α-Mangostin Induces Apoptosis in Human Chondrosarcoma Cells through Down-regulation of ERK/JNK and Akt Signaling Pathway

Article (PDF Available)inJournal of Agricultural and Food Chemistry 59(10):5746-54 · March 2011with31 Reads
DOI: 10.1021/jf200620n · Source: PubMed
Abstract
Chondrosarcoma is a malignant primary bone tumor that is resistant to chemotherapy and radiation therapy. α-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor properties. This study is the first to investigate anticancer effects of α-mangostin in the human chondrosarcoma cell line SW1353. We showed that α-mangostin inhibited cell proliferation of SW1353 cells in a time- and dose-dependent manner by using the trypan blue exclusion method. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that α-mangostin could induce nuclear condensation and fragmentation, typically seen in apoptosis. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. α-Mangostin activated caspase-3, -8, -9 expression, decreased Bcl-2 and increased Bax. This promotes mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm. In addition, total and phosphorylated ERK and JNK were downregulated in α-mangostin-treated SW1353 cells but no changes in p38. α-Mangostin also decreased phosphorylated Akt without altering total Akt. These results suggest that α-mangostin inhinbited cell proliferation and induced apoptosis through downregulation of ERK, JNK and Akt signaling pathway in human chondrosarcoma SW1353 cells.
Published: March 29, 2011
r
2011 American Chemical Society
5746 dx.doi.org/10.1021/jf200620n
|
J. Agric. Food Chem. 2011, 59, 57465754
ARTICLE
pubs.acs.org/JAFC
r-Mangostin Induces Apoptosis in Human Chondrosarcoma Cells
through Downregulation of ERK/JNK and Akt Signaling Pathway
Aungkana Krajarng,
Yukio Nakamura,
Sunit Suksamrarn,
§
and Ramida Watanapokasin*
,
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand
Department of Orthopaedic Surgery, Shins hu University School of Medi cine, Matsumoto, 390-8621, Japan
§
Department of Chemis try, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand
ABSTRACT: Chondrosarcoma is a malignant primary bone tumor that is resistant to chemotherapy and radiation therapy.
R-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor
properties. This study is the rst to investigate anticancer eects of R-mangostin in the human chondrosarcoma cell line SW1353.
We showed that R-mangostin inhibited cell proliferation of SW1353 cells in a time- and dose-dependent manner by using the trypan
blue exclusion method. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that R-mangostin could
induce nuclear condensation and fragmentation, typically seen in apoptosis. Flow cytometry using Annexin V/PI double staining
assessed apoptosis, necrosis and viability. R-Mangostin activated caspase-3, -8, -9 expression, decreased Bcl-2 and increased Bax.
This promotes mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm. In
addition, total and phosphorylated ERK and JNK were downregu lated in R-mangostin-treated SW1353 cells but no changes in p38.
R-Mangostin also decreased phosphorylated Akt without altering total Akt. These re sults suggest that R-mangostin inhinbited cell
proliferation and induced apoptosis through downregulation of ERK, JNK and Akt signaling pathway in human chondrosarcoma
SW1353 cells.
KEYWORDS: R-mangostin, apotosis, chondrosarcoma, MAPK, Akt
INTRODUCTION
Chondrosarcoma is a malignant primary bone tumor that
overproduces chondrocytes and subsequently cartilage matrix.
Chondrosarcoma is the third most common primary malignancy
of bone after myeloma and osteosarcoma.
1
This disease usually
grows within bone or on its surface. The most common sites are
pelvic bones, shoulder bones, and the upper part of the arms and
legs. It develops from slow growing nonmetastasizing lesions to
highly aggressive metastasizing sarcomas.
2
Patients are usually
between 30 and 70 years old.
3
Chondrosarcoma is highly insensi-
tive to conventional chemotherapy and radiation treatment, there-
by the surgical resection remains the mainstay of treatment.
4
However, the development of eective adjuvant chemotherapy is
still required. New therapeutic and preventive strategies targeted at
cell growth inhibition and apoptosis induction may be another
alternative of treatment for chondrosarcoma.
Mangosteen, Garcinia mangostana Linn., is a fruit found in
South East Asia, for example, Thailand, Malaysia , Myanmar,
Philippines, Sri Lanka and India. Mangosteen is known as the
queen of fruits because it is one of the best tasting tropical fruits.
The fruit hull of mangosteen has been used as traditional
medicine for treatment of trauma, diarrhea, skin infection and
wounds for many years.
5
Moreover, xanthones, the secondary
metabolites from pericarp of mangosteen, possess biological
activities, such as antioxidant,
6
antibacterial,
7
antifungal,
8
anti-
inammatory,
9
and cytotoxic activities.
10,11
Previous studies have
reported the anticancer activities of R-mangostin, one of the
major xanthones from mangosteen, on human breast cancer,
12
prostate cancer,
13
leukemia,
14
glioblastoma cells
15
and colon
cancer.
16,17
However, the eects and related molecular mechanism
of R-mangostin on human chondrosarcoma have not been
reported.
In this study, we investigated the hypothesis that R-mangostin
could inhibit the growth of human chondrosarcoma SW1353 cell
line through induction of apoptosis. Our study shows expression
of caspase-3, -8 and -9, increased expression of Bax, decreased
expression of Bcl-2 and promoted cytochrome c release from
mitochondria. The results also show downregulated phos-
phorylation of ERK, JNK and Akt in R-mangostin-treated
chondrosarcoma cells. Altogether, our data demonstrate that
R-mangostin could induce apoptosis involving mitochondrial-
mediated pathway and caspase-8 activation through downregu-
lating the phosphorylated ERK, JNK and Akt in human chon-
drosarcoma SW1353 cells.
MATERIALS AND METHODS
Materials. Dulbeccos modified Eagles medium (DMEM) was
purchased from Gibco BRL (Grand Island, NY). Fetal bovine serum
(FBS), penicillin/streptomycin and trypsin were purchased from PAA
Laboratories (Pasching, Austria). 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT), trypan blue and propidium
iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO).
Hoechst 33342 and Annexin VFITC conjugate were purchased
from Invitrogen Molecular Probes (Eugene, OR). Primary rabbit
polyclonal anti-caspase-3, -caspase-9, -Bax, -BID, rabbit monoclonal
Received: February 14, 2011
Accepted: March 29, 2011
Revised: March 29, 2011
5747 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
anti-Bcl-2, -Bcl-xL, MAPK, Akt and mouse monoclonal anti-caspase-8
antibody were obtained from Cell Signaling Technology (Beverly, MA).
Mouse monoclonal antibody against human cytochrome c was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). ECL reagent and
X-ray film were purchased from Pierce (Rockford, IL)
r-Mangostin Extraction and Isolation. Mangosteen fruit
(G. mangostana) was collected from Kombang District, Chanthaburi
Province, Thailand, in April, 2007. A voucher specimen (Porntip
Wongnapa No. 002) was deposited at the Faculty of Science, Ram-
khamhaeng University. The dried and pulverized fruit hull of
G. mangostana (0.5 kg) was thoroughly extracted with ethyl acetate
(EtOAc) at 50 °C. The combined extract after filtration was concen-
trated under reduced pressure to yield the extract as a yellowish solid
(285 g). A portion of the extract was subjected to repeated column
chromatography over silica gel using a gradient of hexane/acetone and
yielded the pure major compound, R-mangostin, including other minor
xanthones. Purity of R-mangostin exceeded 98% as determined by
LC analysis, and its spectroscopic data (NMR and MS) was consistent with
the reported values.
18
R-Mangostin (C
24
H
26
O
6
) was dissolved in dimethyl
sulfoxide (DMSO; Amresco, OH) at the stock concentration of 100 μg/mL
and further diluted to the desired working concentration before use.
Cell Culture. The chondrosarcoma cell line SW1353 was obtained
from the American Type Culture Collection (ATCC, Manassas, VA).
The cell line was maintained as a monolayer in DMEM supplemented
with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at
37 °C in a humidified atmosphere of 5% CO
2
. The medium was
refreshed every 23 days. After about 90% of confluence, the cultured
cells were detached with 0.25% trypsinEDTA and subcultured.
Cell Proliferation and Cell Viability Assay. The cytotoxictiy of
R-mangostin was initially determined by cell proliferation analysis using
MTT assay. Cells were seeded at a density of 1 10
4
cells/well in a 96-
well plate and allowed to grow for 24 h. Cells were then treated with
R-mangostin at various concentrations of 0.005, 0.05, 0.5, 5, 50, and
500 μg/mL, whereas the control group was treated with DMSO. After
incubation for 24 h, 100 μL of 0.5 mg/mL MTT solution was added to
each well and the plate was further incubated for 2 h at 37 °C. The
supernatant was aspirated and 100 μL of DMSO was added to each well
to solubilize water insoluble purple formazan crystals. The absorbance
at 550 nm wasmeasured using a microplate reader (Multiskan EX, Thermo
electron corporation, Finland), and the IC
50
value was calculated using the
software GraphPad Prism 3.03 (GraphPad Software Inc., San Diego, CA).
The eect of R-mangostin on the cell viability was analyzed by using
a trypan blue exclusion method. Cells were seeded in a 24-well plate at
1 10
5
cells/well and incubated for 24 h. Then, the cells were treated
with the indicated concentrations (0, 5, 10, 15, 20, and 30 μg/mL) of
R-mangostin for 3, 6, 9, 12, and 24 h. Cells were harvested by
trypsinisation, stained with trypan blue and counted with a hemocyt-
ometer. Cell survival was expressed as percentage of viable cells of
treated cells to control cells. Cells were treated in triplicate, and the
experiments were repeated three times.
Cell Cycle Analysis. Cells were seeded in a 6-well plate at 1 10
6
cells/well for 24 h. The cells were then treated with DMSO (control)
and 20 μg/mL R-mangostin for 3 and 6 h. Cells were washed with ice-
cold PBS once and resuspended in 0.5 mL of PI solution (50 μg/mL PI,
0.1% Triton X-100, 0.1% sodium citrate) and incubated at 4 °C overnight in
the dark and analyzed for DNA content on a BD FACScan flow cytometer
(Becton Dickinson, San Jose, CA) in linear scale using BD FACSDiva
Software Version 4.1.2 (BD Science, Becton Dickinson, San Jose, CA
Nuclear Morphology Staining with Hoechst 33342.
SW1353 cells (1 10
6
cells/well in a 6-well plate) were treated with
20 μg/mL R-mangostin for 0, 3, and 6 h. After trypsinization, cells were
washed with 1 PBS and stained with 3 μg/mL of Hoechst 33342 for 15
min. Stained cells were examined using fluorescence microscope
(Olympus, Tokyo, Japan) with an ultraviolet filter.
DNA Fragmentation Analysis. SW1353 cells (1 10
6
cells/well)
were seeded in 6-well plates and treated with 0, 10, 20 μg/mL R-m angostin
for 18 h. Both floating and attached cells were collected by trypsinisation and
washed once with PBS, and DNA was extracted using a Genomic DNA
extraction kit (Gentra Puregene cell kit, QIAGEN) according to the
manufacturers instructions. The sample DNA was analyzed using 1.7%
agarose gel and visualized by ethidium bromide staining of the gel.
Phosphatidylserine Exposure. Cells (1 10
6
) were treated
with 20 μg/mL R-mangostin for 3 and 6 h. Then cells were harvested by
low speed centrifugation and washed once with ice-cold PBS. Cell pellets
were resuspended in 100 μL of binding buffer (10 mM Hepes, 150 mM
NaCl, 5 mM KCl, 1 mM MgCl
2
3
6H
2
O, 1.8 mM CaCl
2
), 5 μLof
Annexin VFITC conjugate and 10 μL of PI were added. The cells were
incubated at room temperature for 15 min in the dark. After adding 400
μL of binding buffer, the cells were analyzed using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA).
Western Blot Analysis. SW1353 cells were incubated for different
times in the presence or absence of 20 μg/mL R-mangostin, harvested
and washed once with ice cold PBS. Then, 2 10
6
cells were lysed for 30
min on ice in 50 μL of RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM
EDTA, 250 mM NaCl, 0.5% Triton X-100) containing complete mini
protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim,
Germany). Clear cell lysate supernatants were prepared by centrifuga-
tion, and the protein content was determined using Bio-Rad protein
assay (Bio-Rad Laboratories, USA). Proteins were separated by 12%
SDSPAGE and transferred onto polyvinylidene fluoride (PVDF)
membranes (Pall Corporation, USA) for 1 h at 100 V with the use of
a Mini Trans-Blot Cell (Bio-Rad). After blocking with TBST (10 mM
Tris, pH 7.5, 150 mM NaCl and 0.1% Tween 20) containing 5% nonfat
milk, the blots were incubated overnight at 4 °C with primary antibody.
The membranes were washed in TBST, and the appropriate secondary
antibody conjugated with horseradish peroxidase was added for 1 h at
room temperature. Immunoreactive protein bands were detected by
chemiluminescence using enhanced chemiluminescence reagent (ECL)
and exposed to X-ray film. The membranes were stripped and reprobed
with β-actin antibody to assess protein loading for each lane.
Detection of Cytochrome c Release. After incubating SW1353
cells for different times in the presence or absence of 20 μg/mL R-
mangostin, cells were harvested and washed once with ice cold PBS.
Cells were resuspended in 50 μL of S-100 lysis buffer (20 mM HEPES,
pH 7.5, 10 mM KCl, 1.5 mM MgCl
2
, 1 mM EGTA, 1 mM EDTA,
250 mM sucrose), homogenized and centrifuged at 4 °C at 500g for 5 min
to eliminate nuclei and unbroken cells. The resulting cell lysates were further
centrifuged at 4 ° C at 10000g for 30 min. The supernatants (cytosol
fractions) were separated by 12% SDSPAGE, and proteins were trans-
ferred onto PVDF membranes. The resulting blots were subjected to
immunodetection of cytochrome c, which had been released from mito-
chondria, using a mouse monoclonal antibody against human cytochrome c
with goat anti-mouse IgG, conjugated to horseradish peroxidase as second-
ary antibody. The peroxidase activity of bound secondary antibodies on the
blots was detected by using ECL and exposed to X-ray film.
Statistical Analysis. All data presented were obtained from at least
three independent experiments and are presented as mean ( standard
deviation (SD). Statistical significance was assessed by Students t-test,
carried out using the software GraphPad Prism 3.03 (GraphPad Soft-
ware Inc., San Diego, CA).
RESULTS
r-Mangostin Inhibited Cell Proliferation and Induced
Apoptosis in Human Chondrosarcoma Cells.
Initially, the
antiproliferative activity of R-mangostin in SW1353 cells was
screened by using MTT assay and the IC
50
value was estimated
to be 10 μg/mL. Then, the effect of R-mangostin on cell viability
5748 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
was reanalyzed for varying concentrations and times by using the
trypan blue exclusion method. As shown in Figure 1A, inhibition
of cell viability by R-mangostin occurred in a dose- and time-
dependent manner. Treatment SW1353 cells with R-mangostin
at 15 μg/mL or higher for 24 h reduced cell viability to
approximately 10% or lower comparing with control cells.
To determine whether antiproliferation and cell death are
associated with apoptosis, SW1353 cells were stained with
Hoechst 33342 dye after exposure to R-mangostin and observed
under uorescence microscopy. The result showed that the
nuclei of untreated SW1353 cell appeared rou nd in shape, while
R-mangostin caused nuclear conde nsation and simultaneously
induced morphological changes in some of the cells at 6 h
(Figure 1B). After prolonged exposure (18 h), DNA samples
were extracted and analyzed by gel electrophoresis to determine
DNA fragmentation. Result clearly showed that DNA from R-
mangostin treated cells exhibited such fragments typical of
apoptosis, whereas control cells did not provide ladders
(Figure 1C). These results suggested that the proliferation
inhibition and the death of target cells upon treatment with
R-mangostin are consequent to the induction of apoptosis.
To furt her investigate the eects of R-mangostin on cell cycle
distribution, we analyzed SW1353 cells treated with R-mangostin
using ow cytometry. The sub-G1 peak formed with reduced
DNA content represented the presence of apoptotic cells. The two
major peaks represented the G0/G1 and G2/M phases of the cell
cycle. It was observed that the mean apoptotic population of SW1353
cells was 3.6% under control condition, while it was increased to 5.3%
and 14.9% after treatment with 20 μg/mL R-mangostin for 3 and 6 h
respectively in a time-dependent manner (Figure 2).
Effect of r-Mangostin on Phosphatidyl Serine Exposure.
One of the early indications of apoptosis is the translocation of
the membrane phospholipid phosphatidylserine from the inner
to the outer leaflet of the plasma membrane. Annexin VPI
double-labeling and FACS analysis were used for the detection of
phosphatidylserine externalization. As shown in Figure 3, the
four quadrants in each panel correspond, respectively, to necrotic
cells (upper left), late apoptotic cells (upper right), viable cells
(lower left), and early apoptotic cells (lower right). After treat-
ment with R-mangostin, the results showed that control cells
were mostly alive whereas the R-mangostin treated cells in-
creased early and late apoptotic cells in a time-dependent
manner.
Effect of r-Mangostin on Capase-3, Caspase-8 and Cas-
pase-9 Activation.
To investigate whether the induction of
apoptosis by R-mangostin treatment is caspase-dependent and
which caspase(s) is involved, we examined the formation of
active forms of caspases in cell lysates by Western blot analysis.
As shown in Figure 4, R-mangostin could induce caspase-3,
caspase-8 and caspase-9 cleavage after 3 and 6 h of treatment.
Thus, R-mangostin-induced apoptosis is mediated by caspase-3
and maybe associated with the activation of both extrinsic (via
caspase-8) and intrinsic apoptosis pathways (via caspase-9).
Effect of r-Mangostin on the Expression of Bcl-2 Family
Proteins and the Induction of Cytochrome c Release.
The
Bcl-2 family of proteins regulates cell death by controlling of the
mitochondrial membrane permeability during apoptosis. The
insertion of Bax into mitochondrial membrane induces the
opening of the mitochondrial voltage-dependent anion channel
(VDAC) and the release of cytochrome c from mitochondria to
cytoplasm, which is a hallmark of the mitochondrial apoptosis
pathway.
19
To determine whether R-mangostin induces apop-
tosis by triggering the mitochondrial apoptotic pathway, we
measured the change in the expression of the Bcl-2 family of
proteins. The results showed that treatment of SW 1353 cells with
R-mangostin induced Bax protein level and decreased Bcl-2
expression but no apparent changes in the expression level of
Bcl-xL (Figure 5). In the case of Bid, its expression apparently
decreased after the treatment, however, the cleaved active
form (tBid) was not produced. In addition, the release of
cytochrome c was detected in cytosolic extract of R-mangostin-
treated SW 1353 at 3 h and 6 h but the control SW1353 cells
(Figure 6). These results indicate that R-mangostin induced
apoptosis via mitochondrial pathway.
Figure 1. Eects of R-mangostin on cell viability and apoptosis induction in SW1353 cells. (A) Time- and dose-dependent eect of R-mangostin was
performed when SW1353 cells were treated with various concentrations of R-mangostin for various times, and their viability was determined by MTT
assay. Results are mean values ( SD of three independent experiments performed in triplicate (n = 3). (B) The morphologic changes of SW1353 treated
with DMSO (control) or 20 μg/mL R-mangostin for 6 h, then stained with Hoechst 33342 and examined under a uorescent microscope (40
magnication). (C) DNA fragmentation of SW1353 cells treated with 10, 20 μg/mL R-mangostin for 18 h visualized by gel electrophoresis (M, DNA
size marker).
5749 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
Effect of r-Mangostin on the Expression of MAPKs and
Akt.
MAPKs are a widely conserved family of serine/threonine
protein kinases involved in many cellular activities such as cell
proliferation, differentiation, motility, survival and death.
20
MAPKs are activated by phosphorylation of specific tyrosine
and threonine residues and the relative levels of phosphorylated
MAPKs in total MAPKs represent the degree of MAPK activa-
tion. Figure 7 shows that R-mangostin decreased the level
of total and phosphorylated ERK1/2 and JNK proteins but it
did not affect p38 activity. It should be noted that the level
of phosphorylated ERK1/2 was increased at 3 h and then
decreased at 6 h after treatment with R-ma ngostin. The
results suggested that ERK and JNK might be involved in R-
mangostin-induced a poptosis in human chondrosarcoma
cells.
Akt plays a key role in controlling survival and apoptosis. It can
be activated by phospholipid binding and activation loop phos-
phorylation at threonine 308 by PDK1 and by phosphorylation
within the carboxy terminus at serine 473.
21
We examined the
phosphorylation of Akt and PDK1 after treatment of R-mangos-
tin. The phosphorylated Akt and PDK1 decreased in a time-
dependent manner, but R-mangostin treatment did not aect
total Akt protein (Figure 8). Our results suggested that R-
mangostin triggers apoptosis by downregulating phosphoryla-
tion of Akt pathway in human chondrosarcoma cells.
DISCUSSION
Chondrosarcoma is a malignant primary bone tumor with a
poor prognosis and resistant to chemotherapy and radiation
treatment. In this study, we investigated the potential of
R-mangostin from pericarp of mangosteen for the treatment of
human chondrosarcoma. We demonstrated the anticancer eect
of R-mangostin in SW1335 cell line. The results show that
R-mangostin could inhibit cell growth of SW1353 cells in a time-
and dose-dependent manner with an IC
50
value of 10 μg/mL.
Several researchers have documented the cytotoxic and antipro-
liferative eects of R-mangostin at concentrations ranging from
1to10μg/mL in various human cancer cell lines.
1013
Our data
are consistent with the IC
50
value of 9 and 9.25 μg/mL in PC3
human prostate cancer cells and SKBR3 human breast cancer
cells, respectively. The dierences in IC
50
values of R-mangostin
in individual cell lines may be attributed to the dierences in the
duration of exposure and the dierential sensitivities of cell lines
to the cytotoxic eects of R-mangostin.
To conrm apoptosis, DNA fragmentation analysis and
characteristic morphological changes, including membrane bleb-
bling, cell shrinkage, chromatin condensation, and formation of
apoptotic bodies were investigated. Hoechst 33342 staining
clearly showed condensed and fragmented nuclei in SW1353
cells treated with R-mangostin (Figure 1B). Cell cycle analysis
Figure 2. Cell cycle analysis of SW1353 cells treated with R-mangostin. The sub-G1 peak formed with reduced DNA content represented the presence
of apoptotic cells. The two major peaks represented the G0/G1 and G2/M phases of the cell cycle. It was observed that the mean apoptotic population of
SW1353 cells was 3.6% under control condition, while it was increased to 5.3% and 14.9% after treatment with 20 μg/mL R-mangostin for 3 and 6 h
respectively.
5750 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
Figure 3. Flow cytometric of phosphatidylserine exposure for SW1353 cells. SW1353 cells were exposed DMSO (control) and 20 μg/mL R-mangostin
for 3 and 6 h. (A) Representative dot plots of Annexin V/PI staining are shown. The upper left quadrant contains the necrotic (Annexin V/PIþ)
population. The upper right quadrant contains the late apoptotic/necrotic (Annexin Vþ/PIþ) population. The lower left quadrant contains the vital
(double negative) population. The lower right quadrant contains the early apoptotic (Annexin Vþ/PI) population. The result is from one experiment
representative of three similar experiments. (B) The percentage of necrotic cells, early apoptotic cells, viable cells and late apoptotic cells at 0, 3, and 6 h
are indicated. The experiment was performed in three independent experiments, and the results are expressed as mean ( SD.
Figure 4. Eects of R-mangostin on activation of caspase-3, -8, and -9 in SW1353 cells. Cells were treated with 20 μg/mL R-mangostin for 3 and 6 h and
examined by Western blot analysis. β-Actin was used as the internal control.
5751 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
was also conducted (Figure 2). As shown in Figure 1C, cleavage
of DNA at the internucleosomal linker sites yielding DNA
fragments in multiple fragments (180200 bp) is regarded as
a biochemical hallmark of apoptosis.
22
Furthermore, in early
apoptosis phosphatidylserine is translocated from the inner to
outer surface of the plasma membrane due to the loss of
membrane asymmetry which can be detected by Annexin V.
Flow cytometry analysis demonstrated phosphatidylserine ex-
posure in R-mangostin treated cells, and the percentage of early
apoptotic and late apoptotic cells increased with the incubation
time (Figure 3).
It is now well-known that apoptotic signaling proceeds
through two main pathways, the extrinsic and intrinsic pathways.
The extrinsic pathway is triggered through the binding of death
Figure 5. Eects of R-mangostin on expression of Bax, Bcl-2, Bcl-xL and Bid in SW1353 cells. Cells were treated with 20 μg/mL R-mangostin for 3 and 6
h and examined by Western blot analysis. β-Actin was used as the internal control.
Figure 7. Eects of R-mangostin on expression of ERK1/2, phosphory-
lated ERK1/2 (p-ERK1/2), p38, phosphorylated p38 (p-p38), JNK,
phosphorylated JNK (p-JNK) in SW1353. Cells were treated with 20
μg/mL R-mangostin for 3 and 6 h and examined by Western blot
analysis. β-Actin was used as the internal control.
Figure 6. R-Mangostin induces cytochrome c release from the mito-
chondria in SW1353 cells. Cells were treated with 20 μg/mL R-
mangostin for 3 and 6 h. Cytosol fractions were separated by
SDSPAGE and subjected to immunodetection of cytochrome c.
5752 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
ligands and death receptors on the cell surface resulting in
activation of initi ator caspase-8. The intrinsic or mitochondrial
pathway is characterized by depolarization of mitochondrial
membrane, leading to the release of cytochrome c and caspase-
9 activation. The intrinsic pathway is regulated by Bcl-2 family of
proteins which are composed of both pro- and antiapoptotic
molecules.
23
Antiapoptotic Bcl-2 and proapoptotic Bax are two
of the major members of the Bcl-2 family. During apoptosis,
antiapoptotic Bcl-2 protects cells from many dierent apoptotic
stimuli. On the other hand, the proapoptotic Bax interacts with
the permeability transition pores to facilitate protein movement
through the mitochondrial membrane, leading to the release of
cytochrome c from the intermemb rane space into cytoplasm.
Subsequently, activated caspase-8 and -9 promote caspase-3
cleavage and activation, resulting in nuclear DNA degradation
and apoptotic death of cells. In our study, activated caspase-3, -8,
and -9 were detected in the presence of R-mangostin (Figure 4),
implying that the induction of apoptosis in SW1353 cells by R-
mangostin may be associated with the activation of both path-
ways. Previous studies report that caspase-8 can be activated by
caspase-3 and caspase-8-mediated Bid cleavage appears to be
downstream of mitochondria-mediated pathway.
24,25
As a result,
tBid could not be detected (Figure 5), therefore further studies
about the binding of death ligands and death receptors are
needed to conrm the extrinsic pathway of apoptosis. However,
R-mangostin could induce the release of cytochrome c, upregula-
tion of Bax and downregulation of Bcl-2 (Figure 5-6). These
results suggested that R-mangostin could induce apoptosis in
SW1353 cells through the mitochondrial pathway.
The MAPK family plays critical roles in cell survival and death.
Three major mammalian MAPK subfamilies, ERK, JNK and p38,
were activated through a specic phosphorylation cascade. It is
well-known that JNK and p38 induc e apoptosis, while ERK
promotes cell survival.
26
Previous studies have shown that
upregulation of MAPK phosphorylation is involved in migration
of chondrosarcoma cells.
27,28
Therefore, downregulation of
MAPK phosphorylation may be an appropriate alternate therapy
for chondrosarcoma patients . In our study, R-mangostin was
shown to downregulate the total and phosphorylated ERK1/2
and JNK proteins but not p38 (Figure 7). However, the
phosphorylated ERK1/2 was increased at 3 h and decreased at
6 h after treatment with R-mangostin. As reported,
17
the
phosphorylated ERK1/2 indicat ed two peaks, one at the early
phase (0.53 h) and the other at the late phase (2448 h) of the
treatment with R-mangostin in DLD-1 cells. ERK1/2 may play
dual roles, as a cellular adaptive response during the initial phase
and a cytotoxic response during the later stages of such stress.
Therefore, the decline in the phosphorylated ERK1/2 may be
associated with the apoptotic machinery.
Akt signaling pathway is essential for cell survival, and the
expression of a constitutively active Akt or an increased activity of
the PI3K/Akt pathway leads to multidrug resistance and prevent
apoptosis in a variety of cell types.
29,30
Our results showed that R-
mangostin could decrease the phosphorylation of Akt in chon-
drosarcoma cells (Figure 8). These results correspond with much
research that reported downregulating of ERK/Akt pathway
induced apoptosis in cancer cells. For instance, Lu et al. showed
that sorafenib induced apoptosis via inhibition of the ERK
pathway in human chondrosarcoma cells.
31
Fei et al. reported
that perifosine induced apoptosis in human hepatocellular
carcinoma cells by inhibition of ERK/AKT phosphorylation.
32
Chai et al. demonstrated tha t downregulation of ERK/Akt is
associated with sorafenib-induced apoptosis in human neuro-
blastoma cells.
33
In conclusion, we demonstrated that R-mangostin inhibited
cell proliferation and induced apoptosis in chondrosarcoma cells,
which may be through downregulating the ERK, JNK and Akt
signaling pathway. These data suggest the potential clinical
Figure 8. Eects of R-mangostin on expression of Akt and phosphorylated Akt (p-Akt) in SW1353 cells. Cells were treated with 20 μg/mL R-mangostin
for 3 and 6 h and examined by Western blot analysis. β-Actin was used as the internal control.
5753 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
application of R -mangostin in the treatment of human
chondrosarcoma.
AUTHOR INFORMATION
Corresponding Author
*Tel: þ662-649-5369. Fax: þ662-649-5834. E-ma il: ramidawa@
yahoo.com.
Funding Sources
This work was supported by Srinakharinwirot Research Budget,
Research Division, Srinakharinwirot University and the pro-
gram Strategic Scholarships for Frontier Research Network for
the Join Ph.D. Program Thai Doctoral degree from the Oce of
the Higher Education Commission, Thailand (to A.K.).
REFERENCES
(1) Gelderblom, H.; Hogendoorn, P. C.; Dijkstra, S. D.; van Rijswijk,
C. S.; Krol, A. D.; Taminiau, A. H.; Bovee, J. H. The clinical approach
towards chondrosarcoma. Oncologist 2008, 13, 320329.
(2) Marco, R. A.; Gitelis, S.; Brebach, G. T.; Healey, J. H. Cartilage
tumors: evaluation and treatment. J. Am. Acad. Orthop. Surg. 2000,
8, 292304.
(3) Ollivier, L.; Vanel, D.; Leclere, J. Imaging of chondrosarcomas.
Cancer Imaging 2003, 4,3638.
(4) Wesolowski, R.; Budd, G. T. Use of chemotherapy for patients
with bone and soft-tissue sarcomas. Cleveland Clin. J. Med. 2010, 77
s23s26.
(5) Mahabusarakam, W.; Wiriyachtra, P.; Taylor, W. Chemical
constituents of Garcinia mangostana. J. Nat. Prod. 1987, 50, 474478.
(6) Jung, H. A.; Su, B. N.; Keller, W. J.; Mehta, R. G.; Kinghorn, A. D.
Antioxidant xanthones from the pericarp of Garcinia mangostana
(Mangosteen). J. Agric. Food Chem. 2006, 54, 20772082.
(7) Suksamrarn, S.; Suwannapoch, N.; Phakhodee, W.; Thanuhiranlert,
J.; Ratananukul, P.; Chimnoi, N.; Suksamrarn, A. Antimycobacterial
activity of prenylated xanthones from the fruits of Garcinia mangostana.
Chem. Pharm. Bull. 2003, 51, 857859.
(8) Gopalakrishnan, G.; Banumathi, B.; Suresh, G. Evaluation of the
antifungal activity of natural xanthones from Garcinia mangostana and
their synthetic derivatives. J. Nat. Prod. 1997, 60, 519524.
(9) Chen, L. G.; Yang, L. L.; Wang, C. C. Anti-inammatory activity
of mangostins from Garcinia mangostana. Food Chem. Toxicol. 2008, 46,
688693.
(10) Suksamrarn, S.; Komutiban, O.; Ratananukul, P.; Chimnoi, N.;
Lartpornmatulee, N.; Suksamrarn, A. Cytotoxic prenylated xanthones
from the young fruit of Garcinia mangostana. Chem. Pharm. Bull. 2006
,
54, 301305.
(11) Zhang, Y.; Song, Z.; Hao, J.; Qiu, S.; Xu, Z. Two new prenylated
xanthones and a new prenylated tetrahydroxanthone from the pericarp
of Garcinia mangostana. Fitoterapia 2010, 81, 595599.
(12) Moongkarndi, P.; Kosem, N.; Kaslungka, S. Antiproliferation,
antioxidation and induction of apoptosis by Garcinia mangostana
(mangosteen) on SKBR3 human breast cancer cell line. J. Ethnophar-
macol. 2004, 90, 161166.
(13) Hung, S. H.; Shen, K. H.; Wu, C. H.; Liu, C. L.; Shin, Y. W. R-
mangostin suppresses PC-3 human prostate carcinoma cell metastasis
by inhibiting matrix metalloproteinase-2/9 and urokinase-plasminogen
expression through the JNK signaling pathway. J. Agric. Food Chem.
2009, 57, 12911298.
(14) Matsumoto, K.; Akao, Y.; Emi, K.; Ohguchi, K.; Ito, T.; Tanaka,
T.; Iinuma, M.; Nozawa, Y. Induction of apoptosis by xanthones from
mangosteen in human leukemia cell lines. J. Nat. Prod. 2003, 66,
11241127.
(15) Chao, A. C.; Hsu, Y. L.; Liu, C. K.; Kuo, P. L. R-Mangostin, a
dietary xanthone, induces autophagic cell death by activating the AMP-
activated protein kinase pathway in glioblastoma cells. J. Agric. Food
Chem. 2011, 59, 20862096.
(16) Matsumoto, K.; Akao, Y.; Ohguchi, K.; Ito, T.; Tanaka, T.;
Iinuma, M.; Nozawa, Y. Xanthones induce cell-cycle arrest and apoptosis
in human colon cancer DLD-1 cells. Bioorg. Med. Chem. 2005, 13
60646069.
(17) Nakagawa, Y.; Iinuma, M.; Naoe, T.; Nozawa, Y.; Akao, Y.
Characterized mechanism of R-mangostin-induced cell death: Caspase-
independent apoptosis with release of endonuclease-G from mitochon-
dria and increased miR-143 expression in human colorectal cancer DLD-
1 cells. Bioorg. Med. Chem. 2007, 16, 56205628.
(18) Watanapokasin, R.; Jarinthanan, F.; Jerusalmi, A.; Suksamrarn,
S.; Nakamura, Y.; Sukseree, S.; Uthaisang-Tanethpongtamb, W.;
Ratananukul, P.; Sano, T. Potential of xanthones from tropical fruit
mangosteen as anti-cancer agents: caspase-dependent apoptosis
induction in vitro and in mice. Appl. Biochem. Biotechnol. 2010, 162,
10801094.
(19) Shimizu, S.; Narita, M.; Tsujimoto, Y. Bcl-2 family proteins
regulate the release of apoptogenic cytochrome c by the mitochondrial
channel VDAC. Nature 1999,
399, 483487.
(20) Boutros, T.; Chevet, E.; Metrakos, P. Mitogen-activated protein
(MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth,
death, and cancer. Pharmacol. Rev. 2008, 60, 261310.
(21) Alessi, D. R.; Andjelkovic, M.; Caudwell, B.; Cron, P.; Morrice,
N.; Cohen, P.; Hemmings, B. A. Mechanism of activation of protein
kinase B by insulin and IGF-1. EMBO J. 1996, 15, 65416551.
(22) Rello, S.; Stockert, J.; Moreno, V.; Gamez, A.; Pacheco, M.;
Juarranz, A.; Canete, M.; Villanueva, A. Morphological criteria to
distinguish cell death induced by apoptotic and necrotic treatments.
Apoptosis 2005, 10, 201208.
(23) Gross, A.; McDonnell, J. M.; Korsmeyer, S. J. BCL-2 family
members and the mitochondria in apoptosis. Genes Dev. 1999,
13, 18991911.
(24) Chen, C. J.; Makino, S. Murine coronavirus-induced apoptosis
in 17Cl-1 cells involves a mitochondria-mediated pathway and its
downstream caspase-8 activation and Bid cleavage. Virology 2002,
302, 321332.
(25) Oh, S. H.; Lee, B. H. A ginseng saponin metabolite-induced
apoptosis in HepG2 cells involves a mitochondria-mediated pathway
and its downstream caspase-8 activation and Bid cleavage. Toxicol. Appl.
Pharmacol. 2004, 194, 221229.
(26) Xia, Z.; Dickens, M.; Raingeaud, J.; Davis, R. J.; Greenberg,
M. E. Opposing eects of ERK and JNK-p38 MAP kinases on apoptosis.
Science 1995, 270, 13261331.
(27) Chen, Y. J.; Wei, Y. Y.; Chen, H. T.; Fong, Y. C.; Hsu, C. J.; Tsai,
C. H.; Hsu, H. C.; Liu, S. H.; Tang, C. H. Osteopontin increases
migration and MMP-9 up-regulation via alphavbeta3 integrin, FAK,
ERK, and NF-kappaB-dependent pathway in human chondrosarcoma
cells. J. Cell Physiol. 2009, 221,98108.
(28) Tan, T. W.; Lai, C. H.; Huang, C. Y.; Yang, W. H.; Chen, H. T.;
Hsu, H. C.; Fong, Y. C.; Tang, C. H. CTGF enhances migration and
MMP-13 up-regulation via alphavbeta3 integrin, FAK, ERK, and NF-
kappaB-dependent pathway in human chondrosarcoma cells. J. Cell
Biochem. 2009, 107, 345356.
(29) McCubrey, J. A.; Steelman, L. S.; Abrams, S. L.; Lee, J. T.;
Chang, F.; Bertrand, F. E.; Navolanic, P. M.; Terrian, D. M.; Franklin,
R. A.; DAssoro, A. B.; Salisbury, J. L.; Mazzarino, M. C.; Stivala, F.;
Libra, M. Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT path-
ways in malignant transformation and drug resistance. Adv. Enzyme
Regul. 2006, 46, 249279.
(30) Knuefermann, C.; Lu, Y.; Liu, B.; Jin, W.; Liang, K.; Wu, L.;
Schmidt, M.; Mills, G. B.; Mendelsohn, J.; Fan, Z. HER2/PI-3K/Akt
activation leads to a multidrug resistance in human breast adenocarci-
noma cells. Oncogene 2003, 22, 32053212.
(31) Lu, X.; Tang, X.; Guo, W.; Ren, T.; Zhao, H. Sorafenib induces
growth inhibition and apoptosis of human chondrosarcoma cells by
blocking the RAF/ERK/MEK pathway. J. Surg. Oncol. 2010,
102, 821826.
5754 dx.doi.org/10.1021/jf200620n |J. Agric. Food Chem. 2011, 59, 5746–5754
Journal of Agricultural and Food Chemistry
ARTICLE
(32) Fei, H. R.; Chen, G.; Wang, J. M.; Wang, F. Z. Perifosine
induces cell cycle arrest and apoptosis in human hepatocellular carci-
noma cell lines by blockade of Akt phosphorylation. Cytotechnology
2010, 62, 449460.
(33) Chai, H.; Luo, A. Z.; Weerasinghe, P.; Brown, R. E. Sorafenib
downregulates ERK/Akt and STAT3 survival pathways and induces
apoptosis in a human neuroblastoma cell line. Int. J. Clin. Exp. Pathol.
2010, 3, 408415.
    • "Recently, the stem bark of G. malaccensis has been shown to contain xanthones, namely, í µí»¼-mangostin as the major compound [26]. Interestingly, the most studied xanthone of Garcinia species is í µí»¼-mangostin for which antibacterial [27] , antiox- idant [28], anti-inflammatory293031, antiproliferative [32], proapoptotic333435, and anticarcinogenic36373839 activities have already been reported. This paper reports the effect of í µí»¼-mangostin isolated from the stem bark of G. malaccensis on 3T3-L1 adipocytes via the adipogenesis and glucose uptake activity. "
    [Show abstract] [Hide abstract] ABSTRACT: Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 diabetes, and cancer. The development of obesity is also accompanied by significant differentiation of preadipocytes into adipocytes. In this study, we investigated the activity of α-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and adipocyte differentiation of 3T3-L1 cells focusing on PPARγ, GLUT4, and leptin expressions. α-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50 μM of α-mangostin reduced intracellular fat accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[3H] glucose uptake activity showed that α-mangostin significantly improved the glucose uptake ( P < 0.05 ) with highest activity found at 25 μM. In addition, α-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50 μM of α-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPARγ gene expression. Induction of glucose uptake and free fatty acid release by α-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that α-mangostin might be possible candidate for the effective management of obesity in future.
    Full-text · Article · Feb 2015
    • "The ERKs pathway is upregulated in human tumors and, as such, represents an attractive target for anticancer therapy [39, 40]. The phosphorylation of ERKs results in their nuclear translocation and activation of numerous substrates, promoting proliferation and inhibiting proapoptotic signals [41]. "
    [Show abstract] [Hide abstract] ABSTRACT: Liquiritigenin (LQ), separated from Glycyrrhiza radix, possesses anti-inflammatory, antihyperlipidemic, and antiallergic effects. Our present study aims to investigate the antihepatocellular carcinoma effects of LQ both in cell and animal models. LQ strikingly reduced cell viability, enhanced apoptotic rate, induced lactate dehydrogenase over-release, and increased intracellular reactive oxygen species (ROS) level and caspase 3 activity in both PLC/PRL/5 and HepG2 cells. The expression of cleaved PARP, the hall-marker of apoptosis, was enhanced by LQ. LQ treatment resulted in a reduction of the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), and an increase of the phosphorylation of c-Jun N-terminal kinases (JNK) and P38. LQ-mediated cell viability reduction, mitochondrial dysfunction, apoptosis related protein abnormal expressions, and JNK and P38 activation were partially abolished by N-Acetyl-L-cysteine (a ROS inhibitor) pretreatment. Moreover, LQ suppressed the activation of extracellular signaling-regulated kinase (ERKs) and reduced the translocation of phosphor-ERKs from cytoplasm to nucleus. This antitumor activity was further confirmed in PLC/PRL/5-xenografted mice model. All these data indicate that the antihepatocellular carcinoma effects of LQ are related to its modulation of the activations of mitogen-activated protein kinase (MAPKs). The study provides experimental evidence supporting LQ as a potential therapeutic agent for hepatocellular carcinoma treatment.
    Full-text · Article · Mar 2014
    Di WangDi WangJiahui LuJiahui LuYan LiuYan Liu+1more author...[...]
    • "In our study, the toxicity in cultured Hela and SW1353 cells induced by gliotoxin was found to be due to apoptosis, since cells changed nuclear morphology by chromatin condensation using DAPI stain (Figure 3A,B). Several studies reported that apoptosis in the Hela and SW1353 cells presented typical apoptotic morphological changes with cell shrinkage, nuclei blebbing, chromatin condensation, and formation of apoptotic bodies [5,14]. Because the appearance of apoptotic bodies is regarded as the apoptotic hallmark, the results suggested that gliotoxin induced the death of Hela and SW1353 cells mainly through apoptosis. "
    Full-text · Dataset · Dec 2013 · Evidence-based Complementary and Alternative Medicine
Show more