Journal of Ethnopharmacology 153 (2014) 375385

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Swietenia macrophylla King induces mitochondrial-mediated apoptosis

through p53 upregulation in HCT116 colorectal carcinoma cells
Bey Hing Goh 1, Chim Kei Chan, Muhamad Noor Alfarizal Kamarudin, Habsah Abdul Kadir n
Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Swietenia macrophylla King is a traditional herb used to treat various
Received 10 September 2013 diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect
Received in revised form but the potential mechanisms have not been clearly dened. The current study was to further investigate
10 January 2014
the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-
Accepted 16 February 2014
Available online 5 March 2014
proliferative effect and apoptosis in HCT116 colorectal carcinoma cell.
Materials and methods: Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay.
Keywords: Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular
Apoptosis reactive oxygen species (ROS) was analyzed by ow cytometry. The apoptotic gene and protein
Bax/Bcl-2
expression were determined by Real-time quantitative PCR (q-PCR) and immunouorescence staining
Caspase
using ow cytometry, respectively.
p53
ROS Results: SMEAF signicantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent
Swietenia macrophylla manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive
oxygen species (ROS) production. Moreover, the signicant increase in p53 was accompanied by a
decrease murine double minute 2 (MDM2) expression. SMEAF signicantly increased the expression of
the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the
mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation.
Conclusion: These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating
proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-
mediated pathway via p53 activation.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Colorectal cancer (CRC) is currently one of the most prevalent
malignancies in the world making it a major public health concern
(Johnson, 2012). In the last 10 years, CRC has overtaken lung
cancer as the second most common cancer among Malaysians,
raising serious concerns about the poor dietary habits, especially
the consumption of foods high in saturated fats. Although early
diagnosis often leads to a complete cure, in most cases the polyps
go undetected. In such cases, therapies such as surgical interven-
tion, chemotherapy and radiation are often insufcient to treat the
disease, thus needing other preventive or unconventional ther-
apeutic strategies. Consequently, there is an emerging interest
in exploring the use of phytochemicals in cancer therapy. Phyto-
chemicals have been widely reported to mediate diverse machineries
n
Corresponding author. Tel.: 60 3 79674363; fax: 60 3 79614178.
E-mail address: habsah@um.edu.my (H. Abdul Kadir).
1
Present address: Brain Research Institute, Jeffrey Cheah School of Medicine
and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, Bandar
Sunway, Selangor 46150, Malaysia.
against cancer, comprising cell cycle arrest, immune response, induc-
tion of apoptosis and DNA damage (Chiang et al., 2010). Therefore,
controlling the growth and proliferation of cancerous cells and
inducing apoptosis are major goals in cancer chemoprevention.
The most classic feature of cancer cells is to evade apoptosis, a
form of programmed cell death which regulates the development
and homeostasis of mammalian cells under a variety of physiolo-
gical and pathological conditions including oxidative stress. Thus,
promotion of apoptotic cell death is a potential therapeutic
approach for prevention and treatment of cancer (Hanahan and
Weinberg, 2000). The mechanisms implicated are diverse and
appeared to involve a combination of cell signaling pathways at
multiple levels including inhibition of NF-kB activation, upregula-
tion of the proapoptotic protein levels, increase in cytochrome c
release, cell cycle arrest, DNA damage, activation of p53 and
caspase-3 (Shishodia et al., 2005; Sahu et al., 2009). Apoptosis
can be initiated by two major signaling pathways, namely, the
death receptor (extrinsic) pathway and the mitochondrial (intrin-
sic) pathway (Fulda and Debatin, 2006) that involves the activation
of a series of molecular events leading to cell death. Stimulation
of intrinsic mitochondrial mediated pathway by reactive oxygen

http://dx.doi.org/10.1016/j.jep.2014.02.036
0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
376 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
species (ROS) will eventually activate the apoptotic cascade,
consequently facilitates further release of apoptogenic factors
(Pourova et al., 2010).
p53 is a tumor suppressor protein encoded by the TP53 gene in
human. It has been described as the guardian of the genome
since its activation induces cell cycle arrest, senescence, differ-
entiation and response to cellular stress such as DNA damage,
oncogene activation, telomere erosion and hypoxia (Vousden,
2000). Thus, p53 is especially related to the initiation of apoptotic
cell death and its activation in tumor cells has been recognized as
a promising strategy for cancer treatment and several drugs
targeting p53 are recently tested in preclinical and clinical trials
(Cheok et al., 2010). One of the crucial events for initiating p53
activation is through ROS generation, where the ROS generated by
stressed cells would trigger expression of p53 which is sensitive to
redox change and eventually leads to apoptosis (Cardaci et al.,
2008). Studies also have shown physical and functional interac-
tions of p53 with various members of Bcl-2 family providing the
basis for an alternative route of p53-mediated cell death (Speidel,
2010).
Swietenia macrophylla is an endangered forest timber plant,
locally known as sky fruit, belonging to the Meliaceae family (50
genera and 1400 species) and growing natively throughout the
tropical regions of the Americas, in particular Central and South
American countries such as Mexico and Bolivia and in west India,
Malaysia, and southern China (Moghadamtousi et al., 2013).
Swietenia macrophylla has been used in Asia and many other
countries to treat diverse ailments based on its antimicrobial,
anti-inammatory, antioxidant effects, antimutagenic, anticancer,
antitumor and antidiabetic activities. Almost all parts of the plant
are used in traditional medicine for the treatment of various
human ailments. The seed of Swietenia macrophylla in particular
has signicant medicinal properties and has been reported to
possess anti-inammatory, antimutagenic and antitumor activity
(Goh and Kadir, 2011; Moghadamtousi et al., 2013). Various
species of Swietenia particularly Swietenia macrophylla, Swietenia
humilis and Swietenia mahagony have been used traditionally to
treat cancer in countries such as Mexico and Malaysia (Graham
et al., 2000; Camacho et al., 2003; Divya et al., 2012; Roy et al.,
2013). In Malaysia, Swietenia macrophylla is considered as a
common and well known medicinal plant for treatment of various
cancers including colon cancer (Jin, 2012). Previous pharmacolo-
gical investigations of these species have provided the scientic
basis for the ethnomedicinal use of these plants for cancer
treatment. The methanolic extract of the leaves and bark of
Swietenia macrophylla, and the bark of Swietenia humilis showed
cytotoxic effect on nasopharyngeal carcinoma (KB). In addition,
Khaya senegalensis (syn: Swietenia senegalensis) bark extract was
reported to possess anti-proliferative, anti-inammatory and pro-
apoptotic effects on HT-29, HCT-15 and HCA-7 human colorectal
cells and hypothesized to be useful in the prevention and treat-
ment of colorectal cancer (Androulakis et al., 2006). Limonoids is
the major class of phytochemicals that are commonly puried
and identied from Swietenia plants and has proven to be
responsible for majority of their bioactivities including cytotoxi-
city (Moghadamtousi et al., 2013). The limonoids derived from
Swietenia humilis, namely, humilinolide A, B, C and D when tested
against lung carcinoma (A-549), breast carcinoma (MCF-7) and
colon adenocarcinoma (HT-29) cell lines revealed selective cyto-
toxic activity against HT-29 cells (Jimenez et al., 1997).
The above studies have demonstrated that Swietenia species
and their bioactive constituents have a signicant role in disease
prevention including cancer and especially colon cancer. Our
previous study revealed that Swietenia macrophylla ethyl acetate
fraction was selectively cytotoxic against HCT116 cells as evident
by the apoptotic characteristic features such as cell shrinkage,
chromatin condensation, membrane blebbing and phosphatidyl-
serine externalization (Goh and Kadir, 2011), suggesting that
the ethyl acetate fraction may induce apoptosis in HCT116 cells.
However, the underlying mechanisms for the antiproliferative
and apoptosis-inducing effects of Swietenia species and Swietenia
macrophylla in particular have not been investigated. These mechan-
istic data are crucial not only in validating the ethnomedicinal uses
of these plants as anticancer agents but more importantly to provide
novel molecular target sites that are imperative to the current
targeted chemopreventive strategies. In this context, this study
was conducted for the rst time to analyze potential apoptosis-
inducing mechanisms on human adenocarcinoma cells HCT116.
2. Materials and methods
2.1. Preparation of ethyl acetate fraction from Swietenia macrophylla
seeds
From our previous report (Goh and Kadir, 2011), seeds of
Swietenia macrophylla were purchased from a local supplier and
upon authentication, a voucher specimen (No. KLU046901) was
deposited at the herbarium in the Institute of Biological Sciences,
University of Malaya, Kuala Lumpur, Malaysia. In brief, the crude
ethanol extract of Swietenia macrophylla seeds was prepared by
decanting and concentrating the ethanol that was used in soaking
the powdered seeds for 72 h. The crude ethanol extract was
further extracted repeatedly using hexane. The hexane insoluble
residue was then partitioned by using ethyl acetate and water
(1:1). Finally, the Swietenia macrophylla ethyl acetate fraction
(SMEAF) was obtained by evaporating the ethyl acetate soluble
fraction to dryness.
2.2. Cell culture
The human colorectal carcinoma (HCT116, ATCC number CCL-
247) was purchased from American Type Culture Collection (ATCC,
USA). HCT116 cells were maintained in RPMI 1640 Medium (Sigma).
The medium was supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (PAA Lab, Austria), 100 mg/mL streptomycin and
100 unit/mL penicillin (PAA Lab, Austria) and 50 mg/mL amphoter-
icin B (PAA Lab, Austria). The medium was lter-sterilized by using
a 0.2 mm lter membrane (Minisart, Sartorius Stedim). The cells
were cultured and conditioned in 5% CO2 incubator at 37 1C in a
humidied atmosphere.
2.3. Cell viability and morphological change analysis by trypan blue
dye exclusion assay
Cell viability was determined by a trypan blue dye exclusion
assay. Cells (1  106 cells) in 60 mm2 culture dishes were incu-
bated overnight, and then the medium was replaced with new
medium with various concentrations of SMEAF (0.051 mg/mL)
and incubated for 24 h. After the incubation period, each cell
suspension was mixed with an equal volume of 0.4% Trypan blue
solution for 5 min. The cells were then observed under a micro-
scope, and viable cells were counted using hemocytometer. For
morphological observation, cells were seeded and incubated for
24 h before further incubated with or without 0.05 mg/mL of
SMEAF for another 6, 12, 24, 48 and 72 h. Following incubation
period, the morphological changes were observed using phase
contrast inverted microscope (Leica DMI 3000B, Germany) at
400  magnications.
 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
2.4. Apoptotic detection by Hoechst 33342/propidium iodide (PI)
staining
Hoechst 33342/PI double staining allowed apoptotic cells to be
distinguished from necrotic cells and was carried out to further
conrm apoptosis-inducing effect of SMEAF on HCT116 cells.
Cells with homogeneously stained nuclei were considered viable,
whereas the presence of chromatin condensation and/or fragmen-
tation was indicative of apoptosis. Briey, 1  106 of HCT116
cells were seeded in 60 mm2 culture dishes and allowed to adhere
for 24 h. Cells were then treated with or without 0.05 mg/mL
of SMEAF and were incubated for 6, 12, 24, 48 and 72 h at 37 1C.
Cells were then harvested after designated incubation period
and washed twice with PBS before stained with Hoechst 33342
(10 mM)/PI (10 mM) at 37 1C for 10 min. The cells were then
inspected under inverted uorescence microscope (Leica Inverted
Fluorescence Microscope, DM16000B). Approximately 100 cells
from ve random microscopic elds were counted. Images were
taken by using (Leica, Germany) camera.
2.5. Measurement of intracellular reactive oxygen species (ROS) by
ow cytometry
The production of intracellular of ROS was determined by ow
cytometric analysis using DCFH-DA (6-carboxy-20 ,70 -dichlorodihy-
drouorescein diacetate) assay. DCFH-DA passively enters the cells
and then reacts with ROS to form a highly uorescent compound
dichlorouorescein (DCF). HCT116 cells were seeded in 60 mm2
culture dishes and allowed to adhere for 24 h. The cells were then
incubated with various concentrations of SMEAF for 4 h. Following
the incubation period, the cells were then washed twice with PBS
before further incubated 1 h in dark with 100 mM of DCFH-DA. The
medium was then aspirated and cells were washed twice with ice-
cold PBS before analyzed by ow cytometry. The generation of ROS
was measured by uorescence intensity of DCF detected using BD
Accuri C6 ow cytometer with the excitation and emission
wavelengths at 488 nm and 525 nm, respectively. Relative DCF
uorescence intensity of treated cells was expressed as a percen-
tage of control (as 100%).
2.6. Quantication of PCNA, Bax and Bcl-2 gene expression by
Real-time quantitative PCR (q-PCR)
The gene expression levels of Bcl-2 family members and
proliferating cell nuclear antigen (PCNA) in SMEAF-induced apop-
tosis were examined by using qPCR. HCT116 cells were seeded into
60 mm2 culture dishes and allowed to adhere for 24 h. The cells
were then incubated with or without 0.05 mg/mL of SMEAF for 6,
12 and 24 h for PCNA and 6 and 12 h for Bax and Bcl-2 gene
expression. After the designed incubation period, the cells were
then harvested and the total RNA was puried using QIAamps
RNA Blood Mini kit (Qiagen). On-column DNase treatment was
performed by using RNase-Free DNase set (Qiagen). The expres-
sion of Bcl-2 (B-cell CLL/lymphoma 2) and Bax (Bcl-2 associated X
protein) genes were measured by one-step SYBR Green relative Q-
PCR (RotorGene-6000 System, Qiagen) and normalized to PBGD
(Porphobilinogen deaminase) reference gene. The PCNA gene was
assessed and normalized to GAPDH (Glyceraldehyde 3-phosphate
dehydrogenase) reference gene. The primer sequences for Bcl-2,
Bax, PCNA and GAPDH were forward 50 -TTGGCCCCCGTTGCTT-30 ,
reverse 50 -CGGTTATCGTACCCCGTTCTC-30 ; forward 50 -GTCGCCCT-
TTTCTACTTTGCCAG-30 , reverse 50 -TCCAGCCCAACAGCCGCTCC-30 ;
forward 50 -GCCTGCTGGGATATTAGCTC-30 , reverse 50 -CATACTGGT-
GAGGTTCACGC-30 and forward 50 -CCAGGGCTGCTTTTAACTCTG-30 ,
reverse 50 -CGTTCTCAGCCTTGACGGTG-30 , respectively. The reac-
tions were carried out in a total volume of 25 mL by using Sensimix
377
One-Step Kit (Quantace). The PCR amplication for Bcl-2, was 45
cycles of 20 s at 95 1C, 40 s at 42 1C and 10 s at 72 1C, Bax was 45
cycles of 20 s at 95 1C, 40 s at 60 1C and 10 s at 72 1C and PCNA was
30 cycles of 45 s at 95 1C, 45 s at 56 1C and 120 s at 72 1C. The mean
uorescence threshold value (CT) of each sample was obtained
according to the manufacturer's guidelines, and used to determine
CT values whereby CT CT target gene (PCNA/Bcl-2/Bax)  CT reference
gene (GAPDH/PBGD). The relative fold change in the expression of
genes of interest in the treated sample over the untreated control
was calculated based on the comparative CT method where
CT CT sample  CT untreated and determined using formula
2  CT.
2.7. Measurement of p53, Bax and Bcl-2 proteins by ow cytometry
The expression level of p53, Bax and Bcl-2 proteins was
determined by immunouorescence staining using ow cytome-
try. 1  106 of HCT116 cells were seeded into 60 mm2 culture
dishes and allowed to adhere for 24 h. The cells were then
incubated with or without 0.05 mg/mL of SMEAF. After the
incubation period, cells were then washed twice with PBS, xed
and subsequently permeabilized using Cytox/Cytoperm kit (BD
Biosciences). A total of 1  106 cells were resuspended in 500 mL
of xation/permeabilization solution and incubated at 4 1C for
20 min. The cells were then washed twice with Perm/Wash
buffer and incubated for another 15 min in 1 mL of Perm/Wash
buffer. For the detection of Bax, Bcl-2 or p53 proteins, the xed and
permeabilized cells were incubated with 100 mL of Perm/Wash
buffer containing corresponding antibodies. For p53, the cells were
either stained directly with 10 L FITC-conjugated mouse anti-
human p53 monoclonal antibody or IgG2b isotype control (BD
Biosciences) at 4 1C in the dark for 30 min. Similarly, for Bcl-2
protein, the cells were either stained directly with 10 L FITC-
conjugated mouse anti-human Bcl-2 monoclonal antibody or IgG1
isotype control (BD Biosciences) at 4 1C in the dark for 30 min. For
indirect Bax staining, the cells were incubated with either rabbit
anti-human Bax polyclonal antibody or IgG1 isotype control (BD
Biosciences) at 4 1C in the dark for 30 min. After the washing
procedure, the cells were further incubated with FITC-conjugated
goat anti-rabbit F(ab0 )2 polyclonal secondary antibody (Abcam) at
4 1C in the dark for 30 min. Finally, the cells were then washed
with Perm/Wash buffer before analyzed by using BD Accuri C6
ow cytometer.
2.8. Measurement of caspase-3/7 and -9 activities
Caspase-3/7 and -9 activities were assessed by Carboxyuor-
escein FLICA Apoptosis Detection Kit based on the manufacturer's
protocol (Immunochemistry Technologies, LLC). HCT116 cells were
seeded in 60 mm2 culture dishes and incubated for 24 h. The cells
were then further incubated with or without 0.05 mg/mL of
SMEAF for 3, 6, 12 and 24 h. After the designed incubation period,
the cells were harvested and 10 mL of 30X FAM-DEVD-FMK
(Caspase-3/7) or FAM-LEHD-FMK (Caspase-9) solution was added
directly into 300 mL cell suspension containing 1  106 cells and
allowed to incubate for another 1 h at 37 1C. The cells were then
washed twice with washing solution before resuspended in 400 mL
of washing solution. The caspase activities of cells were analyzed
by using C6 ow cytometer.
2.9. Determination of TP53 and MDM2 genes by PCR array analysis
The Human Cancer Pathway Finder PCR Arrays was used
(PAHS-033A, SABiosciences Qiagen) in order to elucidate the
possible molecular mechanisms of the apoptotic inducing effects
of SMEAF on HCT116 cells. Briey, total RNA of the cells was
378 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385

isolated by using RNeasy Mini Kit (Qiagen) before being reverse
transcribed by using RT2 PCR array First Strand Kit (SABiosciences).
PCR analysis was performed using RT2 Realtime SYBR Green PCR
mix (SABiosciences) based on the manufacturer's instructions on
Rotor-Gene 6000 (Qiagen). Data normalization was based on
correcting all cycle threshold values for the average cycle thresh-
old values of the reference gene (ACTB) and analyzed with Excel-
based PCR Array Analysis Software (SABiosciences).
2.10. Statistical analysis
All values are expressed as mean 7 standard deviation (S.D.) or
standard error (S.E.) of three independent experiments. Statistical
analysis was performed to compare the difference between control
and SMEAF treated group by either using one-way analysis of
variance (ANOVA) followed by Tukey's test or Student's t-test and
considered signicant (n) when p o0.05.
conrming that SMEAF inhibited the proliferation of the HCT116
cells in a time-dependent manner.
3.3. Morphological changes induced by SMEAF
In order to examine the morphological changes in response to
SMEAF treatment, HCT116 cells were visualized by phase contrast
microscopy. As shown in Fig. 2a, a typical image of untreated cells
featuring elongated attached cells with round intact nuclei was
observed. In contrast, the SMEAF-treated cells showed morpholo-
gical changes characteristic of apoptosis, including cell shrinkage
and chromatin condensation were observed as early as 6 h. In
addition, the results also showed a time-dependent increase in
characteristic apoptotic morphological changes, including bleb
formation.
3.4. Apoptosis detection by Hoechst 33342/PI staining

To elucidate whether the HCT116 cell death induced by SMEAF

3. Results
3.1. SMEAF inhibited the proliferation of HCT116 cells
Evaluation of SMEAF for potential anticancer activity on
HCT116 cells was carried out by studying the growth inhibitory
effects of SMEAF using trypan blue exclusion assay. As shown in
Fig. 1a, the viability of HCT116 cells was signicantly inhibited by
SMEAF in a dose-dependent manner. The inhibitory rate of HCT116
cells treated with SMEAF at 0.05, 0.10, 0.20, 0.5 and 1.00 mg/mL for
24 h was 27.10 73.20%, 34.90 71.90%, 64.45 71.73%, 72.95 71.03%
and 75.63 71.11%. It was therefore evident that SMEAF possessed
anti-proliferative activity on HCT116 cells.
3.2. SMEAF inhibited cell proliferation by downregulating PCNA in
HCT116 cells
Proliferating cell nuclear antigen (PCNA) is a well-known marker
in proliferating cells as it is involved in a vast number of cellular
events including cell cycle regulation, DNA synthesis and DNA repair
and thus it is greatly expressed in most of the proliferating cancer
cells (Chan et al., 1983). In this study, the involvement of PCNA in
SMEAF inhibitory effect was conrmed using q-PCR. As shown
in Fig. 1b the expression level of PCNA mRNA in cells treated
with 0.05 mg/ml of SMEAF gradually decreased with time, further
occurred through apoptosis induction, we performed the apoptotic
characterization in the SMEAF-treated cells by Hoechst 33342/PI
double staining and uorescent microscopy. As shown in Fig. 2b,
the cells underwent remarkable nuclear morphological changes
after treatment with SMEAF indicating apoptosis. Control untreated
cells were uniformly stained with faint blue uorescence. In con-
trast, treatment with SMEAF (0.05 mg/mL) for 6 h showed an
increase in the intensity of blue uorescence indicating nuclear
shrinkage or condensation which is one of the early signs of
apoptosis. Dual-stained cells were considered to be in the late
apoptosis stage. Prolonged treatment of 24 h with SMEAF resulted
in marked increase in the intensity of blue and red uorescence
signals indicating increased number of cells undergoing apoptosis.
Overall, these results clearly indicate that SMEAF exerted its anti-
proliferative effect via the induction of apoptotic cell death.
3.5. SMEAF induced apoptosis via induction of reactive oxygen
species (ROS)
Reactive oxygen species (ROS) have been shown to be involved
in cell proliferation and apoptosis. The accumulated intracellular
ROS level in mitochondria is implicated in the induction of
apoptosis by several phytochemicals (Trachootham et al., 2009).
We speculated that the cell growth inhibition of HCT116 cells
treated with SMEAF might be partly due to elevated intracellular
ROS level, perturbing cellular redox homeostasis. Elevation of

Fig. 1. Antiproliferative effects of SMEAF on HCT116 cells measured by trypan blue exclusion analysis. (a) Effect of SMEAF on cell viability of HCT116 cells. Cells were treated
with (0.051.0 mg/mL) for 24 h. Percentage inhibition (%) of HCT116 cell growth after treatment with SMEAF. (b) Effect of SMEAF on gene expression of PCNA in HCT116 cells.
Cells were treated with 0.05 mg/mL of SMEAF for 6, 12 and 24 h. Each value represents means 7 S.E. from three independent experiments. Asterisks indicate a signicant
difference when compared to control (nPo 0.05).
 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
intracellular ROS level could lead to lipid peroxidation, oxidation
of proteins, DNA damage and consequently induced apoptosis in
cells (Lu et al., 2012). In order to measure oxidative stress induced
379
by SMEAF, uorescent dye DCFH-DA was used to examine ROS
generation. Intracellular ROS was measured in terms of uores-
cence by DCF. As shown in Fig. 3a, the green uorescence
intensities of DCF signals were signicantly enhanced with the
addition of SMEAF (a shift of the histogram to right hand side),
relative to the control. 0.05 mg/mL SMEAF signicantly increased
the intracellular ROS level by 259 7 2.7 folds, at 4 h incubation.
These results suggest that SMEAF elevated intracellular ROS level,
implying that the cells were undergoing oxidative stress.
3.6. SMEAF modulated expression of TP53 mRNA/p53 and MDM2
mRNA
MDM2 binds to the p53 tumor suppressor protein with high
afnity and negatively modulates its transcriptional activity and
stability. Overexpression of MDM2, found in many human tumors,
effectively impairs p53 function. Inhibition of MDM2p53 inter-
action can stabilize p53 and may offer a novel strategy for cancer
therapy. In this study, we found that SMEAF inhibited MDM2 at
the transcriptional level by suppressing its mRNA synthesis. This
MDM2 inhibition in turn led to increased levels of p53 protein and
subsequently triggered the signaling pathway leading to apoptosis.
As indicated in Fig. 3b, TP53 mRNA level was markedly increased
by 1.94 70.5 folds (compared to control) whereas the MDM2 gene
expression was downregulated to 0.49 70.14 of the control during
exposure to 0.05 mg/mL of SMEAF. These outcomes shifted the
balance of TP53/MDM2 ratio in favor of apoptosis. Immunouor-
escence assay was carried out to conrm the translation of the
expressed TP53 into its encoded p53 protein. As shown in Fig. 3c,
the histogram shifted from left to right for the SMEAF-treated cells
in comparison to the untreated control indicating a markedly
upregulated p53 protein expression. The p53 protein expression
was increased by 1.48 70.03 folds at 12 h SMEAF incubation
(Fig. 3d). These data were in accordance with the q-PCR data
revealing marked increase of TP53 mRNA expression level at 12 h
following exposure to 0.05 mg/mL SMEAF. Taken together, these
results suggest that p53 activation is an important factor in
SMEAF-induced apoptosis of HCT 116 cells.
3.7. SMEAF augmented the ratio of Bax to Bcl-2
Expressions of the intracellular proteins related to apoptosis,
such as Bax and Bcl-2, were investigated to understand the
mechanisms by which SMEAF induces apoptosis in HCT116 cells.
As shown in Fig. 4a and b, the 6 and 12 h treatment of HCT 116
cells with SMEAF (0.05 mg/mL) led to a signicant increase in
mRNA levels of Bax and a decrease in expression of Bcl-2, thus
elevating the Bax/Bcl-2 ratio (Fig. 4c). Immunouorescence stain-
ing using ow cytometry analysis was performed to determine
whether the Bax protein level induced by SMEAF was due to
increased level of mRNA. In line with these results, the level of the
proapoptotic protein Bax was increased signicantly while the
Fig. 2. Effect of SMEAF on the morphological changes and apoptosis induction of
HCT116 cells. Cells were treated with 0.05 mg/mL of SMEAF for 6, 12, 24, 48 and
72 h and visualized for apoptotic bodies under inverted microscope (magnication
400  ). (a) After treatment with SMEAF, cells exhibited progressive loss of normal
elongated shape, cell shrinkage and bleb formation. (b) Effect of SMEAF on
apoptosis induction was indicated by chromatin condensation when cells were
treated with the 0.05 mg/mL of SMEAF for 24 h and stained with Hoechst 33342
and propidium iodide. The nuclear morphology was observed by uorescent
microscopy (magnication 400  ); the cells that were stained with an intense
and bright blue uorescence signals indicated early apoptotic cells whereas double
stained (blue and red uorescence) cells were considered as late apoptotic cells.
The untreated group cells 72 h showed normal structure without prominent
apoptosis. The data shown are representative of three independent experiments
with similar results. (For interpretation of references to color in this gure, the
reader is referred to the web version of this article.)
380 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385

Fig. 3. Effect of SMEAF treatment on ROS, gene and protein expression level of MDM2, Tp53 and p53 in HCT116 cells. (a) Effect of SMEAF on ROS level in HCT116 cells
represented by an overlay of histograms showing ROS-associated uorescence intensity. (b) 24 h exposure of SMEAF enhanced the gene expression of TP53 while decreasing
MDM2 gene expression in HCT116 cells. The fold change of the genes was normalized by ACTB. (c) Representative overlay of histograms showing p53-associated uorescence
intensity after exposure to SMEAF for 3, 6, 12 and 24 h. (d) The relative expression levels of p53 protein. Values are mean7 S.D. of three independent experiments. Asterisks
indicate a signicant difference compared to control (nPo 0.05).

level of an antiapoptotic protein Bcl-2 was increased slightly in at 12 h SMEAF incubation (Fig. 4d). However, an insignicant
SMEAF treated HCT116 cells (Fig. 4d and e). Particularly, Bax increase in Bcl-2 protein expression, in coincident with marked
showed a marked increase of 6.30 70.11 folds in its expression increase in Bax protein expression has resulted in signicantly
 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385 381

Fig. 4. Effect of SMEAF on gene and protein expression of Bax and Bcl-2 in HCT116 cells. Cells were treated with 0.05 mg/mL of SMEAF for 6 and 12 h and were harvested
prior to the q-PCR analysis and ow cytometric analysis. (a) The relative gene expression level of Bax as compared to the control. (b) The relative gene expression of Bcl-2 as
compared to the control. The fold change of the gene was normalized against PBGD expression using the formula 2  CT. (c) Bax/Bcl-2 ratio. (d) Time-dependent Bax protein
expression as compared to control. (e) Time-dependent Bcl-2 protein expression as compared to control. (f) Ratio of Bax/Bcl-2. Values are mean 7S.D. of three experiments.
Asterisks indicate a signicant difference compared to control (nP o0.05).

elevated Bax/Bcl-2 ratio (Fig. 4f). In agreement with the upregula- toward apoptotic stimuli which are in accordance with the growth
tion of Bax, a signicant increase in Bax/Bcl-2 ratio of 5.787 0.05 inhibitory effect of SMEAF. Collectively, these results indicated that
was observed at 12 h of SMEAF incubation (Fig. 4f). These results the augmentation of the ratio of Bax/Bcl-2 might be the primary
suggest that increased Bax/Bcl-2 ratio may sensitize the cells contributor to the apoptosis mediated by SMEAF.
382 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385

Fig. 5. Effect of SMEAF on caspase (caspase-3/7 and -9) activities in HCT116 cells. Cells were treated with 0.05 mg/mL for 3, 6, 12 and 24 h. (a) Represented the ow
cytometric overlay histogram of caspase-3/7 activity in HCT116 treated cells, (b) SMEAF stimulated the activity of caspase-3/7 in HCT116 cells. (c) Represented the ow
cytometric overlay histogram of caspase-9 activity in HCT116 treated cells (d) SMEAF stimulated the activity of caspase-9 in HCT116 cells. The values presented are the
mean 7S.D. from three independent experiments. Asterisks indicate a signicant difference when compared to control (nPo 0.05).
 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
3.8. SMEAF induced activation of caspase-3/7 and -9 activities
Caspase protease family is believed to play a central role in
mediating various apoptotic responses (Son et al., 2010). To
analyze the apoptotic pathway in SMEAF-treated HCT116 cells,
we examined the caspase-3/7 and caspase-9 activities. As shown
in Fig. 5a and b, both the initiator caspase-9 and executioner
caspase-3/7 were activated signicantly after 24 h treatment with
SMEAF. Compared to untreated cells, the activities of caspase-3/7
in HCT116 cells treated with 0.05 mg/mL of SMEAF increased by
2.78 70.14 folds at 3 h and reached 4.087 0.080 folds at 24 h
Fig. 5a. Similarly, the intracellular caspase-9 activity also escalated
with the SMEAF treatment. Particularly, the caspase-9 activity
attained 1.667 0.01-fold and 19.19 70.73-fold increase at 3 h and
24 h, respectively, as shown in Fig. 5c and d. Thus, SMEAF-induced
cell death was accompanied by an increase in caspase activities,
which then stimulated the molecular cascade of apoptosis. These
results indicate that SMEAF-induced apoptosis involves a caspase-
dependent pathway in HCT116 cells.
4. Discussion
Phytochemicals affect intracellular targets and this character-
istic often makes them desirable on tumor cells as chemopreven-
tive agents against cancer (Mates et al., 2011). Our previous study
demonstrated that Swietenia macrophylla ethanolic extract and its
fractions exerted cytotoxic effects on multiple human cancer cells
and the ethyl acetate fraction exhibited signicant anti-tumor
activity (Goh and Kadir, 2011) but the mechanism of cell death still
remained undened. In this study, the ethyl acetate fraction
signicantly inhibited cell proliferation and induced apoptosis in
HCT116 cells. This is the rst report to demonstrate anti-
proliferative effect of SMEAF on human colorectal cells and
elucidate its underlying mechanisms.
Trypan blue is a diazo dye which can selectively stain dead cells
making it a well accepted vital stain used in differentiating the
viable and dead cells. In this study, trypan blue assay was used in
evaluating the anti-proliferative effect of SMEAF. Our present ndings
indicated that SMEAF exerts a signicant anti-proliferative effect on
HCT116 cells. The number of viable cells decreased signicantly after
treatment with increasing concentrations of SMEAF indicating a
dose-dependent inhibitory effect on the proliferation of HCT116 cells
(Fig. 1a). In addition, as low as 0.05 mg/mL of SMEAF was shown to
reduce the proliferating marker signicantly in a time-dependent
manner (Fig. 1b). Taken together, these ndings support the anti-
proliferative effect of SMEAF on HCT116 cells as evidenced by the
downregulation of PCNA gene expression.
Apoptosis is an active physiological process of cell destruction
that involves specic morphological and biochemical changes in
the nucleus and cytoplasm (Lim et al., 2009). DNA fragmentation is
one of the hallmarks of apoptosis. To elucidate whether SMEAF-
induced anti-proliferative activity against HCT116 cells was attri-
butable to apoptosis, we carried out a nuclear morphological
evaluation of apoptosis by double staining with Hoechst 33342/
PI. As shown in Fig. 2, the control cells exhibited normal nuclear
morphology whereas the cells treated with SMEAF displayed
chromatin condensation. These results clearly indicated that
SMEAF exerted its anti-proliferative effect via the induction of
apoptotic cell death.
Investigation of apoptosis induction as a potential mechanism
demonstrated a high correlation with the activation of a variety of
intracellular signaling pathways leading to apoptosis. The inu-
ence of SMEAF on the expression of apoptosis related proteins was
determined by ow cytometric analysis. Treatment of HCT116 cells
with 0.05 mg/mL of SMEAF caused a signicant increase in the
383
levels of p53 protein with a concomitant decrease in MDM2.
SMEAF showed a signicant increase in Bax mRNA and protein
expression, resulting in the elevation of Bax /Bcl-2 ratio. In
addition, SMEAF also increased the activity of caspase-3/7 and -9
and stimulate the formation of ROS. These results presented in this
study suggest that SMEAF-induced apoptosis is mediated by the
mitochondrial apoptotic pathway through p53 activation.
Several studies have shown that the Bcl-2 family of proteins is
the principal regulator of apoptosis (Green and Kroemer, 2004;
Kim et al., 2006). The proapoptotic and antiapoptotic members
of Bcl-2 family of proteins have opposing regulatory effects
on apoptosis through the control of cytochrome c release from
mitochondria, which in turn induces caspase-9 and -3 activation
(Karbowski et al., 2006). Overexpression of Bcl-2 and Bcl-XL is
known to repress the apoptotic response while Bax, Bid and Bak
activity promotes cell death (Green and Kroemer, 2004). Our
current ndings demonstrated that SMEAF increased signicantly
the Bax mRNA and protein expression in a time-dependent
manner (Fig. 4a and d), decreased the expression of Bcl-2 mRNA
(Fig. 4b) accompanied by an insignicant change in its protein
expression (Fig. 4e). The antiapoptotic marker, Bcl-2, was probably
downregulated since it was only detected at low levels of expres-
sion. In this regard, it is particularly noteworthy that the overall
modulatory effect of SMEAF has resulted in a signicantly elevated
Bax/Bcl-2 ratio (Fig. 4c and f), which is accepted as an important
index of apoptotic cell death, indicating that the balance between
Bax and Bcl-2 shifted in favor of Bax. Although overexpression of
Bax alone has been shown to trigger the commitment of a cell
towards apoptosis, the ratio between Bax and Bcl-2 plays a more
important role in regulating apoptosis than the level of individual
Bcl-2 family member protein (Weng et al., 2009). Collectively, these
data revealed that while the antiapoptotic Bcl-2 showed no sig-
nicant change, the proapoptotic marker, Bax protein, was signi-
cantly upregulated, thus resulting in an elevation of Bax/Bcl-2 ratio
in HCT116 cells, suggesting the involvement of mitochondrial-
mediated apoptotic pathway by which SMEAF induces apoptosis
in HCT116 cells.
p53 protein is a critical regulator of the cellular response to DNA
damage and oncogenic stress. Loss of p53 function through mutation
or deletion, is a frequent occurrence in human malignancies. Our
current ndings demonstrated that SMEAF treatment resulted in an
increase in the levels Tp53 mRNA and p53 protein expression
(Fig. 3bd). p53 is proven to regulate apoptosis through activation
of the mitochondria-mediated death pathway by increasing the
expression of proapoptotic genes in the Bcl-2 family and suppressing
the expression of antiapoptotic genes (Oren, 2003; Green and
Kroemer, 2004). In addition, p53 also induces apoptosis in a non-
transcription manner, by acting directly on mitochondria to form
complexes with the proapoptotic Bcl-2 family proteins which in turn
increases the outer mitochondrial membrane permeability (Chipuk
et al., 2004). p53 protein interacts with Bcl-2 to enhance Bax-
promoted outer-mitochondrial membrane permeabilization and p53
also activates the Bax gene transcription directly (Toshiyuki and
Reed, 1995; Butt et al., 2000), thus, p53 has been reported to
mediate Bax upregulation (Karpinich et al., 2002), a result we have
also observed in SMEAF-treated HCT116 cells. Taken together, these
results support a role of p53 in SMEAF-induced apoptosis.
Furthermore, increased p53 expression has been correlated
with an increase in the Bax/Bcl-2 ratio, resulting in cytochrome-c,
caspase activation, and ultimately, apoptosis. Our present ndings
seem to suggest that SMEAF induced apoptosis via p53 activation
with concomitant elevation of Bax that leads to an increase in the
Bax/Bcl-2 ratio in HCT116 cells, which in turn alters the mitochon-
drial membrane permeability. Such altered mitochondrial mem-
brane permeability releases cytochrome c into the cytosol (Kang
and Reynolds, 2009) that triggers caspase-9 activation, which
384 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
accelerates apoptosis by activating other caspases such as caspase-
3. In this study, immunouorescence staining analysis revealed
that SMEAF enhanced the expression of Bax protein resulting in
elevated ratio of Bax/Bcl-2 and upregulating p53 expression (Fig. 5),
which promoted the activity of caspase-3/7 to induce apoptosis.
Since proapoptotic Bax is known to be a p53 downstream target,
our result of the higher expression of Bax protein also supports the
involvement of p53 during this cell death mechanism. The increased
expression of p53 and Bax; and decreased expression of Bcl-2 may
confer higher sensitivity to apoptosis or favors the onset of
apoptosis when treated with SMEAF. Collectively, these results
suggested that SMEAF-induced apoptosis may be associated with
upregulation of Bax and an increase in Bax /Bcl-2 ratio taking part in
mitochondrial-mediated apoptosis pathway and further support the
involvement of p53 in apoptotic-induction of HCT116 by SMEAF.
To conrm the apoptotic pathway involved in SMEAF-induced
apoptosis, we measured the activities of caspases and intracellular
ROS production. Caspases, represented by a family of cysteine
proteases, are the key proteins that play a crucial role in the process
of apoptotic signaling pathway (Yin et al., 2009). The present
ndings showed that SMEAF activated caspase-9, which is an
initiator of caspase cascade, and caspase-3/7, an executioner caspase
in apoptosis signaling in concentration-dependent manner (Fig. 5a
d). The activation of caspase-3 was likely to be responsible for the
proapoptotic effect exhibited by SMEAF. Activation of caspase-9 is
an important inducer of apoptosis through the mitochondrial-
mediated pathway. The present ndings showed that SMEAF
signicantly enhanced the activities of caspase-9 and caspase-3/7,
suggesting that the mitochondrial pathway of apoptosis was
involved in SMEAF-induced apoptosis in human colorectal carci-
noma HCT116 cells.
Multiple studies have suggested that cancer chemotherapeutic
drugs induce apoptosis of tumor cells, partially, by enhancing the
production of intracellular reactive oxygen species (ROS) (Hou
et al., 2005; Low et al., 2010). In this study, it was illustrated that
SMEAF signicantly stimulated ROS generation in HCT116 cells,
leading to apoptotic signaling (Fig. 3). Interestingly, the elevation
of intracellular ROS production was also observed in SMEAF-
treated cells in comparison with the control cells. ROS has been
known to collaborate with the mitochondrial apoptotic pathway
by changing the permeability of the mitochondrial outer mem-
brane, resulting in the translocation of apoptosis transducers,
including cytochrome C (Ryter et al., 2007). Therefore, we can
infer that the activation of caspase-9 and caspase-3/7, together
with the elevation of intracellular ROS, collectively promote the
occurrence of mitochondrial apoptotic pathway in SMEAF-treated
cells, leading to the cell death (Son et al., 2010).
Moreover, ROS generation is also correlated with the activation
of p53. ROS accumulation and its effect on mitochondrial function
can contribute to p53-mediated apoptosis as evidenced by several
studies indicating that ROS inducers such as bioactive phytochem-
icals function in conjunction with p53 to inuence apoptosis (Hou
et al., 2005). Therefore, one of the striking features of SMEAF-
induced apoptosis in HCT116 cells was the accumulation of
intracellular ROS with a concomitant increase in p53 levels leading
to apoptosis in the SMEAF-treated HCT116 cells as demonstrated
in our study. Also, studies have implied the importance of the
cooperation involving the Bax, Bcl-2 and ROS production in tumor
cells (Chen and Pervaiz, 2007). For example, Bcl-2 overexpression
was shown to suppress apoptosis induced by resveratrol by a
pathway that involves ROS production (Low et al., 2010). ROS also
acts by promoting mitochondrial membrane permeability in
inducing apoptosis via the caspase cascade (Hou et al., 2005), in
association with elevated expression of the Bax protein. As shown
in Fig. 4a and b the 6 and 12 h treatment of HCT 116 cells with
SMEAF (0.05 mg/mL) led to an increase in mRNA levels of Bax and
a decrease in Bcl-2. Thus, the present ndings also suggest that
SMEAF elevates intracellular ROS level and ultimately apoptosis
through a pathway that involves interaction between p53, Bax and
Bcl-2 (Liu et al., 2008; Low et al., 2010). Based on the hypothesis,
the expression of p53, Bcl-2 and Bax proteins in HCT116 cells after
the treatment with SMEAF was examined by immunouorescence
methods. The results showed that SMEAF enhanced the expression
of Bax and p53 while the Bcl-2 expression was not altered, which
incited the activation of caspase cascade to induce apoptosis. These
results are in accordance with the proposed mitochondrial-mediated
apoptotic pathway shown to be responsible for the observed
apoptosis. Collectively, the above results suggest that SMEAF is a
potent inducer of oxidative stress in HCT116 cells, which plays a
signicant role in triggering the apoptotic process through the
activation of mitochondrial-mediated pathway via p53 upregulation.
MDM2 oncogenic protein is the principal cellular antagonist of
the p53 tumor suppresser gene, involved in catalyzing ubiquitina-
tion of p53 to downregulate the p53 level (Shi and Gu, 2012). As
such, a decrease in MDM2 expression leads to a reduction in p53
degradation resulting in accumulation of p53 which subsequently
triggers caspase-3 and ultimately apoptosis. Besides, the down-
regulation of MDM2 is essential to enhance the proapoptotic
proteins of Bcl-2 family and suppresses their antiapoptotic effects
(Zhang et al., 2010). The manipulation of p53-mediated pathway is
therefore, an ongoing focus for the development of effective anti-
cancer agents. In the present study, the role of MDM2 and p53
expression levels in the SMEAF-induced apoptotic pathway was
further veried based on their expression proles. Interestingly,
the ndings showed that SMEAF treatment has resulted in a
marked increase of Tp53 mRNA and its encoded protein p53
together with a concomitant decrease in MDM2 gene expression.
Collectively, these results suggested that SMEAF may induce
apoptosis in HCT116 cells by shifting the balance of TP53/MDM2
ratio in favor of apoptosis.
In conclusion, the present study showed that ethyl acetate
fraction of Swietenia macrophylla inhibited the proliferation of
HCT116 cells in a dose-dependent manner and caused apoptotic
cell death through p53-mediated pathway. The induction of apop-
tosis by SMEAF in HCT116 cells was associated with an increase in
intracellular ROS, upregulation of p53 protein and downregulation
of its negative regulator, MDM2, followed by an increase in Bax/Bcl-
2 ratio leading to the activation of caspase-3/7 and -9. These
ndings provide a rationale to explore SMEAF as a preventive and
perhaps as a chemotherapeutic agent in the management of color-
ectal cancer.
Acknowledgment
This study was supported by grant from the Institute of
Research Management and Consultancy of University of Malaya
through Institute of Research Management and Consultancy of
University of Malaya through UM High Impact Research Grant UM-
MOHE UM.C/625/1/HIR/MOHE/SC/02, Postgraduate Research
Fund, No. PV022-2011B and the University Malaya Research Grant
(RP001-2012C).
References
Androulakis, X.M., Muga, S.J., Chen, F., Koita, Y., Toure, B., Wargovich, M.J., 2006.
Chemopreventive effects of Khaya senegalensis bark extract on human color-
ectal cancer. Anticancer Res. 26 (3B), 23972405.
Butt, A.J., Firth, S.M., King, M.A., Baxter, R.C., 2000. Insulin-like growth factor-
binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-
independent radiation-induced apoptosis in human breast cancer cells. J. Biol.
Chem. 275, 3917439181.
 B.H. Goh et al. / Journal of Ethnopharmacology 153 (2014) 375385
Camacho, M., Phillipson, J., Croft, S., Solis, P., Marshall, S., Ghazanfar, S., 2003.
Screening of plant extracts for antiprotozoal and cytotoxic activities. J. Ethno-
pharmacol. 89, 185191.
Cardaci, S., Filomeni, G., Rotilio, G., Ciriolo, M.R., 2008. Reactive oxygen species
mediate p53 activation and apoptosis induced by sodium nitroprusside in SH-
SY5Y cells. Mol. Pharmacol. 74, 12341245.
Chan, P.-K., Frakes, R., Tan, E.M., Brattain, M.G., Smetana, K., Busch, H., 1983. Indirect
immunouorescence studies of proliferating cell nuclear antigen in nucleoli of
human tumor and normal tissues. Cancer Res. 43, 37703777.
Chen, Z.X., Pervaiz, S., 2007. Bcl-2 induces pro-oxidant state by engaging mitochon-
drial respiration in tumor cells. Cell Death Differ. 14, 16171627.
Cheok, C.F., Verma, C.S., Baselga, J., Lane, D.P., 2010. Translating p53 into the clinic.
Nat. Rev. Clin. Oncol. 8, 2537.
Chiang, J.-H., Yang, J.-S., Ma, C.-Y., Yang, M.-D., Huang, H.-Y., Hsia, T.-C., Kuo, H.-M.,
Wu, P.-P., Lee, T.-H., Chung, J.-G., 2010. Danthron, an anthraquinone derivative,
induces DNA damage and caspase cascades-mediated apoptosis in SNU-1
human gastric cancer cells through mitochondrial permeability transition pores
and Bax-triggered pathways. Chem. Res. Toxicol. 24, 2029.
Chipuk, J.E., Kuwana, T., Bouchier-Hayes, L., Droin, N.M., Newmeyer, D.D., Schuler,
M., Green, D.R., 2004. Direct activation of Bax by p53 mediates mitochondrial
membrane permeabilization and apoptosis. Science 303, 10101014.
Divya, K., Pradeep, H., Kumar, K., KR, H.V., Jyothi, T., 2012. Herbal drug Swietenia
mahagoni jacq.a review. Global J. Res. Med. Plants Indigenous Med. 1,
557567.
Fulda, S., Debatin, K., 2006. Extrinsic versus intrinsic apoptosis pathways in
anticancer chemotherapy. Oncogene 25, 47984811.
Goh, B.H., Kadir, A., 2011. In vitro cytotoxic potential of Swietenia macrophylla King
seeds against human carcinoma cell lines. J. Med. Plants Res. 5, 13951404.
Graham, J., Quinn, M., Fabricant, D., Farnsworth, N., 2000. Plants used against
canceran extension of the work of Jonathan Hartwell. J. Ethnopharmacol. 73,
347377.
Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death.
Science 305, 626629.
Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 5770.
Hou, D.-X., Tong, X., Terahara, N., Luo, D., Fujii, M., 2005. Delphinidin 3-sambubio-
side, a Hibiscus anthocyanin, induces apoptosis in human leukemia cells
through reactive oxygen species-mediated mitochondrial pathway. Arch. Bio-
chem. Biophys. 440, 101109.
Jimenez, A., Mata, R., Pereda-Miranda, R., Calderon, J., Isman, M., Nicol, R., Arnason, J.,
1997. Insecticidal limonoids from Swietenia humilis and Cedrela salvadorensis.
J. Chem. Ecol. 23, 12251234.
Jin, C.B., 2012. Pembunuh Pelbagai Jenis Kanser. Dewan Kosmik, pp. 3637.
Johnson, I.T., 2012. Impact of diet on risk of colorectal cancer: how strong is the
evidence? Colorectal Cancer 1, 8588.
Kang, M.H., Reynolds, C.P., 2009. Bcl-2 inhibitors: targeting mitochondrial apoptotic
pathways in cancer therapy. Clin. Cancer Res. 15, 11261132.
Karbowski, M., Norris, K.L., Cleland, M.M., Jeong, S.-Y., Youle, R.J., 2006. Role of Bax
and Bak in mitochondrial morphogenesis. Nature 443, 658662.
Karpinich, N.O., Tafani, M., Rothman, R.J., Russo, M.A., Farber, J.L., 2002. The course
of etoposide-induced apoptosis from damage to DNA and p53 activation to
mitochondrial release of cytochrome c. J. Biol. Chem. 277, 1654716552.
Kim, R., Emi, M., Tanabe, K., 2006. Role of mitochondria as the gardens of cell death.
Cancer Chemother. Pharmacol. 57, 545553.
Lim, H.-K., Moon, J.Y., Kim, H., Cho, M., Cho, S.K., 2009. Induction of apoptosis in
U937 human leukaemia cells by the hexane fraction of an extract of immature
Citrus grandis Osbeck fruits. Food Chem. 114, 12451250.
385
Liu, B., Chen , Y., Clair, D.K., 2008. ROS and p53: a versatile partnership. Free Radic.
Biol. Med. 44, 15291535.
Low, I.C.C., Chen, Z.X., Pervaiz, S., 2010. Bcl-2 modulates resveratrol-induced ROS
production by regulating mitochondrial respiration in tumor cells. Antioxid.
Redox Signal. 13, 807819.
Lu, K.H., Lee, H.J., Huang, M.L., La, S.C., Ho, Y.L., Chang, Y.S., Chi, C.W., 2012.
Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide and
Mucuna macrocarpa Stem Extract in Human Leukemic Cells via a Reactive
Oxygen Species-Dependent Mechanism. Evid-Based Complement. Altern. Med.
2012, 921430.
Mates, J.M., A Segura, J., Alonso, J., Mrquez, J., F., 2011. Anticancer antioxidant
regulatory functions of phytochemicals. Curr. Med. Chem. 18, 23152338.
Moghadamtousi, S.Z., Goh, B.H., Chan, C.K., Shabab, T., Kadir, H.A., 2013. Biological
activities and phytochemicals of Swietenia macrophylla King. Molecules 18 (9),
1046510483.
Oren, M., 2003. Decision making by p53: life, death and cancer. Cell Death Differen.
10, 431442.
Pourova, J., Kottova, M., Voprsalova, M., Pour, M., 2010. Reactive oxygen and
nitrogen species in normal physiological processes. Acta Physiol. 198, 1535.
Roy, S., Banerjee, B., Vedasiromoni, J.R., 2013. Anti-tumor activity of Swietenia
mahagoni (L.) Jacq. leaf extract against Ehrlich's ascites carcinoma in mice.
Oriental Pharm. Exp. Med., 112
Ryter, S.W., Kim, H.P., Hoetzel, A., Park, J.W., Nakahira, K., Wang, X., Choi, A.M., 2007.
Mechanisms of cell death in oxidative stress. Antioxid. Redox Signal. 9 (1),
4989.
Sahu, R., Batra, S., Srivastava, S., 2009. Activation of ATM/Chk1 by curcumin causes
cell cycle arrest and apoptosis in human pancreatic cancer cells. Br. J. Cancer
100, 14251433.
Shi, D., Gu, W., 2012. Dual roles of MDM2 in the regulation of p53 ubiquitination
dependent and ubiquitination independent mechanisms of MDM2 repression
of p53 activity. Genes Cancer 3, 240248.
Shishodia, S., Sethi, G., Aggarwal, B.B., 2005. Curcumin: getting back to the roots.
Ann. NY Acad. Sci. 1056, 206217.
Son, Y.-O., Hitron, J.A., Wang, X., Chang, Q., Pan, J., Zhang, Z., Liu, J., Wang, S., Lee, J.-C.,
Shi, X., 2010. Cr (VI) induces mitochondrial-mediated and caspase-dependent
apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41
cells. Toxicol. Appl. Pharmacol. 245, 226235.
Speidel, D., 2010. Transcription-independent p53 apoptosis: an alternative route to
death. Trends Cell Biol. 20, 1424.
Toshiyuki, M., Reed, J.C., 1995. Tumor suppressor p53 is a direct transcriptional
activator of the human bax gene. Cell 80, 293299.
Trachootham, D., Alexandre, J., Huang, P., 2009. Targeting cancer cells by ROS-
mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov.
8, 579591.
Vousden, K.H., 2000. p53: death star. Cell 103, 691694.
Weng, C.-J., Yang, Y.-T., Ho, C.-T., Yen, G.-C., 2009. Mechanisms of apoptotic effects
induced by resveratrol, dibenzoylmethane, and their analogues on human lung
carcinoma cells. J. Agric. Food Chem. 57, 52355243.
Yin, M.-C., Lin, C.-C., Wu, H.-C., Tsao, S.-M., Hsu, C.-K., 2009. Apoptotic effects of
protocatechuic acid in human breast, lung, liver, cervix, and prostate cancer
cells: potential mechanisms of action. J. Agric. Food Chem. 57, 64686473.
Zhang, W., Konopleva, M., Burks, J.K., Dywer, K.C., Schober, W.D., Yang, J.-Y.,
McQueen, T.J., Hung, M.-C., Andreeff, M., 2010. Blockade of mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase and murine double
minute synergistically induces apoptosis in acute myeloid leukemia via BH3-
only proteins Puma and Bim. Cancer Res. 70, 24242434.