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Resveratrol Induces Apoptosis of
Human Nasopharyngeal
Carcinoma Cells Via Activation of
Multiple Apoptotic Pathways
TSUNG-TENG HUANG,
1
HUNG-CHI LIN,
2
CHANG-CHIEH CHEN,
2
CHIA-CHEN LU,
3
CHIA-FONG WEI,
2
TING-SHU WU,
4,5
FU-GUO LIU,
1
*AND HSIN-CHIH LAI
2
*
1
Department of Life Science, National Central University, Jhongli, Taoyuan, Taiwan, Republic of China
2
Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Kweishan, Taoyuan, Taiwan,
Republic of China
3
Department of Respiratory Therapy, Fu Jen Catholic University, Sinjhuang, Taipei, Taiwan, Republic of China
4
Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kweishan, Taoyuan, Taiwan, Republic of China
5
Department of Internal Medicine, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan, Republic of China
Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been reported to trigger a variety of cancer cell
types to apoptosis. Whether resveratrol shows any activity on human nasopharyngeal carcinoma (NPC) cells remained to be determined.
The aim of this study was to investigate the effect and mechanism of resveratrol on human NPC cells. Treatment of resveratrol resulted in
significant decrease in cell viability of NPC cell lines in a dose- and time-dependent manner. A dose-dependent apoptotic cell death was
also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled resveratrol treatment resulted in
a significant loss of mitochondrial transmembrane potential, release of cytochrome c, enhanced expression of Fas ligand (FasL), and
suppression of glucose-regulated protein 78 kDa (GRP78). These were followed by activation of caspases-9, -8, -4, and -3, subsequently
leading to DNA fragmentation and cell apoptosis. Furthermore, up-regulation of proapoptotic Bax and down-regulation of antiapoptotic
Bcl-2 protein were also observed. Taken together, resveratrol induces apoptosis in human NPC cells through regulation of multiple
apoptotic pathways, including death receptor, mitochondria, and endoplasmic reticulum (ER) stress. Resveratrol can be developed as an
effective compound for human NPC treatment.
J. Cell. Physiol. 226: 720–728, 2011. ß2010 Wiley-Liss, Inc.
Nasopharyngeal carcinoma (NPC) is a unique malignant
epithelial carcinoma of the head-and-neck region, with a distinct
racial and geographic distribution across the world (Lo et al.,
2007). Most cases of NPC are found in South East Asia,
Southern China, North Africa, and the Middle East (Lictera
et al., 2003). Three major etiological factors, including
genetic susceptibility, Epstein–Barr virus (EBV) infection,
environmental (including chemical carcinogens) and dietary
factors are all believed to play important roles in NPC
carcinogenesis (Tao and Chan, 2007). Although the concurrent
combination of radiotherapy and chemotherapy has improved
outcome for patients with advanced stage of NPC, side effects
such as fatal toxicity and the poor outcome of recurrent disease
were observed (Erkal et al., 2001; O’Sullivan, 2007). Therefore,
this indicates that the development of more effective and less
toxic alternatives is urgently needed.
Resveratrol (trans-3, 40, 5-trihydroxystilbene) is a natural
polyphenolic compound which has been found in a wide variety
of plant species, including the grapes, peanuts, berries, and
herbs (Jang et al., 1997). Its potential chemopreventive and
chemotherapeutic activities have been demonstrated in all
three stages of carcinogenesis (initiation, promotion, and
progression) in a variety of cancer cell culture systems and
animal models (Athar et al., 2007; Bishayee, 2009). Many studies
have also shown that the chemopreventive activity of
resveratrol is related to its ability to trigger apoptosis in a
number of cancer cell lines (Pervaiz, 2004).
While an imbalance between cell death and proliferation
may result in tumor formation, apoptosis plays a crucial role
in regulation of tissue homeostasis (Lowe and Lin, 2000).
Generally, killing of tumor cells by diverse cytotoxic approaches
including anticancer drugs, g-irradiation, suicide genes, or
immunotherapy is predominantly mediated through induction
of cell apoptosis (Johnstone et al., 2002). In the death receptor-
mediated pathway, after engagement of the specific ligand FasL,
the cytoplasmic domain of death receptor Fas (APO-1/CD95)
trimerizes and recruits the adaptor molecule Fas associated
death domain (FADD). In turn, FADD recruits pro-caspase-8 to
form the death-inducing signaling complex (DISC) in which
caspase-8 is activated, leading to caspase-3 activation and
apoptosis (Nagata, 1997). Caspase-8 can also activate Bcl-2
Contract grant sponsor: National Science Council;
Contract grant number: NSC96-2628-B-033-MY3.
Contract grant sponsor: Chang Gung Memorial Hospital;
Contract grant number: CMRPD160273.
*Correspondence to: Fu-Guo Liu and Hsin-Chih Lai,
Department of Life Science, National Central University,
No. 300, Jhongda Road, Jhongli, Taoyuan 320, Taiwan; Department
of Medical Biotechnology and Laboratory Science, Chang Gung
University, No. 259, Wen-Hwa 1st Road, Kweishan, Taoyuan 333,
Taiwan. E-mail: liur@cc.ncu.edu.tw, hclai@mail.cgu.edu.tw
Received 27 June 2010; Accepted 5 August 2010
Published online in Wiley Online Library
(wileyonlinelibrary.com), 17 August 2010.
DOI: 10.1002/jcp.22391
ORIGINAL ARTICLE 720
Journal of
Journal of
Cellular
Physiology
Cellular
Physiology
ß2010 WILEY-LISS, INC.
interacting domain (Bid), a proapoptotic member of the Bcl-2
family, by converting it to its truncated form, promoting
apoptosis (Roy and Nicholson, 2000). In the mitochondrial-
mediated pathway, activation of mitochondria is accompanied
by the translocation of cytochrome cfrom mitochondria
intermembrane space into the cytoplasm. Cytochrome c,
apoptosis protease-activating factor-1 (Apaf-1), adenosine
triphosphate and procaspase-9 form a supramolecular complex
termed ‘‘apoptosome’’ that activates caspase-9 through
autocatalysis, and the latter then activates caspase-3, resulting
in apoptosis (Green and Reed, 1998). The mitochondria-
initiated apoptotic pathway is also tightly regulated by Bcl-2
family proteins Bax and Bak. Both are proapoptotic proteins
activated by a variety of apoptotic stimuli, resulting in
oligomerization and insertion into mitochondrial outer
membrane to release cytochrome c(Ferri and Kroemer, 2001).
Besides the two pathways, recent studies indicated the
endoplasmic reticulum (ER) stress as a third subcellular
compartment implicated in apoptotic execution (Rao et al.,
2004). ER stress activates unfolded protein response (UPR) and
the ER-resident cysteine protease caspase-12, leading to
caspase-3 activation and apoptosis (Zhang et al., 2006).
However, although murine caspase-12 is an active enzyme, the
human caspase-12 homologue contains several loss-of-function
mutations (Fisher et al., 2002). In comparison, human caspase-4,
another resident of the ER, is a counterpart of murine caspase-
12 and activated by ER stress (Hitomi et al., 2004). Induction of
glucose-regulated protein GRP78, also referred as BiP
(immunoglobulin heavy-chain binding protein), has been used as
a marker for ER stress and the onset of UPR (Lee, 2001). Due to
its antiapoptotic property, stress induction of GRP78
represents an important prosurvival component of the
evolutionarily UPR. Recent evidences also showed that the
microenvironment of tumor represents physiological ER stress,
and GRP78 is up-regulated in many types of cancer cells lines
and tumor biopsies (Li and Lee, 2006). Moreover, CCAAT/
enhancer-binding protein (C/EBP) homologous protein/growth
arrest and DNA damage-inducible gene 153 (CHOP/
GADD153), a unique transcription factor induced by ER stress,
plays a critical role in facilitating ER stress-induced apoptosis
(Martin and Green, 1995). It was reported that overexpression
of CHOP promotes apoptosis, including the dephosphorylation
of the proapoptotic protein Bad (Tombal et al., 2000), down-
regulation of Bcl-2 expression (McCullough et al., 2001).
Whether resveratrol induces growth inhibition in human
NPC cells through apoptotic mechanisms was not determined
and was the main aim of this study. Resveratrol was shown to
induce apoptosis in human NPC cells. The mechanism can be
due to disruption of mitochondrial membrane potential, release
of cytochrome cfrom mitochondria, up-regulation of FasL,
and Bax, and down-regulation of GRP78 and Bcl-2 expression.
These may lead to activation of caspases-9, -8, -4, and -3
activities. Therefore, resveratrol may have the potential for
human NPC treatment.
Materials and Methods
Reagents
Resveratrol (trans-3, 40, 5-trihydroxystilbene), dimethyl sulfoxide
(DMSO), bovine serum albumin, protease inhibitor cocktail were
obtained from Sigma (St Louis, MO). Z-DEVD-FMK (caspase-3
inhibitor), Z-LEVD-FMK (caspase-4 inhibitor), Z-IETD-FMK
(caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) were
purchased from BioVision (Mountain View, CA). Resveratrol was
dissolved in DMSO to make a stock solution of 50 mM stored at
208C and diluted to the desired concentrations before use. Cell
culture medium (DMEM, F-12), fetal bovine serum (FBS), penicillin,
streptomycin, and trypsin were purchased from Gibco BRL (Grand
Island, NY).
Cell lines and cell culture
Three different types of human NPC cell lines were studied,
including TW076 (type 1 NPC; squamous cell carcinoma, EBV
negative), CG-1 (type 2 NPC; non-keratinizing carcinoma, EBV
positive), and TW04 (type 3 NPC; undifferentiated carcinoma, EBV
negative). TW04 and TW076 cells were cultured in DMEM and
CG-1 in DMEM/F-12 (3:1, v/v). Both media were supplemented
with 10% (v/v) FBS and 100 U/ml penicillin and 100 mg/ml
streptomycin. Cells were incubated at 378C in a 5% CO
2
incubator
with saturated humidity.
Cell viability assay
Cell viability was determined using MTT Based in Vitro Toxicology
Assay Kit (Sigma) which colorimetrically measures a purple
formazan compound produced by viable cells. NPC cells were
seeded in 96-well plates (5 10
3
/well) and allowed to adhere for
24 h. The medium was then substituted by a fresh one containing
different concentrations (1–100 mM) of resveratrol for another
24 h. For the time course assay, the incubation time with
resveratrol was 6, 12, 24, 36, and 48 h, respectively. After
incubation, 10 ml of 5 mg/ml of MTT was add to each well and
incubated for 4 h at 378C. This was followed by elution of the
precipitate with 100 ml of solubilization solution. Cell viability was
calculated from absorption values obtained at 570 nm using an
automated ELISA reader (Cape Code, UK). All experimental
concentrations were tested in triplicate.
Annexin V-PI binding assay
To measure apoptotic cell death, an Annexin V-FITC Apoptosis
Detection Kit (BioVision) together with flow cytometry was used
according to the recommended protocol to quantify the
externalization of inner membrane phosphatidylserine. Briefly,
510
5
cells grown to about 60% confluence were treated with 50
or 75 mM of resveratrol for 24 h. For Annexin V/PI binding assay,
both floating and adherent cells were collected and washed with
serum-containing media. Cells collected were washed with serum-
containing media before being resuspended in 500 mlof1binding
buffer, followed by addition of 5 ml of Annexin V-FITC and 5 mlofPI
to cell suspension. Cells used as controls (0 mM resveratrol) were
incubated with the vehicle (DMSO) alone. The mixture was then
incubated for 5 min at room temperature in the dark and
immediately analyzed with the flow cytometer (Becton Dickinson,
San Jose, CA) and Cell Quest 3.3 software (Becton Dickinson)
analysis.
Detection of DNA fragmentation
The induction of apoptosis was evaluated by assessing the
enrichment of nucleosomes in the cytoplasm, compared with
control cells. Cytoplasmic DNA fragments were quantified with a
cell death detection ELISA
PLUS
kit (Roche, Mannheim, Germany).
Briefly, cells were plated in triplicate in a 96-well plate at a density of
110
4
cells per well and were treated with resveratrol at
concentrations (10–100 mM) for 24 h. The occurrence of apoptosis
is expressed using the following formula: enrichment
factor ¼absorbance of the sample/absorbance of the
corresponding control.
Caspase activity assay
The activity of caspase-3, -4, -8, and -9 was measured using a
colorimetric assay kit (BioVision) according to the manufacturer’s
instructions. Cells were incubated for 24 h with different
concentrations of resveratrol. Cell lysate from 1 to 5 10
6
cells
was incubated at 378C for 2 h with 200 mM DEVD-p-nitroanilide
(pNA) (caspase-3 substrate), LEVD-pNA (caspase-4 substrate),
IETD-pNA (caspase-8 substrate), or LEHD-pNA (caspase-9
substrate). Spectrophotometric detection of the chromophore
pNA after cleavage from the labeled caspase substrates was then
JOURNAL OF CELLULAR PHYSIOLOGY
RESVERATROL INDUCES APOPTOSIS OF NPC CELLS 721
performed. Samples were read at 405 nm and the enzyme activity
was expressed as folds over untreated control samples.
Mitochondria membrane potential detection assay
The MitoCapture
TM
Mitochondrial Apoptosis Detection kit
(BioVision) for detection of mitochondrial transmembrane
transition events in live cells was used. Growing cells (60%
confluence) were treated with 75 mM of resveratrol for 24 h. Cells
collected were suspended in 1 ml of the diluted MitoCapture
solution, followed by incubation at 378C for 20 min. After
centrifugation, the cell pellet was resuspended in 1 ml of the
pre-warmed incubation buffer before by flow cytometry.
Western blot analysis
Twenty-four hours after resveratrol treatment, cells were washed
twice with cold PBS before incubation in ice-cold lysis buffer
containing 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 0.5% Nonidet
P-40, 1 mM PMSF, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM
DTT, 10 mM b-glycerophosphate, and 4 mg/ml complete protease
inhibitor cocktail over ice for 30 min. Cell suspensions were then
centrifuged for 30 min at 15,000gat 48C, and the supernatant (total
cell lysates) was collected and stored at 708C. Cytosolic and
mitochondrial extracts were prepared using a Mitochondrial/
Cytosol Fractionation kit (BioVision). The protein concentration
was determining using Bradford assay (Bio-Rad, Richmond, CA).
Equal amounts of protein were separated by electrophoresis in 10–
15% SDS–polyacrylamide gels. Proteins were transferred onto
PVDF membranes (Millipore, Billerica, MA), and detected by the
proper primary and secondary antibodies before visualization using
an enhanced chemiluminescence detection kit (Millipore). The
antibodies anti-Bid and anti-cytochrome cwere from Cell Signaling
(Beverly, MA); anti-Bcl-2, anti-Bax, anti-CHOP, anti-Fas, anti-FasL,
and anti-GRP78 were from Santa Cruz Biotechnology (Santa Cruz,
CA); the anti-b-actin was from Novus Biologicals (Littleton, CO).
The secondary antibodies were horseradish peroxidase-conjugated
anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology).
RNA isolation and reverse transcriptase-polymerase chain
reaction (RT-PCR)
Total RNA from TW04 cells was extracted with the TRIzol
reagent, according to the manufacturer’s instructions (Invitrogen,
Carlsbad, CA). Two micrograms of RNA was reversely transcribed
in 20 ml reaction volumes containing an oligo (dT) primer
(Invitrogen) and the M-MLV reverse transcriptase (Promega,
Madison, WI). The cDNA for Bax, Bcl-2, CHOP, GRP78, Fas, FasL,
and b-actin were amplified by PCR with specific primers: Bax
forward primer 50-TCTGACGGCAACTTCAACTG-30, and
reverse primer 50-TTGAGGAGTCTCACCCAACC-30; Bcl-2
forward primer 50-TCCATGTCTTTGGACAACCA-30, and
reverse primer 50-CTCCACCAGTGTTCCCATCT-30; CHOP
forward primer 50-GCACCTCCC-AGAGCCCTCACTCTCC-30,
and reverse primer 50-GTCTACTCCAAGCCTTCCCCCTGCG-
30; GRP78 forward primer 50-GCTCTCGAATTCCAAAG-30, and
reverse primer 50-TTTGTCAGGGG-TCTTTCACC-30; Fas
forward primer 50-GGATGAACCAGACTGCGTG-30, and
reverse primer 50-CTGCATGTTTTCTGTACTTCC-30; FasL
forward primer 50-CTCTGGAATGGGAAGACACC-30, and
reverse primer 50-ACCAGAGAGAGCTCAGATACG-30;b-actin
forward primer 50-GAGACCTT-CAACACCCCAGCC-30, and
reverse primer 50-GGATCTTCATGAGGTAGTCAG-30. The
PCR products from the same sample were electrophoresed in a 2%
agarose gel and visualized by ethidium bromide staining on an image
system.
Statistical analysis
Data were expressed as mean SE. Comparisons between
untreated control cells and resveratrol-treated cells were made
using Student’s t-test. The statistically significant levels were set at
P<0.05.
Results
Resveratrol reduces the viability of human NPC cells
The effects of resveratrol on the viability of human NPC cells
were first studied. Three different cancer types of NPC cell
lines were treated with increasing concentrations of
resveratrol for 24 h, followed by MTT assay. Compared to
that of controls, the cell viability of TW076, CG1, and
TW04 cells was significantly reduced by resveratrol treatment
in a dose-dependent manner (Fig. 1A–C). Besides, a time-
dependent inhibition on the viability of TW04 cells was also
observed (Fig. 1D). Briefly, reduction of cell viability in different
histological types of NPC cell lines was observed by resveratrol
treatment.
Resveratrol induces apoptosis and DNA fragmentation
in TW04 cells
The undifferentiated carcinoma cell line TW04 cells were used
for subsequent mechanistic studies. To determine whether
reduction of cell viability against NPC cells by resveratrol was
mediated through apoptosis, annexin V-FITC/PI double staining
was performed. As shown in Figure 2A,B, results of flow
cytometry analysis indicated the percentage of apoptotic TW04
cells increased from 6.6% in control cells to 24% and 38% after
treatment with 50 and 75 mM of resveratrol, respectively.
A prominent feature of apoptosis is the degradation of
chromatin DNA at internucleosomal linkages. To determine
whether resveratrol also induces DNA fragmentation in NPC
cells, a Cell Death Detection ELISA kit that specifically detects
cytoplasmic histone-associated DNA fragments (mono- and
oligonucleosomes) was used. Compared with untreated
control cells, treatment of TW04 cells by resveratrol resulted
in a dose-dependent increase in enrichment of nucleosomes in
the cytoplasm, with a maximal increase of DNA fragmentation
of up to 12-fold at 100 mM (Fig. 2C).
Resveratrol activates caspase-mediated apoptosis in
TW04 cells
Apoptosis is executed by a family of cysteine-dependent
aspartate-specific proteases known as caspases. As caspase-3 is
a key protease associated with the DNA fragmentation and
apoptosis, we investigated the activity of caspase-3 in
resveratrol-treated TW04 cells by colorimetric assay. The
enzyme activity of caspase-3 was measured by ELISA using a
specific substrate DEVD-pNA. Caspase-3 specific activity
was markedly increased by resveratrol, displaying 1.87-, 2.81-,
and 6.23-fold increase after exposing to 25, 50, and 75 mM
resveratrol for 24 h, respectively, compared with untreated
controls (Fig. 3A).
To further elucidate whether activation of caspase-3
can be mediated by extrinsic (death receptor), intrinsic
(mitochondria) or ER stress-mediated apoptotic pathway,
the activity of caspases-9, -8, and -4, representing the apical
proteases in the intrinsic, extrinsic, and ER-mediated pathway,
respectively, were also analyzed by colorimetric assay in TW04
cells. As shown in Figure 3A, resveratrol increased the activity
of capase-8 and -9 in a dose-dependent manner compared with
untreated cells. A respective increase in enzyme activities
between 1.23- to 1.99-fold and 1.38- to 1.96-fold was observed.
While caspase-4 activity remained not significantly affected
by 25 and 50 mM resveratrol treatments, the activity was
significantly increased up to 1.93-fold at 75 mM resveratrol,
compared with untreated cells. We also showed that treatment
of TW04 cells with 50 mM resveratrol and 10 mM caspase-3, -8,
JOURNAL OF CELLULAR PHYSIOLOGY
722 HUANG ET AL.
or -9 inhibitor resulted in significantly increase of cell viability
(Fig. 3B). Altogether, resveratrol induced apoptosis through
activation of multiple apoptotic pathways, mainly through the
activation of caspase-8 and -9 activities, and partly associated
with the activation of caspase-4 activity. However, we cannot
exclude existence of other resveratrol activated mechanisms
which regulate cell survival and apoptosis (Fulda and Debatin,
2006).
Resveratrol induces loss of mitochondrial membrane
potential and release of cytochrome cto cytosol in
TW04 cells
The activation of caspase-9 is often the result of disruption
of mitochondrial transmembrane potential and the release of
cytochrome c. Therefore, after resveratrol treatment, the
status of mitochondrial membrane potential was first measured
by flow cytometry after staining with MitoCapture
TM
, a cationic
dye. In healthy cells, the mitochondria appear red due to
aggregation of MitoCapture reagent. In contrast, the dye
remains in its monomeric form and appears green in apoptotic
cells. As shown in Figure 4A,B, exposure of TW04 cells to
75 mM resveratrol caused a 34.6% shift from red fluorescence
to green fluorescence, indicating the disruption of
mitochondrial transmembrane potential. The release of
cytochrome cwas next examined by Western blot analysis.
While cytochrome cwas hardly detectable in the cytosol of
untreated NPC cells, it was detected in cytosol after treatment
with 50 or 75 mM of resveratrol for 24 h (Fig. 4C). These results
indicated resveratrol induces loss of mitochondrial membrane
potential and release of cytochrome cinto cytoplasm.
Resveratrol induces FasL/CD95L expression in
TW04 cells
Stimulation of the tumor necrosis factor receptor such as
Fas/CD95 or TRAIL receptor by FasL/CD95L may result in
activation of caspase-8, leading to apoptosis (Fulda and Debatin,
2004). To determine whether resveratrol also affects
expression of Fas or FasL in TW04 cells, the mRNA and protein
levels were measured by RT-PCR and Western blot
analysis, respectively. Compared with untreated control cells,
expression of FasL mRNA, together with the amount of FasL
protein were increased in a dose-dependent manner following
24 h of treatment with resveratrol at 50 or 75 mM. In contrast,
Fas expression remained not affected (Fig. 5). Briefly,
resveratrol treatment increased FasL expression.
Resveratrol modulates the level of Bcl-2 family proteins
in TW04 cells
The release of cytochrome cis tightly regulated by members of
Bcl-2 family known to play an important role in regulation
of apoptosis (Adams and Cory, 2001). To examine whether
resveratrol also modulates expression of Bcl-2 family proteins
in TW04 cells, cells were treated with 50 or 75 mMof
resveratrol for 24 h, and mRNA and protein levels of Bcl-2 and
Bax were examined. As shown by RT-PCR, Western blot
analysis and its densitometric quantitation (Fig. 5), resveratrol
treatment of TW04 cell lines resulted in decrease in
antiapoptotic protein Bcl-2 and a concomitant increase in
proapoptotic Bax proteins, thereby causing a significant
increase in the Bax/Bcl-2 ratio that favors apoptosis. In addition,
the expression level of full-length Bid, a proapoptotic Bcl-2
Fig. 1. Effect of resveratrol on the viability of human NPC cells. A: TW076 cells, (B) CG1 cells, and (C) TW04 cells were treated with various
concentrations (1–100 mM) of resveratrol for 24 h. D: TW04 cells were treated with 50 mM resveratrol for the indicated time. Cell viability was
measured by MTT assay. The data shown are the means WSE of three individual experiments (
M
P< 0.05 vs. 0 mM control).
JOURNAL OF CELLULAR PHYSIOLOGY
RESVERATROL INDUCES APOPTOSIS OF NPC CELLS 723
Fig. 2. Resveratrol treatment induced apoptosis and DNA fragmentation in TW04 cells. Cells were untreated or treated with 50 and 75 mM
resveratrol for 24 h and stained with Annexin V-FITC and PI. A: Cells in the upper-right (UR) portion are late apoptosis cells, whereas cells in
the lower-left (LL) and lower-right (LR) portions are viable and early-apoptotic cells, respectively. B: The data indicate the percentage of
annexin V-positive cells (apoptosis). C: TW04 cells were treated with various concentrations of resveratrol for 24 h in a 96-well plate, and the
enrichment of nucleosomes in the cytoplasm was determined using the Cell Death Detection ELISA
PLUS
kit. Results are shown as means WSE of
four independent experiments (
M
P< 0.05 vs. 0 mM control). [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Fig. 3. Resveratrolinduced caspase-dependent apoptosis in TW04 cells. A: Cells were treated with various concentrations of resveratrol for 24 h,
and then the cytosolic fraction of cells was analyzed for changes in the activity of caspase-3, -4, -8, and -9 using caspase colorimetric assay kits
(
M
P< 0.05 vs. 0 mM control). B: Cells were treated with 50 mM resveratrol for 24 h in the presence of 10 mM caspase-3, -4, -8, or -9 inhibitor, and then
cell viability was determined by MTT assay. Data shown are the means WSE of three individual experiments (
M
P< 0.05 vs. control cells treated with
resveratrol alone).
JOURNAL OF CELLULAR PHYSIOLOGY
724 HUANG ET AL.
family member, which can be activated by caspase-8-mediated
cleavage to produced truncated Bid (tBid) before translocation
to mitochondria to induce cytochrome crelease and
breakdown of mitochondrial membrane potential (Li et al.,
1998), was reduced 20% in cytosolic fraction of 75 mM
resveratrol-treated NPC cells (Fig. 4C).
Differential regulation of GRP78/BiP and CHOP/
GADD153 in resveratrol-induced apoptosis of
TW04 cells
Under ER stress condition, GRP78/BiP is recruited to misfolded
proteins to facilitate their proper folding, thus serving as a major
player in the survival program (Lee, 2007). On the other hand,
CHOP/GADD153 expression mediates ER stress-induced
apoptosis (Martin and Green, 1995). Thus, to further clarify
whether the expression level of GRP78 and CHOP is affected
by resveratrol in TW04 cells, the mRNA and protein levels of
GRP78 and CHOP were evaluated by RT-PCR and Western
blot analysis, respectively. As shown in Figure 5, treatment of 50
and 75 mM resveratrol down-regulated the expression of
GRP78 in a dose-dependent manner. In comparison, the level of
CHOP was unchanged in cells untreated and treated with
different concentrations of resveratrol.
Discussion
During the recent years, natural antioxidants present in
food and beverages have earned extensive attention because of
their cancer prevention function (Surh, 2003). Among these,
resveratrol has gained interests as a nontoxic chemopreventive
agent capable of inducing apoptosis in many cancer cell lines
(Ulrich et al., 2005; Fulda and Debatin, 2006). Moreover,
resveratrol treatment seems not to cause apoptotic effect
in normal cells and tissues (Baur and Sinclair, 2006; Bishayee,
2009). Whether resveratrol inhibits human NPC cell growth
was not characterized, neither did the mechanism of
resveratrol-induced apoptosis. Here we found that resveratrol
shows reduction of cell viability effect on all three different
types of human NPC cell lines (Fig. 1A–C). This occurs in a
concentration-dependent manner. Besides, using the type 3
NPC cell line TW04 cells as the study model, resveratrol also
showed a time-dependent inhibition effect against the cell
viability (Fig. 1D). Subsequent studies showed induction of cell
apoptosis is responsible for the resveratrol effect (Fig. 2). These
results basically agree with the prevailing response of most
cancer cells to chemotherapeutic agents.
Previous studies had indicated several mechanisms for
resveratrol-mediated apoptosis of cancer cells (Aziz et al.,
Fig. 4. Resveratrolcaused disruption of mitochondrial membrane potential and release of cytochrome cinto cytosol in TW04 cells. A: Cells were
treated with 75 mM of resveratrol for 24 h, and then stained with MitoCapture
TM
, a cationic dye. The mitochondrial membrane potential was
measured by flow cytometry as described in the Materials and Methods Section. The shift-down of fluorescence from red to green indicates the
collapse of mitochondrial membrane potential. Camptothecin was used as a positive control for the disruption of mitochondrial membrane
potential. The percentage of cells with the disruption of mitochondrial membrane potential was indicated. B: Data indicated percentage of cells
with disrupti on of mitochondria membrane potential. Results are shown as means WSE of four independent experiments (
M
P< 0.05 vs. 0 mM
control). C: Proteins prepared from cells treated with 50 mMor75mM of resveratrol for 24 h were subjected to Western blot for measuring Bid and
cytochrome cin cytosol. b-Actin was used as internal control to ensure that equal amount of proteins was loaded in each lane. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
JOURNAL OF CELLULAR PHYSIOLOGY
RESVERATROL INDUCES APOPTOSIS OF NPC CELLS 725
2003). For example, resveratrol induces apoptotic cell death in
HL60 leukemia cells as well as in T47D breast carcinoma cells
through enhancing CD95L/FasL expression and activating the
CD95-CD95 ligand pathway (Cle
´ment et al., 1998). FasL is a
TNF-related type II membrane protein. Cleavage of membrane-
bound Fas ligand (mFasL) by a metalloprotease-like enzyme
results in the formation of soluble Fas ligand (sFasL) (Kayagaki
et al., 1995). Both mFasL and sFasL can bind to Fas, and
subsequently trigger the Fas/FasL system, albeit sFasL was
reported to be a weaker inducer of apoptosis than mFasL
(Schneider et al., 1998). Our results indicated that up-
regulation of mFasL was involved in resveratrol-inducing
apoptosis of NPC cells (Figs. 3 and 5). The increased FasL may
stimulate Fas receptor in an autocrine or paracrine manner.
Whether the amount of sFasL synthesized is also increased
by resveratrol treatment remains to be further characterized.
Interestingly, while treatment of resveratrol triggered an
increased expression in mFasL, expression of Fas was not
affected. Even so, previous studies also found that
redistribution of Fas in the plasma membrane rafts is induced
by resveratrol in colon cancer cells (Delmas et al., 2003).
These results suggested that expression of Fas and FasL may
be differentially regulated by different pathways, and that
resveratrol may regulate the signaling of death receptor
pathway by acting at different components.
Resveratrol also activates the mitochondrial apoptotic
pathway in many other cancer cells. It was reported to induce
apoptosis in B-linkage leukemic cells (acute lymphoblastic
leukemias) that are resistant to CD95 signaling, through the
CD95-independent, mitochondria/caspase-9-specific pathway
(Do
¨rrie et al., 2001). Such effect was also supported by Madan
et al. (2008) that resveratrol stimulates apoptosis through
mitochondria mediated pathway in human epidermoid
carcinoma A431 cells. The Bcl-2 family is composed of a number
of proteins that play crucial roles in the control of apoptosis
under mitochondrial pathway. This family comprises
antiapoptotic members such as Bcl-2 and Bcl-x
L
, as well as
proapoptotic members including Bax and Bad (Konopleva et al.,
2002; Panaretakis et al., 2002). Overexpression of Bax
promotes the cell death; conversely, overexpression of Bcl-2
represses the function of Bax and promotes cell survival
(Pettersson et al., 2002). It was reported that resveratrol
induces the apoptosis in human esophageal carcinoma cells,
which may be mediated by down-regulating the expression
of Bcl-2 and up-regulating the expression of Bax (Zhou et al.,
2003). Similar to these results, resveratrol treatment of NPC
Fig. 5. Effectof resveratro l on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153, and Bcl-2 family proteins in TW04 cells.
Cells were treated with various concentrations of resveratrol for 24 h. A: RNA was isolated from cells treated with 50 mM and 75 mM of resveratrol
for 24 h, and 2 mg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using specific primers for
Fas, FasL, GRP78, CHOP, Bax, Bcl-2 and also the internal control gene, b-actin. B: Cell lysates were prepared for SDS–PAGE followed by Western
blot for Fas, FasL, GRP78, CHOP, Bax, and Bcl-2 with b-actin being used as a loading control. C–E: Results are presented as the relative densities
of protein bands normalized to b-actin. Data shown are the means WSE of three individual experiments (
M
P< 0.05 vs. 0 mM control).
JOURNAL OF CELLULAR PHYSIOLOGY
726 HUANG ET AL.
cells is accompanied by down-regulation of Bcl-2 and up-
regulation of Bax (Fig. 5). We also found that resveratrol results
in a significant increase of the Bax/Bcl-2 ratio that is regarded as
a driving force for apoptosis in TW04 cells. Furthermore,
disruption of mitochondrial transmembrane potential, release
of cytochrome cfrom mitochondria, and activation of caspase-9
and caspase-3 activity were also observed (Figs. 3 and 4). Thus,
activation of the mitochondrial pathway by resveratrol may
be through modulation of Bax and Bcl-2 expression, leading
to NPC cell death. A branch signaling from the death receptor
may also be linked to mitochondrial pathway, and this involves
cleavage of the Bcl-2 family member Bid by caspase-8
(Nagata, 1997). Our data showed a 20% decrease of full-length
Bid protein in cytosolic fraction of NPC-TW04 cells after
treatment with 75 mM resveratrol for 24 h (Fig. 4C). However,
no cleaved Bid was detected by Western blot analysis after
three independent experiments. Thus cleaved Bid was not
shown in the Results Section. Previous studies have shown that
tBid is rapidly degraded by the ubiquitin proteolytic system and
inhibition of the proteasome increase apoptosis by increasing
tBid levels (Breitschopf et al., 2000). It may be possible that 24 h
after resveratrol treatment, tBid is already degraded.
Activation of ER stress-mediated apoptotic pathway can be
induced by resveratrol treatment. Park et al. (2007) reported
that the involvement of ER stress in the induction of apoptosis in
resveratrol treated HT 29 colon carcinoma cells. ER stress
leads to proteolytic cleavage of caspase-12 in mouse and
caspase-4 in human, both of which localize to the cytoplasmic
side of the ER membrane (Hitomi et al., 2004). Kim et al. (2006)
found that neuronal apoptosis in infantile neuronal ceroid
lipofuscinosis (INCL) is caused by ER stress-mediated caspase-4
activation leading to caspase-3 activation and apoptosis. In this
study, our results showed that resveratrol treatment activates
caspase-4 and caspase-3 activity (Fig. 3), indicating activation of
ER stress-mediated apoptotic pathway in NPC. Many
downstream components of ER stress signaling involve
regulation of apoptosis. The transcription regulator protein
CHOP is usually present at low levels under normal conditions
but is robustly increased in expression in response to ER stress
(Wang et al., 1996). Woo et al. (2007) also showed that
resveratrol-induced apoptosis may be involved, at least in part,
through the CHOP up regulation in human colon cancer cells.
Besides CHOP, the ER chaperone GRP78/BiP also involves
regulation of ER stress induced apoptosis. GRP78 that has
antiapoptotic property is a central regulator of ER homeostasis,
and its expression is increased in human cancers (Li and Lee,
2006). Recent studies also revealed GRP78 can directly inhibit
ER stress-induced apoptosis through direct binding and sequent
inhibition of caspase-7 activation (Reddy et al., 2003). GRP78
also possesses the capacity to bind Ca
2þ
, which helps to
immobilize Ca
2þ
and maintain ER calcium homeostasis (Lee,
2001). The Ca
2þ
release from the ER enters mitochondria
leading to depolarization of the inner membrane, cytochrome c
release, and activation of Apaf-1/procaspase-9-regulated
apoptosis (Boya et al., 2002). In addition, previous studies also
showed that resveratrol induces a rapid and sustained elevation
of intracellular Ca
2þ
, which compromised the mitochondrial
membrane potential and triggered the process of human
hepatoblastoma HepG2 cell apoptosis (Ma et al., 2007). Thus
resveratrol may increase cytoplasmic Ca
2þ
concentration,
resulting in apoptosis of NPC cells. Taken together, GRP78
serves as a buffering agent in the ER, preventing Ca
2þ
efflux
from the ER to the cytosol and alleviating the ER stress-induced
apoptotic stimuli. Our results showed that resveratrol
treatment down-regulates the expression of GRP78 protein in
NPC cells (Fig. 5). However, the level of CHOP protein is
unchanged in resveratrol-treated NPC cells. Thus, resveratrol
may down-modulate GRP78 expression in ER stress-mediated
apoptosis of NPC cells.
In conclusion, our results demonstrated that similar to the
effects against many other cancer cell types, resveratrol results
in an effective decrease in cell viability of human NPC cells by at
least three different apoptotic pathways (Fig. 6). These findings
indicated a general resveratrol killing effect and mechanism on
cancers, suggesting resveratrol might be potentially used as a
promising agent for prevention or treatment of human NPC.
Acknowledgments
This work was supported by the grants from the National
Science Council (NSC96-2628-B-033-MY3) and Chang Gung
Memorial Hospital (CMRPD160273) which was really
appreciated.
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