of 10 μM 1 (a) and 2 (b) for 2 h at 37°C. Lanes 1 and 16, 1 kb
DNA ladder; Lane 2, λ DNA alone (0.5μg); Lane 3, λ DNA + 10μM compound; Lane 4, λ DNA + 5units Tsp 509I (control); Lane 5, λ DNA + 5 units Tsp 509I+ 10 μM compound; Lane 6, λ
DNA + 5 units Hae III (control); Lane 7, λ DNA + 5 units Hae III + 10 μM compound; Lane 8, λ DNA + 5 units Sal I (control); Lane 9, λ DNA + 5 units Sal I + 10 μM compound; Lane 10, λ
DNA + 5 units Pst I (control); Lane 11, λ DNA+5units Pst I+ 10μM compound; Lane 12, λ DNA+5units Pvu II (control); Lane 13, λ DNA+5units Pvu II+ 10μM compound; Lane 14, λ
DNA + 5 units Sca I (control); and Lane 15, λ DNA + 5 units Sca I + 10 μM compound.

44
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
Fig. 6. Electrophoresis results after incubating λ DNA (0.5μg/μL) with 5units of restriction enzyme in the presence or absence of 10μM 1 (a) 2 (b) for 2h at 37°C. Lanes 1 and 16, 1kb DNA
ladder; Lane 2, λ DNA alone (0.5 μg); Lane 3, λ DNA + compound; Lane 4, λ DNA+ 5 units Ssp I (control); Lane 5, λ DNA + 5 units Ssp I + compound; Lane 6, λ DNA + 5 units Ase I (con-
trol); Lane 7, λ DNA+5units Ase I+compound; Lane 8, λ DNA + 5 units Mun I (control); Lane 9, λ DNA+5units Mun I+compound; Lane 10, λ DNA+5units EcoR I (control); Lane 11, λ
DNA + 5 units EcoR I + compound; Lane 12, λ DNA + 5 units Nde I (control); Lane 13, λ DNA + 5 units Nde I + compound; Lane 14, λ DNA + 5 units Bst 11071 (control); and Lane 15, λ
DNA + 5 units Bst 11071 + compound.
membrane region [44]. Cytochrome c, which binds to apoptotic activating
factor-1 (APAF-1), was also significantly expressed in HepG2 cells treated
with 1 and 2, and is known to activate caspase-9 leading in turn to the ac-
tivation of caspase-3 which is the most potent effector caspase (Table 2).
The increase in ΔΨm also allows for the release of AIF, which activates a
DNase leading to apoptosis [45-47]. Downward shifts (from M2 to M1)
in the fluorescence response due to cytochrome c in HepG2 cells after
treatment of 1 and 2 after 12 and 24h are illustrated in Fig. 9; the negative
control samples show low levels of cytochrome c loss.
Another outcome from the increase in the mitochondrial membrane
potential is the generation of ROS. The generation of ROS is reportedly
involved in apoptosis induced by chemotherapeutic agents through
the activation of the mitochondria-mediated apoptotic pathways
[48,49]. ROS are thought to play a role in TNFR (tumour necrosis factor
receptor) and FAS (tumour necrosis factor superfamily, member
6) receptor-mediated apoptosis even though the exact mechanism
remains undefined [50]. In the presence of 1 and 2, cellular ROS gener-
ation exceeded about 32% and 21% that of the negative control (untreat-
2 2
ed cells), indicating that the trial compounds do induce significant H O
generation (Fig. 10). These findings suggest that 1 and 2 activate the in-
trinsic pathway by enhancing ROS production and repressing mito-
chondrial function, and contributes to apoptotic cell death in HepG2
cells. Drugs which are able to compromise the functional integrity of mi-
tochondria could be of significant relevance in anti-cancer therapy [51].
The present study revealed that treatment of HepG2 cells with 1 and
2 also induced an extrinsic apoptotic pathway by triggering death
Fig. 7. Proposed signalling pathway of apoptosis induced by 1 and 2. This diagram represents a collation of the results obtained from the PCR microarray analysis, caspase activity study,
DNA fragmentation and ROS production measurements.

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
Table 2 (continued)
45
Table 2
Apoptosis gene expression level in HepG2 cell lines after treatment with 1 and 2 compared
to untreated cells.
Up–down regulation fold regulation
comparing to control group)
a
(
Up–down regulation fold regulation
1
2
(comparing to control group)
CD27
−1.88
1.01
2.95
2.93
7.47
−1.32
1
2
TNFRSF9
TNFSF10
CD70
TNFSF8
TP53
TP53BP2
TP73
TRADD
TRAF2
TRAF3
TRAF4
1.01
2.03
ABL1
AKT1
APAF1
BAD
BAG1
BAG3
BAG4
BAK1
BAX
58.13
72.06
33.17
253.96
241.94
12.62
49.81
21.51
172.26
236.96
164.11
14.04
26.51
−6.33
18.75
61.33
1.01
3.29
5.42
113.08
4.41
90.63
22.76
8.62
92.31
77.11
73.46
51.98
34.56
−11.62
−54.99
−14.13
64.50
34.99
11.64
−16.63
9.59
−5.70
−8.41
3.08
18.79
35.29
−1.05
−3.14
−11.74
BCL10
BCL2
Bold face: up-regulated genes; italic: down-regulated genes.
Data represent mean of samples 1 and 2-induced fold-change in gene expression
relative to control-treated cells (n = 3), p b 0.05.
a
BCL2A1
BCL2L1
BCL2L10
BCL2L11
BCL2L2
BCLAF1
BFAR
BID
BIK
NAIP
8.16
35.54
93.78
1.55
13.37
7.52
24.44
123.51
393.03
2.88
2.16
8.22
67.11
1.85
−30.94
4.90
receptors of the tumour necrosis factor receptor superfamily, member 6
(FAS) and the tumour necrosis factor receptor (TNFRSF1A) gene expres-
sion. Caspases-8, -10, -9 and -3 were activated subsequently after FAS
expression with an increase in their enzymatic activities (Tables 2–4).
In general, FAS and TNFR1 recruit Fas-associated protein with death do-
main (FADD) and procaspase-8 and -10 to the receptor. FADD controls
the recruiting of procaspase-8 and -10 leading to its auto-cleavage and
activation, and subsequently activates effector caspases in initiating
cell death [52,53]. Alternatively, caspase-8 and -10 could cleave the
BCL-2 family member Bid, a molecular linker bridging death receptor
and mitochondria pathways to truncated BH3 interacting-domain
death agonist (tBID), that binds to pro-apoptotic protein BAX, resulting
in the disruption of mitochondrial membrane potential and the release
of cytochrome c [54–56]. Compounds 1 and 2 also induced expression of
BID in HepG2 cells (Table 2), which provides additional supporting
evidence for the proposed apoptotic pathways.
Interestingly, apart from the mitochondrial and FAS death receptor
apoptotic pathway, 1 was also found to induce another death
receptor-dependent pathway by induction of a series of pro-apoptotic
genes, such as CD40 (CD40 gene), CD40L (CD40LG), TNF-α (TNF) and
TNF-R1 (p55/TNFRSF1A) (Table 2). CD40 is a TNF receptor family
member that is widely recognized for its prominent role in immune reg-
ulation and homeostasis [57]. However, accumulating evidence sug-
gests that the CD40 pathway can be exploited for cancer therapy as it
can stimulate the host's anti-tumour immune response, followed by
normalisation of the tumour microenvironment and arrest of the
growth of CD40-positive tumours [57]. Further, CD40 also contains a
cytoplasmic motif reminiscent of the death domain which is involved
in the initiation of TNF-R1 and CD95-dependent apoptosis, stimulating
cell death in cells of mesenchyme origin, tumour cells and certain trans-
formed cell lines [58–60]. Moreover, membrane- anchored TNF-α and
TNF-R1 (p55) may be activated by CD40 followed by stimulation of
the death receptor-dependent pathway involving caspases-8 and -3
[61].
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
−1.35
−185.95
−68.65
−56.06
−18417.65
75.12
227.73
−87.96
55.28
−22.92
22.03
1.01
−24.28
−4842.45
179.58
595.72
−49.92
22.65
−8.18
99.63
1.09
3.10
44.67
1.01
3.50
54.51
4.43
33.32
304.12
4.91
1.01
58.94
97.69
8.64
9.31
3.36
3.41
38.35
27.69
37.57
103.34
12.33
8.64
28.47
−1.19
23.77
37.04
42.54
103.34
7.42
42.85
6.03
4.26
10.06
−212.13
9.52
215.45
36.53
82.21
1.04
109.78
159.62
14.32
160.95
1.01
1.01
46.48
−1.19
134.22
112.87
1.01
59.16
18.23
131.46
8.39
FADD
FAS
FASLG
GADD45A
HRK
IGF1R
LTA
LTBR
MCL1
NOL3
PYCARD
RIPK2
TNF
TNFRSF10A
TNFRSF10B
TNFRSF11B
TNFRSF1A
TNFRSF21
TNFRSF25
2.62
18.11
−86.91
11.22
395.76
69.00
160.73
1.01
180.83
−7.95
65.73
928.32
20.80
225.73
51.57
93.60
In another pathway, death-associated protein kinase 1 (DAPK1), a
tumour suppressor, was highly expressed by 1 in HepG2 cells (by
about 43-fold; Table 2). Over-expression of siDAPK1 significantly
rescues the protein expression of transcription factor Rel/nuclear
factor-kappaB (NF-κB)-targeted genes [62]. NF- B plays a crucial role
in regulating gene transcription and is involved in the mechanism of
cell proliferation [63,64]. DAPK1 also suppresses protein expression of
anti-proliferation genes such as COX-2 and ICAM-1, and anti-apoptosis
genes such as XIAP, which is involved in apoptosis inhibition triggered
by multiple varied stimuli that activate both of the principle cell death
pathways [65]. XIAP is an efficient apoptotic inhibitor because it is crit-
ically positioned to act at the point of convergence of both the extrinsic
37.82
−7.94
22.49
250.94
6.28
138.26
12.65
1.01

46
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
Fig. 8. Cell cycle gene expression level in HepG2 cell lines after treatment with compound 1 (a) and 2 (b) at 12 and 24 h compared to untreated cells corresponding to value 1. *p b 0.05.
and intrinsic death pathways on effector caspase activation [66].
Likewise, XIAP directly obstructs the activities of multiple caspases,
particularly, the effector caspases-3, -7 and -9 either by direct enzyme
inhibition or through ubiquitin-mediated proteasome degradation
cell cycle analysis revealed a prominent S and G
cells upon exposure to 1 and 2, and increases cyclin D
cyclin E (CCNE ) gene expression at 24 h after the treatment (Fig. 8),
suggesting that the reduction of cyclin D and cyclin E, mediates the S
2
/M arrest of HepG2
1
(CCND ), and
1
1
1
[
66]. XIAP is highly expressed in many cancer cells and tumour
specimens. Therefore, its inactivation in cancer cells results in apoptosis
67,68]. In the present investigation, XIAP was significantly down-
phase cell cycle arrest by regulating the indispensable S phase passage.
Cdc2 (CDK1) interacts with cyclin B1 (CCNB1) and forms a cdc2-
[
cyclin B
inhibits the transition from the G2 to M phase as this complex plays a
crucial role in G /M phase regulation [75]. The M-phase inducer phos-
1 1
complex. Generally, inactivation of the cdc2-cyclin B complex
regulated by 1 after 24 h of incubation in HepG2 cells (Table 2). Under
the same conditions, 2 induced high expression of XIAP (5-fold)
which defers the cell death process in HepG2 cells. Furthermore, over-
expression of XIAP will cause cancer progression and increase the
resistance-level of cancer cell lines to chemotherapy [67,68].
2
phatase 3, cdc25c, plays an important role in the dephosphorylation of
cdc2, and the activation of cdc25c brings the subsequent activation of
the cdc2-cyclin B
inhibitor p21 (CDKN1A), regulates cell cycle progression. Notably, the
level of expression p21 increased at the G /M transitions in a variety
1
complex [76]. In addition, cyclin-dependent kinase
3
.7. Cell cycle analysis
2
of cancer cells [77–80]. The anti-cancer effect in HepG2 cells exhibited
The cell cycle is a fundamental process that is studied in order to
by 1 and 2 was partially associated with their capacity to trigger growth
comprehend the mechanisms of tumour growth. In the realm of
metal-based compounds, several gold compounds have been reported
to exhibit anti-cancer activities through the modulation of the cell
cycle and the inhibition of the growth of cancer cells [69,70]. In this
study, HepG2 cells were treated with 1 and 2 for various time periods
inhibition at the G
2
/M phase by down-regulation of gene expression of
cdc2, cyclin B and p21 (Fig. 8 and Table 2).
1
ATM and Rad3-related protein (ATR) are two protein kinases of the
PI-3 PIKK family that activate signalling networks to freeze cell cycle
progression followed by termination of DNA replication and initiation
of DNA damage. The serine/threonine-protein kinase (Chek1) is an en-
zyme that in humans is encoded by the CHEK1 gene is a downstream
effecter of ATR and ATM, and is crucial for mediating G2/M arrest
following double-strand breaks [81,82]. In response to replication
stress, Chek1 delays both mitotic entry and S-phase replication delays
mitotic entry, and stabilizes stalled replication forks [83]. In this study,
CHEK1 was significantly up-regulated by 1 and 2, approximately 23-
and 11-fold, respectively, after incubation for 24 h, together with up-
regulation of ATR and ATM (Fig. 8). These results are corroborated
with the flow cytometry analysis, in which 1 induced approximately
(
6, 12, and 24 h) to determine whether the growth-inhibitory effect
was associated with the induction of cell cycle arrest and/or apoptosis;
the distribution of cells in the different phases of the cell cycle was ana-
lyzed using flow cytometry. Both 1 and 2 inhibited the growth of HepG2
cancer cells via cell cycle arrest at the S and G
lation of cells in the S and G /M phases was accompanied by synchro-
nous decreases in the percentage of cells in the G /G phase in a time-
2
/M phases. The accumu-
2
0
1
dependent manner up to 24 h (Figs. 11 and 12). Significant changes in
the population of cells in the G1 phase or in the levels of proteins related
to G phase, such as Ckd4, were observed (Fig. 8). Further, the exposure
1
of 1 and 2 was associated with cellular phenotypic changes, such as cell
shrinkage and density (Fig. 3b,c).
24% of cell cycle arrest at the G
value for 2 is 18% (Fig. 12).
2
/M checkpoint while the comparable
Cell cycle arrest and apoptosis progression are the main machineries
involved in anti-cancer drug treatment [71,72]. In recent studies, certain
Studies have shown that mini-chromosome maintenance (MCM)
proteins of which there are six (MCM2–7), might be useful as prolifera-
tion markers in various types of cancer [84]. Generally, MCM proteins
are a family of six highly conserved and highly homologous proteins
anti-cancer agents induced cell cycle G1 arrest and decreased cyclin D
and/or cyclin E expression in cancer cells [73,74]. In the present study,
1

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
47
Fig. 9. Histograms depicting cells stained using the FlowCellect Cytochrome c Kit™. (a) untreated HepG2 cells after 12 and 24h (negative control). (b) HepG2 cells treated with 0.53μM 1
for 12 and 24 h. (c) HepG2 cells treated with 55.9 μM 2 for 12 and 24 h. (B) and (C) show downward shifts (from M2 to M1) in the fluorescence due to reduced levels of cytochrome c.
Negative control samples show low levels of cytochrome c loss.
that are indispensable in forming pre-replication complexes which are
responsible for allowing DNA replication [85]. Consequently, increased
expression of MCM proteins has been recognised in most pre-
malignant proliferative states and solid tumours. The present study
showed a down-regulation of the MCM2–5 genes after treatment with
approximately 15- and 7-fold, respectively, compared to the control
(Fig. 8). Moreover, in vitro, GADD45 also efficiently destabilizes cdc2-
cyclin B complexes, suggesting that it may mediate Cdc2/cyclin inacti-
1
vation in vivo [89].
1
and 2 (Fig. 8), and suggests that they could limit the expression of
MCM proteins and could act as chemo-preventive agents.
3.8. Cell invasion study
Furthermore, recent evidence suggests that p53 may also play a cru-
cial role in the G
cancer cells leads to activation of p53 in response to DNA damage
87,88]. GADD45 was up-regulated by 1 and 2 treatment with
2
/M checkpoint [86]. Induction of GADD45 in various
Metastasis, the main cause of death of cancer patients, is the spread
of tumour cells from a primary tumour source to other organs, and
remains as one of the ultimate challenges in cancer treatment [90].
[

48
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
Table 4
Quantification of AFC in HepG2 cell cultured in the presence of sample 1 and 2 upon cleav-
a
age of the AEVD-AFC substrate by caspase-10.
Absorbance
Control
Caspase
1
2
Caspase 10
124.0 ± 0.3
1483.0 ± 0.2
1276.0 ± 0.3
Means and standard deviations from four independent experiments conducted in
triplicate.
a
AEVD-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by
caspase-10, free AFC emits a yellow-green fluorescence (λmax = 505 nm).
relationship was established in that cytotoxicity varied for R =
Me b Et N n-Pr N n-Bu, introducing branching in R reduced cytotoxicity,
and when R was derived from a cyclic amine, smaller rings were
2
Fig. 10. Interaction of 1 and 2 with HepG2 cells recording ROS production after treatment
at doses corresponding to their IC50 value (0.53 and 55.9μM, respectively) for 16h and la-
belled with carboxy-H2DCFDA for 1 h, after which the fluorescence response was
measured.
more potent [22]. In the present study, it is likely that the different cyto-
toxicity exhibited by 1 and 2 is related to lipophilicity which is reduced
for 2 owing to the presence of hydrophilic hydroxyl substituents. As
both 1 and 2 exhibited significant and some activity, respectively,
against HepG2, this cell line was selected for further studies.
The results of the membrane permeability study showed that the
majority of HepG2 cells underwent apoptosis after treatment with
both 1 and 2, but in the case of 2, some evidence was also detected for
necrosis. The DNA fragmentation study was also consistent with
apoptosis. Apoptopic pathways were also indicated by the experimental
observations that treatment with 1 and 2 enhanced levels of cyto-
chrome c release and ROS production.
As approaches that could prevent metastasis have not been found, the
prevention of tumour metastasis is an urgent topic in cancer therapy.
An invasion assay was performed in the present study and the find-
ings showed that 1 and 2 inhibited cell invasion. As illustrated in Fig. 13,
after treatment with 1 and 2 (IC50=0.53 and 55.9μM, respectively) the
invasion rate of HepG2 cells through matrigel (*p b 0.05) decreased to
3
.96 ± 0.52% and 40.22 ± 2.36% (normalized to 100%), respectively.
A survivin (BIRC5)-XIAP complex activates NF-κB and leads to the
activation of cell motility kinases focal adhesion kinase (FAK) and
c-Src tyrosine kinase (c-Src) that contribute to cancer progression and
metastasis [91]. NF-κB, a heterodimeric DNA-binding protein that
consists of two major subunits, p50 and p65, has been found to play a
crucial role in the evolution and distant metastasis of human cancer
An evaluation of the possible apoptotic pathways was conducted
by quantitative PCR microarray analysis. This showed that induced apo-
ptotic cell death involves both intrinsic and extrinsic pathways.
Compounds 1 and 2 induce a p53-dependent PTP-mediated activation
of the mitochondrial pathway of apoptosis in HepG2 cells allowing for
the release of an apoptosis-inducing factor (AIF), which activates a
DNase leading to apoptosis. The cytotoxicity of 1 was about 100-fold
greater than 2, an observation that is ascribed to the observation that
the caspase-3 expression by 2 is about 20 times less than that induced
by 1.
[92–95]. Activated NF-κB can selectively bind to DNA and leads to the
expression of diverse genes that regulate apoptosis, promote cell prolif-
eration, facilitate angiogenesis, and stimulate invasion and metastasis
[95,96]. However, cell migration and invasion may be increased or de-
creased by XIAP-deficiency in cells depending on the situation [91].
Thus, down-regulation of survivin and XIAP expression by 1 leads to
the inhibition of metastasis in HepG2 cells (Fig. 13).
4
. Discussion
The results from the in vitro cytotoxicity studies indicate that the
IC50 values vary greatly between 1 and 2 in both cisplatin-sensitive
and -resistant cell lines, with 1 being particularly potent and 2 generally
inactive. In fact, 1 promoted significant inhibition of cell proliferation
and potency levels on all tested cancer cell lines at levels comparable
or greater than exhibited by cisplatin, doxorubicin and paclitaxel.
2 2 3
These results are in accord with the original study of Bi(S CNR ) com-
pounds which showed 1 to be the exceptionally cytotoxic against a
range of cancer cells [22]; the role of the dithiocarbamate ligand is the
deliver “bismuth” to the cancer cell. A qualitative structure-activity
Table 3
a
Quantification of caspases -3/7, -8 and -9 in HepG2 cells in the presence of 1 and 2.
Absorbance
Caspase
Control
1
2
Caspase 3/7
Caspase 8
Caspase 9
126.0 ± 0.3
135.0 ± 0.3
130.0 ± 0.2
26946.0 ± 0.3
25782.0 ± 0.4
21805.0 ± 0.3
6365.0 ± 0.2
22018. ± 0.3
13085.0 ± 0.4
Means and standard deviations from four independent experiments were conducted in
triplicate.
Fig. 11. Effect of (a) 1 and (b) 2 on cell cycle distribution in HepG2 cells. Cells were incu-
bated at doses corresponding to the IC50 value (0.53 and 55.9 μM, respectively) for 6, 12
and 24 h, and the distribution of the cell cycle was ascertained by FACS analysis. The
data are representative examples for triplicate independent tests. *p b 0.05 versus control
group.
Excitation wavelength = 490 nm; Emission wavelength = 520 nm.
a
The green fluorescent signal is a direct measure of the amount of active caspases
present in the cells at the time the reagent was added.

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
49
Fig. 12. Effect of 1 and 2 on cell cycle distribution in cultured HepG2 cells. Cells were incubated at doses corresponding to the IC50 value (0.53 and 55.9μM, respectively) for 6, 12 and 24h,
and the distribution of the cell cycle were ascertained by FACS analysis.
In addition to this intrinsic pathway, both 1 and 2 stimulated death
receptor p53 resulting in the activation of an extrinsic pathway. Initially,
p53/p73 is activated in response to the presence of 1 and 2. In turn, p53
promotes expression of Bax, which leads to an imbalance of Bax/Bcl-2
and possible mitochondrial dysfunction. Finally, after activation of
caspase-3, lead-induced apoptosis occurs. Complementing this pathway,
p53 also stimulates the expression of Fas followed by the activation of
receptor-bound FADD and TRADD. FADD is responsible for recruiting
procaspases-8 and -10 and leads to their auto-cleavage and activation,
and subsequently activates effector caspases in initiating cell death.
Apart from the mitochondrial and FAS death receptor apoptotic
pathways, 1 was also found to induce another death receptor-
dependent pathway by induction of CD40, CD40L and TNF-R1 (p55).
In addition, DAPK1, a tumour suppressor, was highly expressed by 1
in HepG2 cells together with down-regulation of XIAP and NF-κB; inac-
tivation of XIAP in cancer cells results in apoptosis. By contrast, XIAP
was significantly expressed by 2 under the same experimental condi-
tions. Over-expression of siDAPk1 significantly rescued the protein
expression of transcription factor NF-κB-targeted genes.
accumulation of cells in these phases being matched by a concomitant
decreases in the percentage of cells in the G /G phase according
0
1
time-dependent experiments conducted up to 24 h. These findings are
supported by the cell cycle RT2 profiler PCR microarray focused on
cyclin-dependent kinase-related pathway genes, PI-3 PIKK family,
serine/threonine-protein kinase and mini-chromosome maintenance
(MCM) proteins.
An invasion assay was also performed with the findings showing
that the cell invasion rate of 2 is 10-fold higher than 1. The down-
regulation of survivin and XIAP expression by 1 leads to the inhibition
of metastasis in HepG2 cells.
The present study showed that 1 and 2 interact with DNA through
different binding motifs. Thus, the reactivity of the Ssp I, Nde I and Bst
11071 (AT or TA specific) restriction enzymes was mitigated after treat-
ment with 1, thereby indicating that the AT or TA specific sites were oc-
cupied by 1 followed by the inhibition of restriction enzyme digestion.
On the other hand, 2 did not interfere with any restriction enzyme dem-
onstrating that they are neither AT- nor GC-specific and therefore, the
compound might not interact with or just randomly bind to DNA. This
finding provides a further explanation as to why 1 is a better cytotoxic
agent than 2 in HepG2 cells.
The inhibition in the growth of HepG2 cancer cells mediated by 1
and 2 occurred via cell cycle arrest at the S and G /M phases with the
2

50
D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
Fig. 13. Matrigel invasion assay showing that 1 and 2 inhibited cell invasion. (a) Photos were taken at 200× magnification, and are representative of three independent experiments.
b) Graphical representation of the numbers of invaded cells per microscopic fields when treated with 1 and 2. Data were shown as mean ± SEM from three independent experiments.
p b 0.05, compared to the DMSO only. The invasion rates of 1 and 2 are 3.96 ± 0.52 and 40.22 ± 2.36%, respectively (normalized to 100%).
(
*
5
. Conclusions
Cdc2
cell division control protein 2 homolog
CDKN1A cyclin-dependent kinase inhibitor 1A
CDKN1A cyclin-dependent kinase inhibitor p21
Bi(S
2
CNEt
2
)
3
(1) is shown to be cytotoxic against a range of human
CNCH CH2OH) (2) is considerably less cytotoxic.
carcinoma but, Bi(S
2
2
3
Chek1
COX-2
c-Src
serine/threonine protein kinase
cyclooxygenase-2
c-Src tyrosine kinase
Against HepG2, mechanisms of cell death induced by 1 and 2 occur by
mitochondrial and FAS apoptotic pathways. In addition, 1 highly
expressed the tumour suppressor DAPK1, down-regulated both XIAP
and NF-κB, and also induced CD40, CD40L and TNF-R1 (p55). Growth
inhibition of HepG2 is correlated with cell cycle arrest at the S and G /
2
M phases. The cell invasion rate of 1 is 10-fold less than 2, and 1 interacts
DAPK1 death-associated protein kinase 1
DFFA
EtBr
FADD
FAK
DNA fragmentation factor subunit alpha
ethidium bromide
Fas-associated protein with death domain
Focal adhesion kinase
with DNA at AT- or TA-specific sites.
FAS
tumour necrosis factor superfamily, member 6
Abbreviations
GADD45 growth arrest and DNA Damage protein 45
ICAM-1 intercellular adhesion molecule-2
AIF
Apoptosis-inducing factor
MCM
MMT
protein Mini-chromosome maintenance
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium
bromide
polymerase chain reaction
PI-3 protein kinase-like kinase
reactive oxygen species
APAF-1 Apoptosis activating factor-1
ATM
ATR
Bax
Bcl-2
BID
gene ataxia telangiectasia mutated gene
protein Rad-3 related protein
Bcl-2 associated X protein
b-cell lymphoma 2
PCR
PIKK
ROS
TAE
tBID
BH3 interacting domain death agonist
Tris-acetate EDTA
truncated BH3 interacting-domain death agonist
BNIP3L protein BCL2/adenovirus E1B 19 kDa interacting protein 3-
like
TNF-R1 tumour necrosis factor receptor superfamily, member 1A
TNFRSF1A tumour necrosis factor receptor
XIAP
BRIC5
c-Abl protein c-abl oncogene 1, non-receptor tyrosine kinase
Carboxy-H DCFDA 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein
diacetate
cyclin B
cyclin D
cyclin E
benign recurrent intrahepatic cholestasis protein 5
X-lined mammalian inhibitor of apoptosis protein
2
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2013.09.018.
CCNB
CCND
CCNE
CD40
1
1
1
1
1
1
Conflict of interest
tumour necrosis factor receptor superfamily, member 5
tumour necrosis factor superfamily, member 5
CD40L
The authors have no conflict of interest to disclose.

D.H.A. Ishak et al. / Journal of Inorganic Biochemistry 130 (2014) 38–51
51
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Acknowledgements
The authors thank the Ministry of Higher Education of Malaysia and
the University of Malaya (UM.C/HIR-MOHE/SC/03) for funding these
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