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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: suvithaavs@gmail.com (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

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

(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).

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

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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