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b-Mangostin induces p53-dependent G2/M cell cyclearrest and apoptosis through ROS mediatedmitochondrial pathway and NfkB suppressionin MCF-7 cells
1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.10.018
* Corresponding authors. Address: UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra MalaysiaSelangor, Malaysia (S. Syam). Tel.: +60 389472120; fax: +60 389472101.
E-mail address: [email protected] (S. Syam).Abbreviations: bM, b-mangostin; MMP, mitochondrial membrane potential; NF-kB, nuclear factor-kappa B; TNF-a, tumou
factor alpha; ROS, reactive oxygen species; DCFH-DA, 2 0,7 0-dichlorofluorescein diacetate; HCS, high content screening; PARPribose polymerase; GADD, growth arrest DNA damage; XIAP, X-linked inhibitor of apoptosis proteiphenylmethanesulfonylfluoride
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dependent G2/M cell cycle arrest and apoptosis through ROSmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.201
Suvitha Syama,d,*, Ahmad Bustamama,*, Rasedee Abdullahb, Mohamed Aspollah Sukaric,Najihah Mohd Hashimd, Mostafa Ghaderiand, Mawardi Rahmanic, Syam Mohane,Siddig Ibrahim Abdelwahabe, Hapipah Mohd Alif
aUPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, MalaysiabDepartment of Veterinary Pathology and Microbiology, Faculty of Veterinary, University Putra Malaysia, Serdang, Selangor, MalaysiacDepartment of Chemistry, Faculty of Science, University Putra Malaysia, Serdang, Selangor, MalaysiadDepartment of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaeMedical Research Centre, Jazan University, P.O. Box 114, Jazan, Kingdom of Saudi ArabiafDepartment of Chemistry, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
A R T I C L E I N F O A B S T R A C T
Article history:
Received 10 July 2013
Received in revised form
23 October 2013
Accepted 24 October 2013
Available online xxxx
Keywords:
Cratoxylum arborescens
b-Mangostin
Apoptosis
p53
NF-kB
Bax/Bcl-2
b-Mangostin (bM) was isolated from Cratoxylum arborescens to investigate its anti-cancer
effect in MCF-7 cells. bM induced apoptosis by down-regulation of Bcl2 and up-regulation
of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of
caspase-9 and -7 and consequently cleaved PARP leading to apoptotic was observed upon
treatment. Reduction of both bid and caspase 8 and the up regulation of Fas showed the
involvement of the extrinsic pathway. Significantly up regulated GADD45A and HRK genes
were observed upon treatment, with concomitant inhibition of NF-kB to nucleus. The pro-
tein array had demonstrated the expression of HSP 70, HSP 60, XIAP, Survivin, p53 and Bax.
Moreover, bM had showed p53-dependent G2/M cell cycle arrest by down regulation of cdc2
and PCNA. Together, the results demonstrated that the bM induced anti-proliferative effect,
leading to G2/M phase cell cycle arrest and apoptosis through both the extrinsic and mito-
chondrial pathways with the involvement of the multiple pro and anti-apoptosis and NF-kB
signalling pathways.
� 2013 Elsevier Ltd. All rights reserved.
, Serdang,
r necrosis, poly ADPn; PMSF,
mediated3.10.018
2 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
1. Introduction
Xanthones are chemical compounds found naturally in a
variety of fruits and vegetables. Moreover, they carries various
biological, biochemical and pharmacological activities sug-
gesting that they significantly affect basic cell functions such
as growth, differentiation and/or programmed cell death
(apoptosis). Among the available xanthones, recently, mango-
stin compounds have gained much attention due to its effi-
cacy in diseases especially cancer and related. Hence foods
containing mangostins have been considered as nutraceuti-
cals in functional foods and dietary supplements (Bumrung-
pert et al., 2009). Among the mangostins a and c mangostins
has been studied extensively. a Mangostin inhibited cell inva-
sion and migration in mammary and prostate cancer cells
(Hung, Shen, Wu, Liu, & Shih, 2009), reduction of rat colon car-
cinogenesis (Shibata et al., 2011), inhibition of prostate cancer,
inhibition of estrogen receptor positive breast cancer (Lee
et al., 2010) and head and neck cancer (Kaomongkolgit,
Chaisomboon, & Pavasant, 2011).
Breast cancer is the most common malignancy in European
women and the fifth most common cause of cancer death in
the world. Approximately one-third of women with breast
cancer developed metastases and ultimately died of this dis-
ease (Ferlay et al., 2010). In Malaysia, incidence of female
breast cancer was 46.2 per 100,000 populations, meanwhile
survival is generally lower with conventional therapeutic
strategies which include surgery, radiation and chemotherapy
(Saxena et al., 2012). Even though breast cancer initially re-
sponds to chemotherapy, they may subsequently survive
and lead to resistance to existing treatment regimen (Li
et al., 2008). Due to their limited effectiveness and side effects,
there is still a pressing need for the development of anti-breast
cancer drugs. Since considered relatively safe and cheaply
available, natural products and their metabolites is the target
of anti-cancer drug discovery now days. Moreover, com-
pounds which regulate apoptosis and overcome the apoptosis
deficiency of cancer cells are of high medical significance
(Daniel, Koert, & Schuppan, 2006).
Significantly increasing evidences suggest that the neo-
plastic alteration, progression and metastasis related pro-
cesses involve the modification of normal apoptotic
pathways (Yang et al., 2006). In this regard, the relation be-
tween malignancy and apoptosis has attracted much atten-
tion, especially in the drug discovery area. Apoptosis is a
cell suicide program essential for controlling cell numbers
in development and for adult tissue homeostasis in all meta-
zoan animals which is a strictly regulated pathway responsi-
ble for the ordered removal of surplus, aged, and injured cells
(Elmore, 2007). The stereotypical death throes of a cell under-
going apoptosis include morphological hallmarks such as loss
of cell volume, cell shrinkage, nuclear condensation, plasma
membrane blebbing, chromosomal DNA fragmentation, and
the formation of apoptotic bodies, followed by cleavage of
the nucleus and cytoplasm into multiple membrane-enclosed
bodies containing chromatin fragments (Isa et al., 2012). Mito-
chondria are dynamic organelles, which is regarded to play a
central role in programmed cell death and can serve as a no-
vel target for chemotherapies. Loss of the mitochondrial
membrane potential, termination of oxygen consumption,
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
and the release of cytochrome c has been the biochemical
hallmark of mitochondrial apoptosis (Zamzami & Kroemer,
2003). Moreover, the involvement of mitochondria in the reg-
ulation of apoptosis is controlled by anti- and pro-apoptotic
members of the Bcl-2 family such as Bcl-2, Bcl-xL, Bax, Bak,
Bad, and Bid. This may then lead to the downstream activa-
tion of caspase cascades and leading to cell death (Gross,
McDonnell, & Korsmeyer, 1999). Recently, considerable atten-
tion has been devoted to the sequence of events referred to as
apoptotic cell death and the role of this process in mediation
of the lethal effects of the diverse antineoplastic agents.
Even though bM is structurally very close to a mangostin,
except very few studies (Matsumoto et al., 2003, 2005; Taher
et al., 2012), there are no detailed reports regarding the anti-
cancer mechanism of bM to the best of our knowledge. Hence,
in the current study, we isolated bM from Cratoxylum arbores-
cens (family: Guttiferae) and evaluated its apoptosis activity
on MCF-7 breast cancer cells. The estrogen receptor (ER)-posi-
tive MCF-7 cell line has been studied longer than any other
breast cancer cell model system. This cell variant is a novel
tool for the study of breast cancer resistance to chemotherapy,
because they appear to mirror the heterogeneity of tumor cells
in vivo (Simstein, Burow, Parker, Weldon, & Beckman, 2003).
2. Materials and methods
2.1. Plant material and compound isolation
The ground air-dried stem bark of C. arborescens (1.0 kg) was
soaked at room temperature in hexane for three days and re-
peated thrice. The extract was filtered and then concentrated
by using rotary evaporator under reduced pressure to give dark
gummy semisolid residue. The plant material was then
sequentially extracted with chloroform, and methanol. The
weights of hexane, chloroform, and methanol crude extracts
obtained were 6.12, 28.18, and 40.27 g, respectively. The hexane
extract (6.12 g) was separated by vacuum column chromatog-
raphy and eluted with hexane and followed by mixtures of sol-
vents, hexane/chloroform, chloroform/ethyl acetate and ethyl
acetate/methanol to give 26 fractions of 200 ml each. Similar
fractions based on TLC and observed under UV light were com-
bined. Fraction 14 was further separated by mini column chro-
matography to give 56 fractions. Fractions 16–52 (eluted with
50% ethyl acetate: 50% methanol) was similarly further
purified by preparative thin layer chromatography as well as
chromatotron to give yellowish solid and was identified as
b-mangostin. Similar separation and fractionation of the
chloroform and methanol extracts with series of column chro-
matography led to the isolation another batch of b-mangostin.
2.2. Isolation of bM
b-Mangostin was initially obtained as yellowish solid and yel-
lowish needle-shaped crystals after being recrystallised with
hot chloroform. IR mmax cm�1 (KBr): 3407 (OH), 2923 (CH),
1642 (C@O) and 1596 (C@C); UV MeOH kmax nm (log e): 374
(1.89), 350 (3.99), 344 (2.19), and 340 (3.29); EIMS m/z (% inten-
sity): 424 (53.79), 409 (5.20), 393 (1.19), 3.81 (19.45), 368 (31.93),
353 (100.00), 335 (20.79), 310 (7.59), 299 (23.66) and 169 (8.41);1H-NMR (500 MHz, acetone-d6): d 13.61 (OH-1), 9.62 (OH-6),
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 3
6.82 (s, 1H, H-5), 6.47 (s, 1H, H-4), 5.25 (t, J = 6.9 Hz, 1H, H-12),
5.18 (t, J = 6.9 Hz, 1H, H-17), 4.10 (d, J = 6.9 Hz, 2H, H-11), 3.94
(OMe-3), 3.77 (OMe-7), 3.29 (d, J = 6.9 Hz, 2H, H-16), 1.80 (s,
3H, Me-14), 1.75 (s, 3H, Me-19), 1.63 (s, 3H, Me-15) and 1.61
(s, 3H, Me-20); 13C-NMR (125 MHz, acetone-d6): d 186.8 (C-9),
168.5 (C-4a), 164.4 (C-1), 161.5 (C-10a), 160.2 (C-6), 160.1 (C-3),
148.5 (C-7), 142.0 (C-8), 135.4 (C-18 and C-13), 128.6 (C-12),
127.2 (C-17), 115.9 (C-8a), 115.7 (C-2), 108.0 (C-9a), 106.6 (C-5),
93.8 (C-4), 65.2 (OMe-7), 60.4 (OMe-3), 30.8 (C-11), 29.8 (C-15),
29.8 (C-20), 25.8 (C-16), 22.2 (C-14) and 21.7 (C-19).
The structure of b-mangostin (Fig. 1A) was established due
to significant correlations in Heteronuclear Multiple Bond
Connectivity, Heteronuclear Multiple Quantum Coherence,
Distortionless enhancement by polarisation transfer together
with the signals displayed by 1H and 13C-NMR spectra. The
fragmentation pattern and molecular mass of the compound
were further confirmed by the electron impact mass spec-
trometry and the typical absorption bands of the functional
groups were displayed from the Infrared spectroscopic data.
In comparison with literature values, the compound was
identified as b-mangostin previously reported to occur in C.
arborescens (Sim, Ee, Lim, & Sukari, 2011).
2.3. Cell culture
MCF-7 cells were obtained from American Type Cell Collec-
tion (ATCC) and were maintained in 37 �C incubator with 5%
CO2 saturation. They were maintained in RPMI-1640 medium
that is supplemented with 10% fetal bovine serum (FBS).
2.4. Cellular viability assay
The inhibitory effect of bM was determined by MTTassay. This
colorimetric assay is based on the conversion of the yellow tet-
razolium bromide (MTT) to the purple formazan derivatives by
mitochondrial succinate dehydrogenase in viable cells. Cells
were seeded at the density of 1 · 105 cells/ml in 96-well plate
and incubated for 24 h at 37 �C with 5% CO2 saturation. After
incubation, the cells were treated with bM (dissolved in 1%
DMSO) at different concentration and incubated for another
24 and 48 h. After the drug treatment, 20 ll of MTT solution
at 5 mg/ml was added and incubated for 4 h. Dimethyl sulfox-
ide (DMSO) in volume of 100 ll is added into each well to dis-
solve the purple formazan formed. The colorimetric assay is
measured and recorded at absorbance of 570 nm.
Results were expressed as percentage of control giving per-
centage cell viability after exposure to test agent. The potency
of cell growth inhibition for test agent was expressed as IC50
value, defined as the concentration that caused a 50% loss of
cell growth. Viability was defined as the ratio (expressed as a
percentage) of absorbance of treated cells to untreated cells.
2.5. Morphological assessment of apoptotic cells byacridine orange (AO) propidium iodide (PI) double staining
bM-induced cell death in MCF-7 cells was quantified using
acridine orange (AO) and propidium iodide (PI) double-stain-
ing according to standard procedures and examined under
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
fluorescence microscope (Lieca attached with Q-Floro Soft-
ware). Briefly, treatment was carried out in a 25 ml culture
flask. The cells were plated at 1 · 105 cells/ml and treated with
bM. The cells were then centrifuged at 300g for 10 min. Super-
natant was discarded and the cells were washed twice using
PBS after centrifuging at 300g for 10 min to remove the
remaining media. Ten microliters of fluorescent dyes contain-
ing AO (10 mg/ml) and PI (10 mg/ml) were added into the cel-
lular pellet at equal volumes. Freshly stained cell suspension
was dropped into a glass slide and covered by a cover slip.
Slides were observed under UV-fluorescence microscope
within 30 min before the fluorescence fade. AO and PI are
intercalating nucleic acid-specific fluorochromes which emit
green and orange fluorescence, respectively, when they are
bound to DNA. Of the two, only AO can cross the plasma
membrane of viable and early apoptotic cells. The criteria
for identification are as follows: (i) viable cells appear to have
green nucleus with intact structure; (ii) early apoptosis exhibit
a bright-green nucleus showing condensation of chromatin in
the nucleus; (iii) dense orange areas of chromatin condensa-
tion showing late apoptosis and (iv) orange intact nucleus
depicting secondary necrosis (Mohan et al., 2011).
2.6. Annexin V assay
Cells (1 · 105 cells/ml) were exposed to 7 lg/ml concentrations
of bM for 12, 24 and 48 h and the Annexin V assay was per-
formed using the BD Pharmingen� Annexin V-FITC Apoptosis
Detection Kit (APO Alert Annexin V, Clon Tech, California,
USA). Briefly, treated cells were centrifuged for 10 min at
200g to remove the media. Later, the cells were rinsed with
1· binding buffer supplied by the manufacturer. The rinsed
cells were resuspended in 200 ll of binding buffer. To this,
5 ll of Annexin V and 10 ll of propidium iodide (Sigma, Saint
Louis, Missouri, USA) were added and incubated at room tem-
perature for 15 min in dark. Flow cytometric analysis was car-
ried out using FACS Canto II Becton–Dickinson flow cytometry
by analyzing at least 10,000 cells per sample. The binding buf-
fer supplied by the manufacturer was used to bring the reac-
tion volume to at least 500 ll for the flow cytometry analysis.
DMSO-treated (0.1%, v/v) cells were used as control.
2.7. Multiple cytotoxicity assay
Cellomics multiparameter cytotoxicity 3 kit was used as de-
scribed in detail previously (Mohan et al., 2012). This kit en-
ables simultaneous measurements in the same cell of six
independent parameters that monitor cell health, including
cell loss, nuclear size and morphological changes, mitochon-
drial membrane potential changes, cytochrome c release, and
changes in cell permeability. Plates were analysed using the
ArrayScan HCS system (Cellomics, PA, USA). Images and data
regarding intensity and texture of the fluorescence within
each cell, as well as the average fluorescence of the cell pop-
ulation within the well were stored in a Microsoft SQL data-
base for easy retrieval. Data were captured, extracted and
analysed with ArrayScan II Data Acquisition and Data Viewer
version 3.0 (Cellomics).
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
C
A
*
**
D
**
**
*
E
VIVI
BLCC
LA
BL
BL
SN
AB
B
Fig. 1 – (A) Structure of b-mangostin [1,6-dihydroxy-3,7-dimethoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one]. (B)
Cytotoxicity of bM on MCF-7 cells measure by MTT assay at 48 and 24 h. (C–F) Fluorescent micrographs of acridine orange and
propidium iodide double-stained MCF-7 cells. Untreated cells after 48 h showed normal structure without prominent
apoptosis and necrosis (C). Early apoptosis features were seen after 12 h representing intercalated acridine orange (bright
green) amongst the fragmented DNA (D), Blebbing and orange color representing the hallmark of late apoptosis were noticed
in 24 h treatment (E), bright red colored secondary necrosis were visible after 48 h. VI, viable cells; BL, blebbing of the cell
membrane; CC, chromatin condensation; LA, late apoptosis; SN, secondary necrosis; AB, apoptosis bodies. Images are
representative of one of three similar experiments. Statistical significance is expressed as *P < 0.05. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
4 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
2.8. Immunofluorescence analysis of Bax/Bcl-2
5000 cells seeded in 96 well plate (Genetix). The cells were
treated with bM and incubated for 12, 24 and 48 h. Then cells
were washed twice with PBS and then fixed in 4% paraformal-
dehyde for 15 min at room temperature. After washing three
times in PBS, the cells were treated by blocking buffer for
60 min incubation in 0.03% Triton X-100/PBS and normal ser-
um then cells were washed again with PBS. Diluted primary
antibody solution contains 1· PBS/1% BSA/0.3% Triton
X-100, was added after aspirate of blocking buffer. The cells
were incubated for overnight at 4 �C. Bcl-2 and Bax
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
flurochrome-conjugated secondary antibody diluted (Santa
Cruz Biotechnology, Santa Cruz, CA) in antibody dilution or
in PBS only was added to the cells and incubated for 1 h. After
washing three times in PBS, the cells were treated with DAPI
to be examined using CellReporter� cytofluorimeter system
(Gentix/Molecular devices, United Kingdom).
2.9. Caspase activity assay
Caspase-3/7, -8 and -9 activity was measured using lumines-
cence-based assay, Caspase-Glo� 3/7, Caspase-Glo� 8 and
Caspase-Glo� 9 Assay (Promega). Cells were cultured in
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 5
96-well culture plates in 50 ll of RPMI 1640 supplemented
with 10% FBS and incubated for 24 h. Cells then were treated
with bM for 12, 24 and 48 h. At the end of incubation, 100 ll of
assay reagent was added to be incubated for 1 h at room tem-
perature. Luminescence was measured using a microplate
reader (Tecan Infinite M 200 PRO, Mannedorf, Switzerland).
2.10. Measurement of reactive oxygen species generation
The production of intracellular reactive oxygen species (ROS)
was measured using 2 0,7 0-dichlorofluorescin diacetate (DCFH-
DA). Briefly, 10 mM DCFH-DA stock solution (in methanol) was
diluted 500-fold in Hank’s balanced salt solution (HBSS) with-
out serum or other additives to yield a 20 lM working solu-
tion. After 24 h of exposure to bM the cells in the 96-well
black plate was washed twice with HBSS and then incubated
in 100 ll working solution of DCFH-DA at 37 �C for 30 min.
Fluorescence was then determined at 485-nm excitation and
520-nm emission using a fluorescence microplate reader
(Tecan Infinite M 200 PRO, Mannedorf, Switzerland).
2.11. Cell cycle analysis
MCF-7 cells at concentration of 1 · 105 cells/ml were seeded
into 25 ml culture flask (TPP Brand, Trasadingen, Switzerland)
which contains RPMI 1640 (PAA Laboratories, Coelbe,
Germany) medium supplemented with 10% FBS and treated
with bM in several incubation time period (12, 24, and 48 h).
After incubation, the cells were spun down at 200g for
10 min. Supernatant was discarded and the pellets were
washed with PBS twice to remove any remaining media. In or-
der to restore cellular integrity, fixation for flow cytometery
analysis was performed. Briefly, cells pellets were fixed by
mixing 500 ll of 70% cold ethanol and 250 ll of cell suspen-
sion and kept at �20 �C overnight. The cells were then spun
down at 200g for 10 min and excess ethanol decanted. After
washing twice with PBS, cells were resuspended in PBS.
Twenty microliters of RNase A (10 lg/ml) and 2 ll of propidi-
um iodide (PI) (2.5 lg/ml) were added to the fixed cells for
30 min in dark on ice. PI has the ability to bind to RNA mole-
cule and hence, RNase enzyme was added in order to allow PI
to bind directly to DNA. The DNA content of cells was then
analysed using a FACS Canto II Becton–Dickinson flow cytom-
etry by analyzing at least 10,000 cells per sample. The per-
centage of cells in G1, S and G2 phases were analysed by
ModFit LT software (Verity Software House, Topsham, ME).
2.12. Western blot analysis
MCF-7 cells in 25 ml culture flask (TPP Brand, Trasadingen,
Switzerland) were treated with bM for 12, 24 and 48 h. The to-
tal proteins of cells were extracted with cell lysis buffer
(50 mM Tris–HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM
PMSF), and 40 lg of protein extract was separated by 12%
SDS–PAGE, then transferred to a polyvinylidenedifluoride
(PVDF) membrane (Bio-Rad), blocked with 5% nonfat milk in
TBS-Tween buffer 7 (0.12 M Tris–base, 1.5 M NaCl, 0.1% Tween
20) for 1 h at room temperature, and incubated with the
appropriate antibody overnight at 4 �C, then incubated with
horseradish peroxidase conjugated secondary antibody for
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
30 min at room temperature. The bound antibody was de-
tected with peroxidase-conjugated anti-rabbit antibody
(1:10,000) or anti-mouse antibody (1:10,000) followed by
chemiluminescence (ECL System) and exposed by autoradiog-
raphy. The following primary antibodies b-actin (1:10,000),
cdc2 (1:500), PCNA (1:1000), p53 (1:1000), PARP (1:1000) were
purchased from Santa Cruz Biotechnology, Inc., California,
USA.
2.13. Detection of NF-kB activity
HCS was used to measure the inhibitory effects of bM on
TNF-a-induced NF-kB activation, i.e., nuclear translocation
of NF-kB. The experiments were performed according to
manufacturer’s instructions for the NF-kB activation kit
(Cellomics). ArrayScan reader was used to quantify the differ-
ence between the intensity of nuclear and cytoplasmic NF-kB-
associated fluorescence, reported as translocation parameter.
2.14. Apoptotic gene expression profiling using real-timePCR array
Total RNA was isolated from treated cells using the RNeasy
Micro Kit – Qiagen. RNAs with an OD260 nm/OD280 nm absor-
bance ratio of at least 2.0 were used. Total RNA was reverse-
transcribed into cDNA using the RT2 First strand Kit (Qiagen),
mixed with RT2 qPCR mastermix containing SYBR Green (Qia-
gen), and aliquoted in equal volumes to each well of the real-
time PCR arrays. The Human Apoptosis RT2 Profiler� PCR Ar-
ray (Qiagen) interrogates 84 genes related to the apoptotic
pathway. The real-time PCR cycling program was run on a
on a StepOne Plus real-time PCR system (Applied Biosystems).
The threshold cycle (Ct) of each gene was determined and
subsequently analysed by RT2 Profiler PCR array data analysis
software; http://pcrdataanalysis.sabiosciences.com/pcr/
arrayanalysis.php.
2.15. Human apoptosis proteome profiler array
To investigate the pathways by which bM induces apoptosis,
we performed determination of apoptosis-related proteins
using the Proteome Profiler Array (RayBio� Human Apoptosis
Antibody Array Kit, Raybiotech, USA), according to manufac-
turer’s instructions. In short, the cells where treated with
7 lg/ml bM. Three hundred micro gram proteins from each
sample were incubated with the human apoptosis array over-
night. The apoptosis array data were quantified by scanning
the membrane on a Biospectrum AC ChemiHR 40 (UVP, Up-
land, CA) and analysis of the array image file was performed
using image analysis software according to the manufac-
turer’s instruction.
2.16. Statistical analysis
Results were reported as mean ± SD for at least three analyses
for each sample. Normality and homogeneity of variance
assumptions were checked. Statistical analysis was per-
formed according to the SPSS-16.0 package and GraphPad
prism 5.0. Analyses of variance were performed using the
ANOVA procedure.
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
6 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
3. Results
3.1. bM inhibited the growth of MCF-7 cells
The cytotoxicity of bM was studied using the MTT assay. The
results are shown in Fig. 1B. The bM inhibited the MCF-7 cells
with an IC50 of 7.3 ± 0.22 lg/ml (16.5 lM) and 11.2 ± 0.61 lg/ml
(26 lM) for 48 and 24 h respectively. Meanwhile, the normal
cells (MCF10A) used in this study did not died significantly
even at the highest concentrations (30 lg/ml) of bM. These re-
sults thus indicate that bM is highly cytotoxic to tumor cells.
3.2. Apoptosis determination using AO/PI double-staining
The cells were observed under fluorescence microscope. We
have observed the early apoptosis by intervened acridine or-
ange within the fragmented DNA with bright green fluores-
cence. At the same time, control cells were observed with a
green intact nuclear structure. At 12 h treatment with bM,
moderate apoptosis were seen by blebbing and nuclear chro-
matin condensation. Furthermore, in the late stages of apop-
tosis, changes such as presence of reddish-orange color due
to the binding of AO to denatured DNA were observed after
24 and 48 h of treatment (Fig. 1C–F). The results showed that
bM generated morphological features that relate to apoptosis
in a time-dependent manner.
3.3. Indication of an early stage of apoptosis wasdetermined using Annexin V
As shown in Fig. 2, the Annexin Vassay revealed that the apop-
totic induction in MCF-7 cells began after being exposed to bM
at 7 lg/ml. For the untreated control, 84% cellswere viable, 2.9%
was in the early stages of apoptotic induction and 7.1% were in
the late stages of apoptosis/dead. After 12 h incubation withbM
at 7 lg/ml, the early apoptotic population (Annexin V-positive,
PI-negative) significantly (p < 0.05) rose to 20%. After 24 h of
exposure to bM, the number of viable cells decreased to 65.9%
with a concomitant increased in both early apoptotic and late
stage of apoptotic/dead cell (positive for both Annexin V and
PI) populations. After 48 h of exposure the number of early
apoptosis cells were decreased, but with significant migration
of cells to late stage of apoptosis (Fig. 2E). These results indi-
cated that the bM allowed the translocation of PS to occur
and hence induced early apoptotic induction in MCF-7 cells.
3.4. bM-induced apoptosis disrupt the MMP and releasecytochrome c
To confirm the presence of apoptosis seen in the AO/PI and
Annexin V assay, we then carried out multi parameter cyto-
toxicity assay, which simultaneously measures 4 important
characteristics of apoptosis. The assay, showed the nuclear
condensation and fragmentation hallmark for apoptosis.
Hoechst 33342 staining showed that a part of the cells dis-
played nuclear condensation at all the treatment concentra-
tions of bM treatment (Fig. 3A). The nuclear intensity which
is directly corresponding to apoptotic chromatin changes:
blebbing, fragmentation and condensation were quantitated
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(Fig. 3B). Meanwhile, concurrent increase in the cell perme-
ability was also observed (Fig. 3C).
MMP was significantly reduced on cells treated with bM
(p < 0.05) (Fig. 3D). Changes of mitochondrial membrane po-
tential in MCF-7 cells treated with bM 7 lg/ml for 24 and
48 h showed a significant reduction of fluorescence intensity,
which reflected the collapse of MMP. Meanwhile, bM signifi-
cantly triggered the cells to translocate the cytochrome c from
mitochondria into cytosol during apoptosis (Fig. 3).
3.5. Expression of Bcl-2 and Bax
The constitutive levels of mitochondrial proteins such as Bcl-2
or Bax and the time course for the effect of bM on Bcl-2 or Bax
expression in MCF-7 cells were studied by immunofluorecent
analysis. The levels of Bcl-2 expression in the MCF-7 cells were
slightly down-regulated with the addition of bM when ex-
posed for 24 and 48 h. In contrast, the expression of Bax was
significantly up-regulated (Fig. 4). The fluorescent intensity
measurement was also conducted to obtain a quantitative va-
lue for the protein expression of Bax and Bcl-2 (Fig. 4B). The fig-
ure shows that both up and down-regulation of bax and bcl-2
were statistically significant (P < 0.05) and time dependent.
3.6. Expression of p53 and PARP
The expression of p53 and PARP clevage on MCF-7 cells with
or without bM treatment was tested by Western blot analysis.
As shown in Fig. 5, after 24 and 48 h of treatment with 7 lg/ml
of bM, the level of p53 protein has decreased significantly.
Since PARP clevage results in cleaved product of 89 kDa, we
have observed the reduction in the PARP (116 kDa) and con-
comitant presence of cleaved product at 24 and 48 h.
3.7. bM inhibits TNF-a-induced NF-kB nucleartranslocation
The role of bM in the inhibition of activated NF-kB induced by
the inflammatory cytokine, TNF-a using Alexa Fluor 488-con-
jugated anti-NF-kB antibody was examined. In control cells
high NF-kB fluorescent intensity was found only in cytoplasm.
At the same time, cells stimulated with TNF-a alone increased
the NF-kB fluorescent intensity in the nuclei. bM significantly
inhibited the activation of NF-kB to nucleus both in 15 and
20 lg/ml treatment in a dose-dependent manner (Fig. 6).
3.8. bM inhibits the MCF-7 cell proliferation and arrestG2/M phase cell cycle
The cell cycle analysis performed to evaluate the presence of
cell cycle arrest and apoptosis. Results of this experiment
demonstrated that the bM arrested the cell cycle progression
at G2/M phase (p < 0.05). Results shown in Fig. 7E indicate that
there is significant G2/M phase arrest in a time depended
manner, accounting for 26%, cells after 48 h treatment
(p < 0.05). The significantly increased population of hypodip-
loid (sub G1/apoptosis) also was observed. In order to confirm
the cell cycle arrest especially at G2/M phase, we have per-
formed the western blot analysis of both PCNA and cdc2
protein. The results shown in Fig. 7F clearly showed
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
A B
C D
E
Fig. 2 – The effect of bM on apoptosis of MCF-7 cells. Cells were exposed to 7 lg/ml and incubated at 37 �C in a CO2 incubator.
After staining with FITC-conjugated Annexin V and PI, cells were analysed by flow cytometry. Control cells received no drug
treatments. The early apoptotic events (Annexin+/PI�) are shown in lower right quadrant (Q4) of each panel. Quadrant Q2
represents Annexin+/PI+ late stage of apoptosis/dead cells. (A) The MCF-7 control (n = 2). (B–D) The effects of bM for 12, 24 and
48 h exposure (respectively). Statistical significance is expressed as *P < 0.05.
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 7
significant down regulation of both the protein at various
time points.
3.9. bM increased activity of caspase-3/7, -8 and -9enzymes
When MCF-7 cells were treated with bM, the caspase-3/7, -8
and -9 enzyme activities was found. All the three caspases
were significantly increased at 24 and 48 h treatment, in par-
ticularly caspase 9 (p < 0.05) (Fig. 8A).
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3.10. bM induced cell death includes increased ROSformation
The generation of intracellular ROS is always associated with
MMP disruption and cell apoptosis. Therefore, we examined
the levels of ROS in MCF-7 cells treated with bM. ROS was
monitored by the oxidation-sensitive fluorescent dye DCFH-
DA. A concentration depended increase in DCF fluorescence
was detected in treated cells (Fig. 8B). Rapid generation of
ROS, up to 2-fold faster than the control, was detected at
20 lg/ml treatment.
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
Hoechst 33342 Cell Permeability Mitochondrial Membrane Potential
Cytochrome c Composite image
Control
βM (7μg/ml)
Tamoxifen
AA
B D
E F
Fig. 3 – Effect of bM on MMP, permeability and cytochrome c release. (A) Representative images of MCF-7 cells treated with
medium alone and 7 lg/ml of bM, and stained with Hoechst for nuclear, cell permeability dye, MMP and cytochrome c. The
images from each row were obtained from the same field of each sample (magnification 20·). (B–E) Average fluorescence
intensities of Hoechst 33342, cell permeability dye, MMP and cytochrome c in MCF-7 cells treated with bM or standard drug
Tamoxifen. Data were mean ± SD of fluorescence intensity readings measured from different photos taken (*P < 0.05).
8 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dependent G2/M cell cycle arrest and apoptosis through ROS mediatedmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
Hoechst FITC Composite
Bcl-2
Bax
Control
Control
βM(48 h)
βM(48 h)
A B
Fig. 4 – bM change the regulation of Bax/Bcl-2 expression. (A) MCF-7 cells were treated with 7 lg/ml of bM for 12, 24 and 48 h.
Cells were stained with Hoechst 33342 for nucleus and FITC flurochrome-conjugated secondary antibody for Bax and Bcl2
expression respectively. (B) Fluorescence intensity of FITC in cells treated with designated concentration of bM. Data were
shown as mean ± SD. Significant differences (*p < 0.05) between bM-treated and untreated control cells.
p53
PARP
B ac n
116 kDa
89 kDa
C 12 24 48 h
**
* *
*
A B
Fig. 5 – The effect of bM on apoptosis regulatory proteins in MCF-7 cells was determined by Western blot. (A) Detection of
protein was done by specific antibodies with beta actin as a loading control. (B) Quantitative band intensity provided as ratio.
The statistical significance is expressed as *P < 0.05.
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 9
3.11. Human apoptosis PCR array
In order to deepen the understanding of the apoptosis process
in bM treated cells, we performed a PCR array designed to
determine the expression profile of a group of 84 genes. We
have restricted the gene which was up or down regulated
more than 4-fold. The treatment with bM significantly up reg-
ulated GADD45A and HRK genes. Meanwhile genes such as
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
CASP14, DAPK1, TNFRSF11B and CD70 were found to be down
regulated, all of which are involved in apoptosis (Table 1).
3.12. Human apoptosis protein array
After bM (7 lg/ml) exposure, cells were lysed and apoptotic
markers where screened using a protein array. As shown in
Table 1, bM treatment significantly increased the expression
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
Hoechst (Blue)
N�B (Green) Composite
TNF 10 ng/ml
Control (Medium)
βM(20 μg/ml)
A
B
Fig. 6 – Photographs (A) and dose–response histogram for quantitative image analysis of intracellular targets (B) of stained
MCF-7 cells were treated with bM for 2 h and then stimulated for 30 min with 10 ng/ml TNF-a (NF-jB activation). Triplicates of
each treatment group were used in each independent experiment. The statistical significance is expressed as *P < 0.05.
10 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
of Fas, HSP 70, HSP 60 XIAP, Survivin, p53 and bax. The treat-
ment also resulted in a reduction in the level of expression of
bcl-2, Bid and livin.
4. Discussion
Amongst the most imperative of advances in cancer is the
understanding that programed cell death (apoptosis) and
the genes that control it have an intense consequence on
the malignant phenotype (Lowe & Lin, 2000). Moreover, con-
vincing evidence shows that oncogenic changes may promote
apoptosis (Lee et al., 2013; McCurrach, Connor, Knudson,
Korsmeyer, & Lowe, 1997); thereby development of successful
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
anticancer agents which induce apoptosis may be of thera-
peutic benefit. Secondary metabolites exhibit an anticancer
effect via cell growth inhibition and apoptogenesis either di-
rectly or by modulating the course of tumor development
through various biochemical pathways (Kintzios & Barberaki,
2004; Singh, Bhat, & Singh, 2003). Xanthones, a class of phyto-
chemicals isolated from the guttifeare family has gained
much attention owing to its significant biological properties
(Bennett & Lee, 1989; Matsumoto et al., 2005; Pedro, Cerqueira,
Sousa, Nascimento, & Pinto, 2002), in which mangostins such
as a- and c- has been evaluated and extensively studied for its
mechanisms in exerting anti-proliferative action via apopto-
sis in colon (Nabandith et al., 2004), prostate (Sato, Fujiwara,
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
PCNA
CDC2
B Ac�n
0 12 24 48 h
(a) (b)
(e) (f)
A B
C D
E F
Fig. 7 – bM induced G2/M phase cell cycle arrest in MCF-7 cells. Histograms for cell cycle from analysis of MCF-7 cells treated
with 7 lg/ml of bM for 12 (B), 24 (C) and 48 h (D), where (A) is control. Results are representative of one of three independent
experiments. (E) Induction of G2/M phase arrest in the cell cycle progression of MCF-7 cells by bM. ‘*’ Indicates a significant
difference p < 0.05). (F) Immunoblot analysis of G2/M phase cell cycle arrest related protein PCNA and cdc2.
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 11
Oku, Ishiguro, & Ohizumi, 2004), head and neck (Kaomongkol-
git, Chaisomboon, & Pavasant, 2011) and breast (Sampath &
Vijayaragavan, 2008) cancers. bM, structurally very similar to
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
the aforementioned xanthones, however, is least studied with
respect to cancer therapy. This study is a report of the anti-
breast cancer effect of bM (from C. arborescens) in vitro. As
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
Table 1 – Analysis of apoptosis pathway-related genes/proteins expression in bM treated MCF-7 cells.
Fold-change Regulation Full name
Genes
GADD45A 8.1413 Up Growth arrest and DNA damage inducible alpha
HRK 13.7979 Up Harakiri, BCL2 interacting protein (contains only BH3 domain)
CASP14 �104.6651 Down Caspase 14, apoptosis-related cysteine peptidase
DAPK1 �16.0513 Down Death associated protein kinase 1
TNFRSF11B �8.1035 Down Tumor necrosis factor receptor superfamily, member 11b
CD70 �6.6588 Down CD 70 molecule
Proteins
BAX 1.4956 Up Bcl-2-associated X protein
BCL-2 �12.1369 Down B-cell lymphoma 2
BID �1.6167 Down BH3 interacting domain
CASPASE3 1.5461 Up Cysteine-aspartic acid protease 3
CASPASE8 1.2740 Up Cysteine-aspartic acid protease 8
FASL 2.2121 Up TNF receptor superfamily, member 6 ligand
FAS 3.3615 Up TNF receptor superfamily, member 6
HSP70 2.2040 Up Heat shock protein 70
HSP60 1.5415 Up Heat shock protein 60
LIVIN �3.4573 Down Inhibitor of apoptosis protein family member
P27 1.0928 Up Cyclin-dependent kinase inhibitor
P53 1.5595 Up The p53 tumor suppressor protein
SMAC �1.0036 Down Second mitochondria-derived activator of caspases
SURVIVIN 1.1090 Up Apoptosis inhibitor survivin
XIAP 1.4118 Up X-linked inhibitor of apoptosis
Fig. 8 – Effects of bM on MCF7 cell’s caspase 3/7, 8 and 9 and ROS generation. (A) Relative luminescence expression of
Caspases in the MCF-7 cells treated with bM (7 lg/ml) for 12, 24 and 48 h. (B) Relative DCF-fluorescence intensity (ROS) after 5,
10 and 20 lg/ml of bM exposure at 24 h. Values are mean ± SD from three independent experiments. Triplicates of each
treatment group were used in each independent experiment. The statistical significance is expressed as *P < 0.05.
12 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
such, it is the first documentation of apoptotic effect of bM to-
wards MCF-7 via mitochondria-dependent activation of cas-
pase cascade, increased ROS, inhibition of Nuclear Factor-
kappa Beta (NF-kB), and p53 mediated G2/M cell cycle arrest.
The time- and dose- dependent inhibition of MCF-7 cells
proliferation was profound with IC50 of 7.3 lg/ml (16.5 lM)
for 48 h. The early and late phases of apoptosis in these cells
treated with bM were morphologically identified with dual
methods involving AO/PI double staining and Hoechst dyes.
Significant (p < 0.05) time-dependent increase in apoptotic
population was recorded in Annexin V flow cytometric analy-
sis for both phases. Thus the mode of cell death was con-
firmed to be apoptosis.
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
There was elevated (2-fold) intracellular reactive oxygen
species (ROS) with bM treatment (20 lg/ml) on MCF-7 cells
and this could be due to the free radical generation during
the cytotoxicity. Mitochondria being the main source of ROS
is itself a prime target for ROS provoked insult. Together with
ROS, mitochondria plays a significant role in apoptosis induc-
tion under both physiologic and pathologic conditions (Jia,
Xiong, Kong, Liu, & Xia, 2012). During the process of ROS in-
duced damage to cell, the MCF-7 cells tried to protect itself
by activating the Heat shock proteins (HSP’s) (Garrido et al.,
2003). The expressed HSP’s were HSP60 and 70; which how-
ever, could not help to overcome the cytotoxicity of bM. Thus,
the ROS triggered mitochondrial damage in MCF-7 cells
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 13
exposed to bM was initiated through the loss of mitochondrial
membrane potential (MMP) (Cai, Yang, & Jones, 1998). Mito-
chondrial structural and functional variation has been viewed
as one of the hallmarks of apoptosis (Desagher & Martinou,
2000) and the loss of MMP is considered as an early phase
of apoptosis (Kroemer, 2003).
Bcl-2 family has the key proteins involved in pro- or anti-
apoptotic activities and regulation of mitochondrial pathway
of apoptosis (Ham et al., 2012). Members of this family such as
Bcl-2/Bax involve in complex interactions with each other to
decide if a cell will die by controlling mitochondrial mem-
brane permeabilisation. The sequestration of Bcl-2 with Bax
helps to prevent apoptosis (Hengartner, 2000) and this was
inhibited by bM treatment by down-regulation and up-regula-
tion of Bcl-2 and Bax proteins respectively. Special genes are
involved in this kind of inhibition. For e.g., Hrk (the proapop-
totic Bcl-2 family member with only the BH3 domain) sup-
ports apoptosis by binding to the antiapoptotic members of
Bcl-2 family proteins to prevent them from inhibiting the
proapoptotic members (Coultas et al., 2007). bM accelerated
the expression of the Hrk gene by 13-fold in MCF-7 cells sug-
gesting that the aforementioned status of Bcl2/Bax was at-
tained through this gene alteration.
Another pro-apoptotic member in the Bcl-2 family is BID,
which is also found to be cleaved and activated in the MCF-
7 cells treated with bM. The active form interacts with Bax,
thus triggering the release of cytochrome c from the mito-
chondria to the cytoplasm (Elmore, 2007). Once released, cyto-
chrome c binds to the cytoplasmic scaffolding protein Apaf-1,
causing a conformational change that allows Apaf-1 to bind
to the prodomain of procaspase-9, which activates the down-
stream executive caspases in the intrinsic pathway. The in-
crease in caspase 9 along with other caspases (-8 and -7)
was observed with further analysis. Caspase-3 is the down-
stream effector caspase, which however, is absent in MCF-7
due to a 47-base pair deletion in the caspase-3 gene (Liang,
Yan, & Schor, 2001). Evidences suggest that caspase-7 activat-
ing apoptosome complex in MCF-7 acts instead of caspase 3
(Walsh et al., 2008) for execution of apoptosis. In this case,
the increase of caspase-7 was 4-folds making a large contri-
bution into apoptosis induction.
The increased expression of caspase-8 (5-folds), however,
suggests the involvement of the extrinsic pathway. The acti-
vation of caspase-8 is attained by the ligation of Fas with FasL
(Li, Zhu, Xu, & Yuan, 1998); both demonstrated to be ex-
pressed in the bM treated MCF-7 cells. It is also noteworthy
that caspase-8 can cleave and activate BID, thus, showing
the flow of one pathway into another (extrinsic to intrinsic).
The intrinsic pathway directs the fragmentation of DNA,
which, the cell tries to prevent and repair with the help of Poly
(ADP-ribose) polymerase (PARP) (Althaus et al., 1999). This at-
tempt of the cell to protect the DNA fragmentation was pre-
vented by bM by cleaving PARP and making it unable to
function properly. Apart from this, various studies indicated
the consequence of DNA strand breaks by anticancer agents
to be sufficient and probably necessary for p53 induction in
cells such as MCF-7 with wild-type p53 (Nelson & Kastan,
1994). The up regulation of p53 upon bM treatment was
clearly evident in this study and hence the apoptosis in also
p53-dependent.
Please cite this article in press as: Syam, S. et al., b-Mangostin induces p53-dmitochondrial pathway and NfkB suppression in MCF-7 cells, Journal of
Despite the intrinsic and extrinsic pathways, survival
pathways also play in determining the fate of cells going
through apoptosis. NfkB, widely recognised as a key positive
regulator of cancer cell proliferation and survival is repressed
from translocation into the nucleus by bM in MCF-7 cells
which is considered as a positive demeanor by an anti-cancer
candidate (Yeung et al., 2004). Other promoters of cell survival
deregulated in MCF-7 cells treated with bM were XIAP and
Survivin; both of which appear to have a more significant
inhibitory effect on caspase-7 activation by the apoptosome
and also forms a stable �200-kDa complex with active cas-
pase-7 (Lin et al., 2004). Both the proteins were up-regulated
for augmenting cell survival against the death signals pro-
duced by bM. But the overall results clearly indicate that this
survival machinery did not mark up to the point where it
could protect the cell from cytotoxicity induced by bM.
Another target of anticancer agents is to instigate cell cy-
cle arrest in the cancer cells. This is because, in many in-
stances, irregularities in cell cycle regulation are associated
with cancer progression (Hartwell & Kastan, 1994). bM in-
duced G2/M phase cell cycle arrest in MCF-7 cells and showed
a slender increase in the sub-G1 phase remarking apoptosis.
The proteins related to cell cycle arrest and G2/M phase, the
PCNA and cdc2 respectively, were analysed and the results
showed the level of both proteins were significantly decreased
upon treatment. It is estimated that the limitation of cdc2
supply for cdc2/cyclin B complex formation, along with the
PCNA down-regulation, altered the regulation of passage of
cells to mitosis (Ando et al., 2001; Bulavin et al., 2001).
Xanthone of similar kind (prenylated xanthone) had previ-
ously shown similar cell cycle arrest behavior in cancer cells.
For instance, in a previous study, a-mangostin arrested G2/M
phase, suppressed cdc2 protein and up-regulated p53 (Gut-
ierrez-Orozco & Failla, 2013). The anticancer property of a-
mangostin is not only limited to cell cycle arrest, but also
accomplishes apoptosis by modulation of signaling pathways
which includes disruption of MMP in mitochondria, eliciting
caspase cascade though Bax/Bcl-2 regulation and suppression
of NfkB (Gutierrez-Orozco & Failla, 2013; Hung, Shen, Wu, Liu,
& Shih, 2009). The effect of bM observed in this study is at par
with a-mangostin in cancer inhibition. While bM, through
this study showed that it does not show cytotoxicity towards
normal cells, there is no literature evidencing such specificity
for aM.
To sum up, through this study, it was established that bM
is a potent compound which can induce apoptosis in meta-
static breast cancer in vitro. The process of apoptosis was at-
tained through multiple cancer signaling pathway inhibition
such as intrinsic, Fas mediated, NfkB and cell cycle arrest.
These findings provide new insights into targeting bM, which
is found in functional foods containing mangostins, for fur-
ther investigation to delineate the exact mechanisms of apop-
tosis in animal model to study its suitability for it to be used
in human cancers.
Acknowledgments
This research is supported by High Impact Research Grant
University of Malaya-MOHE (UM-MOHE UM.C/625/1/HIR/
ependent G2/M cell cycle arrest and apoptosis through ROS mediatedFunctional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.10.018
14 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x
MOHE/SC/09) from the Ministry of Higher Education Malaysia
and Makna Cancer research lab, UPM. The authors would like
to express their utmost gratitude to Late Dato Prof. Hamid A.
Hadi for his support during the research.
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