Content uploaded by Vladimir N Ivanov
Author content
All content in this area was uploaded by Vladimir N Ivanov on Nov 08, 2017
Content may be subject to copyright.
Research Article
Resveratrol sensitizes melanomas to TRAIL through
modulation of antiapoptotic gene expression
Vladimir N. Ivanov
a,
⁎, Michael A. Partridge
a
, Geoffrey E. Johnson
b
, Sarah X.L. Huang
c
,
Hongning Zhou
a
, Tom K. Hei
a,c
a
Center for Radiological Research, Columbia University, New York, NY 10032, USA
b
Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
c
Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY 10032, USA
ARTICLE INFORMATION ABSTRACT
Article Chronology:
Received 22 October 2007
Revised version received
11 December 2007
Accepted 11 December 2007
Available online 23 December 2007
Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/D R4
on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to
TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-κB
that control the expression of antiapoptotic genes, including cFLIP an d Bcl-xL.Ontheotherhand,
the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor)
expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased
STAT3 and NF-κB activation, while activating JNK-cJun that finally suppressed expression of
cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive
melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human
melanomas, while γ-irradiation or sodium arsenite treatment substantially upregulated DR5
expression. Hence, an initial increase in DR5 surface expression (either by γ-irradiation or
arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol),
appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-
resistant human melanomas. In spite of partial suppression of mitochondrial function
and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the
DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an
increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest
that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of
human melanomas.
© 2007 Elsevier Inc. All rights reserved.
Keywords:
Melanoma
TRAIL
Resveratrol
Apoptosis
STAT3
cJun
cFLIP
EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
⁎Corresponding author. Center for Radiological Research, Columbia University, VC11-236, 630 West 168th Street, New York, NY 10032, USA.
Fax: +1 212 305 3229.
E-mail address: vni3@columbia.edu (V.N. Ivanov).
Abbreviations: Ac-IETD-CHO, N-acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde); Ac-LEHD-CHO, N-acetyl-Leu-Glu-His-Asp-CHO (aldehyde); AP-1,
activator protein-1; DR4, death receptor-4; DR5, death receptor-5; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-
regulated kinase; FACS, fluorescence-activated cell sorter; FasL, Fas ligand; GFP, green fluorescent protein; JNK, Jun N-terminal kinase; IκB,
inhibitor of NF-κB; IKK, inhibitor nuclear factor kappa B kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MFI,
medium fluorescence intensity; NF-κB, nuclear factor kappa B; PI, propidium iodide; ROS, reactive oxygen species; RNAi, RNA interference;
RSV, resveratrol; STAT, signal transducers and activators of transcription; TNFα, tumor necrosis factor alpha; TRAIL, TNF-related apoptosis
inducing ligand; TRAIL-R, TRAIL-Receptor; XIAP, X-linked inhibitor of apoptosis
0014-4827/$ –see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2007.12.012
available at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Introduction
Cell death by apoptosis regulates numerous physiological and
pathological processes in the human body and its deficiency is
implicated in tumor development. The inability of advanced
cancer cells to undergo apoptosis may be based on inactiva-
tion of proapoptotic genes, due to mutations or epigenetic
regulatory mechanisms that suppress death signaling path-
ways [1,2]. As a result, both the extrinsic death receptor-
mediated signaling pathway (Fas- or TRAIL-R1/R2-mediated)
and the intrinsic mitochondrial death pathway could be
partially or completely suppressed in cancer cells [3].
Melanoma, the most aggressive form of skin cancer, is
known to be highly resistant to radio- and chemotherapeutic
treatment. In the USA an estimated 60,000 new cases will be
diagnosed, and 8100 deaths will occur in 2007 (ACS). Numer-
ous observations indicate that the incidence of melanoma has
significantly increased over the last ten years in the USA and
worldwide. However, only limited therapies for metastatic
stage of the disease are currently available. Various attempts
have been made to restore high levels of apoptosis in response
to treatment for this type of cancer [4–6]. One of the key
contributors to radio- and chemoresistance of human mela-
nomas is upregulation of transcription factors STAT3 and NF-
κB, which control expression of numerous antiapoptotic genes
in cancer cells, including cFLIP, cIAP, XIAP, Bcl-xL, Survivin,as
well as suppressing proapoptotic genes [7–10]. On the other
hand, via activation of cJun, JNK is involved in the negative
regulation of cFLIP (an inhibitor of caspase-8) expression
[11,12] and, via activation of E3 ubiquitin-protein ligase Itch,
in acceleration of proteasome-dependent cFLIP degradation
[13]. Taken together, these data suggest that agents that
simultaneously downregulate NF-κB and STAT3 activities,
while upregulating JNK-cJun activity, might increase sensitiv-
ity to TRAIL- or Fas-mediated apoptosis. Our recent observa-
tions indicated that sodium arsenite [11] was a useful
candidate for mediating these effects in melanomas.
In the present study we have used resveratrol (trans-3, 4′,5-
trihydroxystilbene) [14], as a non-toxic alternative to sodium
arsenite treatment, which was previously demonstrated to be
a powerful promoter of apoptosis in melanomas [15]. Resver-
atrol (RSV) was previously shown to suppress JAK2-STAT3, Src-
STAT3 and IKK-NF-κB activation and to induce apoptosis in
some cancer cell lines [16–18]. We have demonstrated that, as
with sodium arsenite, RSV suppressed expression of anti-
apoptotic cFLIP protein in human melanomas and dramati-
cally increased sensitivity to exogenous TRAIL in TRAIL-R2/
DR5-positive melanomas. Interestingly, RSV did not increase
the surface expression of DR5, whereas sodium arsenite
treatment or γ-irradiation of melanoma cells substantially
upregulated DR5 expression. We present evidence showing
that sequential treatment of melanoma cells with γ-irradiation
and then RSV, initially upregulated DR5 surface levels [19], and
subsequently downregulated antiapoptotic cFLIP, Bcl-xL and
survivin levels. Under conditions of pronounced deficiency of
mitochondrial function in some human melanomas, RSV
treatment may still activate the extrinsic TRAIL-R-mediated
death pathway, thereby increasing sensitivity to TRAIL and
restoring apoptotic signaling in melanomas.
Methods
Materials
Sodium arsenite, cycloheximide and resveratrol were obtain-
ed from Sigma (St. Louis, MO). Human soluble Fas Ligand
(recombinant) and soluble Killer-TRAIL (recombinant) were
purchased from Alexis (San Diego, CA). JNK inhibitor SP600125
and IKK-NF-κB inhibitor BAY 11-7082 were obtained from Bio-
mol (Plymouth Meeting, PA); MEK inhibitor U0126, MAPK p38
inhibitor SB203580, caspase inhibitors zVAD-fmk, Ac-IETD-
CHO (an inhibitor of caspase-8 and caspase-6) and Ac-LEHD-
CHO (an inhibitor of caspase-9) were purchased from Calbio-
chem (La Jolla, CA).
Cell lines
Human melanoma cell lines LU1205 (also known as 1205lu),
WM9, WM35, and WM793 [20], as well as OM431, FEMX, HHMSX,
LOX and normal human lung fibroblasts TIG-3 were maintained
in a DMEM medium supplemented with 10% fetal bovine serum
(FBS), L-glutamine and antibiotics. Normal human melanocytes
were maintained in TICVA medium.
Irradiation procedures
To determine sensitivity to γ-rays, the plates with melanoma
cells were exposed to radiation from a Gammacell 40
137
Cs
irradiator (dose rate, 0.82 Gy/min) at Columbia University. 6 to
24 h after irradiation, cells were analyzed by the flow
cytometry or underwent additional treatments.
FACS analysis of TRAIL and TRAIL-R2/DR5 levels
Surface levels of TRAIL and TRAIL-R2/DR5 on human mela-
nomas were determined by staining with the PE-conjugated
anti-human-TRAIL or anti-human DR5 mAb (R&D System,
Minneapolis, MN and eBioscience, San Diego, CA) and sub-
sequent flow cytometry. PE-conjugated nonspecific mouse
IgG1 was used as an immunoglobulin isotype control. A FACS
Calibur flow cytometer (Becton Dickinson, Mountain View, CA)
combined with the CellQuest program was used to perform
flow cytometric analysis. All experiments were independently
repeated 3–5 times.
Transfection and luciferase assay
The NF-κB luciferase reporter containing two κB binding sites,
Jun2-Luc reporter and empty vector tk-Luc [21], STAT-Luc re-
porter containing three repeats of GAS sites from the Ly6A/E
promoter were used to determine NF-κB, AP-1 and STAT tran-
sactivation, respectively. Additional reporter constructs used
included: 1.5 kb TRAIL-promoter-Luc [22], 1 kb cFLIP-promo-
ter-Luc [23,24] and p53RE-Luc [25]. Transient transfection of
different reporter constructs (1 μg) together with pCMV-βgal
(0.25 μg) into 5×10
5
melanoma cells was performed using Li-
pofectamine (Life Technologies-Invitrogen). Proteins were
prepared for β-Gal and luciferase analysis 16 h after transfec-
tion. Luciferase activity was determined using the Luciferase
1164 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
assay system (Promega, Madison, WI) and was normalized
based on β-galactosidase levels. Since RSV was known to
partially inhibit enzymatic activity of firefly luciferase [26],a
ratio of the specific luciferase reporter activity to luciferase
activity driven by the empty tk-Luc vector in the mock control
culture was finally determined.
In some experiments, melanoma cells were transfected
with reporter constructs together with a dominant-negative
form of cJun/TAM67 in the presence of pCMV-βGal (ratio
1:3:0.25). Sixteen to 24 h after transfection, luciferase activity
was determined. Expression construct pCMV4-cFLIP was
received from Dr. M. E. Peter (University of Chicago, IL) and
was used for overexpression of cFLIP in melanoma lines.
cFLIP suppression by RNAi
The pSUPER retro RNA interference (RNAi) system (Oligoen-
gine, Seattle, WA), which has been utilized for the production
of small RNAi transcripts used to suppress cFLIP expression.
Two variants of RNAi of 19 nucleotides each were designed to
target human cFLIP were expressed using vector pSUPER.retro.
puro (pSR-puro). RNAi cFLIP-92 (UGUGGUUCCACCUAAUGUC)
was the most efficient in the corresponding mRNA targeting.
Apoptosis studies
Cells were exposed to soluble TRAIL (50 ng/ml) alone or in
combination with cycloheximide (2 μg/mL), TRAIL in combina-
tion with resveratrol (25–100 μM). Apoptosis was then assessed
by quantifying the percentage of hypodiploid nuclei undergoing
DNA fragmentation 16 h–48 h after treatment. Flow cytometric
analysis was performed on a FACS Calibur flow cytometer.
Clonogenic survival assay
Cells (500/well) were placed in triplicate on 6-well plates 16 h
before treatment. For analysis of clonogenic survival of mela-
noma cells after treatment (24 h) with TRAIL, RSV or their
combination, colonies were stained with crystal violet sol-
ution 12 days after treatment. The percentage of colony-
forming efficiency (in relation to values of untreated control
cells) was calculated.
Western blot analysis
Total cell lysates (50 μg protein) were resolved on 10% SDS-
PAGE, and processed according to standard protocols. The
antibodies (Abs) used for Western blotting included: mono-
clonal anti-β-Actin (Sigma), monoclonal anti-FLIP (NF6) (Axxo-
ra, San Diego, CA), monoclonal anti-XIAP (BD Biosciences, San
Jose, CA), monoclonal anti-p21-WAF1 (Cell Signaling Beverly,
MA); monoclonal anti-Dynamin-2 (Upstate, Lake Placid, NY);
polyclonal Abs to TRAIL (human), TRAIL-R1/DR4 and TRAIL-
R2/DR5 (Axxora, San Diego, CA); polyclonal Abs against:
phospho-STAT3 (Tyr705) and STAT3; phospho-c-Jun (Ser73)
and c-Jun; phospho-SAPK/JNK (Thr183/Tyr185) and JNK; phos-
pho-p44/p42 MAP kinase (Thr202/Tyr204) and p44/p42 MAP
kinase; phospho-p38 MAP kinase (Thr180/Tyr182) and p38 MAP
kinase; phospho-FOXO3A (Ser318/321) and FOXO3A; BAX, phos-
pho-p53 (Ser20) and p53; Bid (Cell Signaling, Beverly, MA). Optimal
dilutions of primary Abs were 1:1000 to 1:5000. The secondary Abs
anti-mouse or anti-rabbit) were conjugated to horseradish
peroxidase (dilution 1:5000 to 1:10,000); signals were detected
using the ECL system (Amersham, Piscataway, NJ).
EMSA
Electrophoretic mobility shift assay (EMSA) was performed for
the detection of NF-κB DNA-binding activity as previously
described, using the labeled double-strand oligonucleotide
AGCTTGGGGACTTTCCAGCCG. (Binding site is underlined).
Ubiquitous NF-Y DNA-binding activity was used as an internal
control [15].
Immunofluorescence
Immunofluorescence was performed as described previously
[27]. Cells were grown on glass coverslips in growth medium,
fixed (PBS, 4% paraformaldehyde for 10 min), permeabilized
(PBS, 0.5% Triton X-100 for 5 min) and stained with antibodies
to pyruvate dehydrogenase (Binding Site, United Kingdom).
Secondary antibodies were obtained from Jackson ImmunoR-
esearch. Nuclear staining was done with propidium iodide
(PharMingen). Images were captured using a laser confocal
microscope (Nikon).
Results
Sensitivity of melanoma cell lines to resveratrol
Human melanocytes, normal human fibroblasts and most
metastatic human melanoma cell lines are relatively resistant
to RSV-induced apoptotic stimuli (at RSV doses of 25–100 μM),
demonstrating only a modest S/G2 arrest in the cell cycle (Fig. 1A
and data not shown). The rare example of a moderate sensitivity
to RSV was LOX human metastatic melanoma cells, which dev-
eloped average levels of apoptosis 24 h after treatment (Fig. 1A).
A clonogenic survival assay confirmed death in over 40% of LOX
cells 12 days after treatment with 100 μM RSV. In contrast, LU1205
metastatic melanoma cells were more resistant to RSV (Fig. 1B).
Both LOX and LU1205 melanoma cells express on their surface
FAS, TRAIL-R2/DR5 and TNF-R1 (Fig. 1C and data not shown). RSV
treatment did not change surface expression of DR5 and FAS
death receptors (data not shown), but induced surface expression
of endogenous TRAIL in LOX cells (Fig. 1C). RSV-induced death of
LOX cells could be blocked by the addition of an inhibitory anti-
body to TRAIL, but not to FasL (5 μg/ml) in the culture medium
(Fig. 1D). In contrast, inhibition of TNFα, which was constitutively
produced by many melanoma cell lines and induced basal NF-κB
activity, accelerated RSV-inducedapoptosisinbothLOXand
LU1205 lines (Fig. 1D), linking protection against RSV/TRAIL
apoptotic signaling with high basal levels of NF-κBactivity.
Hence, endogenously produced TRAIL after translocation to cell
surface, via paracrine action, induced a receptor-mediated death
signaling cascade in TRAIL-R-positive LOX cells, ultimately re-
sulting in suicide of subpopulation of these cells (Fig. 1A).
There are at least two possible mechanisms that could
account for the sensitivity of LOX cells to RSV: i) down-
regulation of STAT3- and NF-κB-dependent transcription, which
1165EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
regulate numerous survival functions in the cell (Fig. 2A); ii)
upregulation of TRAIL promoter activity and increased surface
expression of endogenous TRAIL (but not TRAIL-R) induced by
RSV in a dose-dependent manner (Figs. 2A and 1C). In general,
changes in nuclear NF-κB DNA-binding activity and NF-κB-
dependent reporter activity were relatively modest after RSV
(50 μM) treatment in this cell line (Figs. 2A and B). An additional
suppression of NF-κB activity by pharmacological inhibitor Bay
11-7082 (5 μM) upregulated RSV-induced apoptotic levels and
decreased clonogenic survival of LOX melanoma cells (Figs. 2B–D).
Signaling pathways affected by RSV in LU1205 melanoma cells
Effects of RSV on gene transcription directed by AP-1, NF-κB
and STAT3 and on TRAIL-, cFLIP-promoter activities were
relatively similar in both LOX and LU1205 melanoma cells
(Figs. 2A and 3D). In order to determine biochemical back-
ground of RSV-induced changes in the cell cycle and RSV-
induced decrease in cancer cell survival, we evaluated its
effects on the main signaling pathways, the correspondent
transcription factors, proapoptotic and antiapoptotic proteins
in LU1205 cells (Figs. 3A–C). Pronounced downregulation of
NF-κB DNA-binding activity 6 h after RSV treatment was
determined by EMSA in the nuclear fraction of LU1205 cells
(Fig. 3A). Levels of the active form of STAT3, phospho-STAT3
(Tyr705), were substantially decreased 6 h after treatment of
LU1205 cells with RSV. Simultaneously, a decrease in phos-
pho-FOXO3A (Ser318/321) levels was also detected (Fig. 3A).
AKT-dependent phosphorylation of FOXO3A is known to
induce its nuclear export and functional inactivation [28].
Partial suppression of this process by RSV should enhance the
proapoptotic function of FOXO3A.
Fig. 1 –Effects of resveratrol (RSV) on induction of apoptosis in melanocytes and melanomas. (A) Cells were stained by PI
16–24 h after treatment. Apoptosis (Ap) levels were determined as the percentage of cells with hypodiploid content of DNA in
the pre-G0/G1 region using flow cytometry. % Cells at the distinct phases of the cell cycle is indicated. Results of typical
experiments (one of three) are presented. (B) Clonogenic survival assay of LU1205 and LOX cells 12 days after indicated
treatment. Error bars represent mean±S.D. from three independent experiments. (C) Surface expression of TRAIL-R2/DR5 and
TRAIL in LU1205 and LOX cells was determined by immunostainig using PE-labeled mAbs and the FACS analysis. (D) RSV
induced TRAIL-mediated apoptosis in LOX melanoma cells. Introduction of anti-TRAIL (5 μg/ml), but not anti-FasL inhibitory
antibodies suppressed RSV-induced apoptosis in LOX and LU1205 cells, while anti-TNF mAb (5 μg/ml) accelerated apoptosis.
1166 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
In contrast, RSV treatment upregulated levels of active
forms of MAP kinases, phospho-ERK1/2 (Thr202/Tyr204),
phospho-p38 (Thr180/Tyr182) and phospho-JNK-1/2 (Thr183/
Tyr185). This was followed by an increase in phospho-cJun
(Ser73) levels, with a maximum phosphorylation observed
for RSV at 25 μM in LU1205 cells. There was also a strong
increase in phospho-ATF2 (Thr71) levels and a modest
increase in phospho-CREB (Ser133) levels after RSV treatment
at 25-100 μM(Fig. 3A). Additionally, upregulation of phospho-
p53 (Ser20) level, and increased expression of its target, p21-
WAF, was also detected after RSV treatment of LU1205 cells
(Fig. 3B) that was well correlated with a general increase of
p53-dependent transcription after RSV treatment (Fig. 3D).
Furthermore, p53-dependent BAX protein level [29] increased
also after RSV treatment. On the other hand, pronounced
downregulation of cyclin D1 level was caused by RSV (Fig. 3B).
Negative regulation of cFLIP promoter activity by RSV was
accompanied by a rapid decrease of cFLIP protein levels due
to protein degradation, which could increase sensitivity to
TRAIL-mediated apoptotic signaling. Downregulation of pro-
cessed forms of cFLIP-L, FLIP-p43, FLIP-p32 and cFLIP-S (p25)
was observed 6 h after treatment in LU1205 cells (Fig. 3B).
Protein levels of anti-apoptotic Bcl-xL and survivin were also
substantially decreased 16 h after RSV treatment (Fig. 3C).
Since expression of cyclin D1,Bcl-xL and Survivin required
positive transcriptional control of STAT3 [30,31], the observed
changes in these levels were expected. In contrast, levels of
anti-apoptotic protein XIAP did not notably decrease after RSV
treatment in LU1205.
In order to determine a role of elevated activation of the
MAPK pathways (MEK-ERK, MKK6-p38-ATF2, MKK4/7-JNK-
cJun) by RSV treatment in LU1205 cells, we used specific
pharmacological inhibitors: U0126 (10 μM) for MEK-ERK,
SB203580 (10 μM) for p38 and SP600125 (20 μM) for JNK.
Inhibition of RSV-induced MEK-ERK or p38 MAPK activation
substantially accelerated apoptotic response of LU1205 cells,
while inhibition of JNK activity did not cause pronounced
changes in apoptotic levels induced by RSV (Fig. 3E). It ob-
viously demonstrated prosurvival function of ERK1/2 and,
especially, MAPK p38 activation following RSV treatment of
LU1205 melanoma cells. JNK activation by RSV in LU1205 cells
appears to play dual proapoptotic and prosurvival role and
suppression of JNK does not notably change the life-death
balance in RSV-treated LU1205 melanoma cells.
Regulation of the main signaling pathways by RSV human
LU1205 melanoma cells resulted in strong downregulation of
Fig. 2 –Modulation of RSV-induced apoptosis in LOX human melanoma cells. (A) Effects of RSV (25-100 μM; 6 h) on NF-κB-,
AP-1/cJun- and STAT-dependent luciferase reporter activities, TRAIL and FLIP promoter activities. The reporter constructs
used were: 2xNF-κB-Luc, Jun2-Luc, 3xLy6A/E-Luc (STAT-dependent), 1.5 kb TRAILpr-Luc, 1 kb cFLIPpr-Luc and tk-Luc, as the
control vector with tk-promoter. Luciferase reporter activity was normalized based on β-gal activity; β-gal expression
construct was cotransfected at a ratio of 1/4. A ratio of the specific luciferase reporter activity to luciferase activity driven by the
empty tk-Luc vector in the mock control culture is indicated. Error bars represent mean ± S.D. from three independent
experiments. (B) Effects of RSV (50 μM) and Bay11–7082 (5 μM) on basal nuclear NF-κB DNA binding activity in LOX cells
determined by EMSA. Positions of DNA-binding complexes and free labeled probe (FP) are indicated. (C, D) Effects of
pharmacological inhibitor of NF-κB (Bay 11–7082, 5 μM), on apoptosis and clonogenic survival of LOX cells after RSV treatment.
Error bars represent mean±S.D. from three independent experiments.
1167EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
antiapoptotic protein levels that could sensitize these cells to
TRAIL-mediated apoptosis. On the other hand, direct RSV-
dependent induction of cell killing additionally required an
efficient surface expression of endogenous TRAIL. In contrast to
LOX cells, RSV did not notably induce surface expression of
endogenous TRAIL in LU1205 cells, in spite of high levels of
intracellular TRAIL (Fig. 3B), demonstrating a possible deficiency
in TRAIL protein translocation from the intracellular pools to
cell surface. Consequently, RSV treatment did not initiate pro-
nounced TRAIL-mediated suicide of these melanoma cells.
Cell death in human melanomas induced by either
recombinant TRAIL or combination of TRAIL and RSV
TRAIL-mediated cell death can be induced in TRAIL-R-positive
cancer cells either via stimulation of expression and translo-
cation of endogenous death ligand to cell surface with sub-
sequent paracrine action (as we observed for RSV stimulation
of LOX cells) or by exogenous TRAIL secreted by natural
immunocompetent cells, or finally by pharmacological recom-
binant TRAIL. In all cases, surface levels of death receptors in
Fig. 3 –Effects of resveratrol (RSV) on cellular proteins controlling cell survival and apoptosis in LU1205 human melanoma cells.
(A, B) Effects of RSV on basal nuclear NF-κB and NF-Y activities in SW1 and LU1205 cells determined by EMSA 6 h after
treatment. Positions of DNA-binding complexes are indicated. Free labeled probes are not shown. Western blot analysis was
performed for detection of total and phospho-protein levels of STAT3, AKT, FOXO-3A, ERK1/2, JNK1/2, cJun, p38MAPK, ATF2,
CREB, TRAIL, TRAIL-R2/DR5, cFLIP, P-(Ser20) p53, total p53, p21-WAF, BAX, Bcl-xL and of Cyclin D1 6 h after treatment with
indicated concentrations of RSV. (C) Western blot analysis of Bcl-xL, survivin, XIAP and β-actin levels in LU1205 cells 16 h after
treatment with RSV. (D) Effects of RSV (25–100 μM; 6 h) on AP-1/cJun-, NF-κB-, STAT- and p53-dependent luciferase reporter
activities, TRAIL and FLIP promoter activities. The reporter constructs used were: Jun2-Luc, 2xNF-κB-Luc, 3xLy6A/E-Luc
(STAT-dependent), p53RE-Luc, 1.5 kb TRAILpr-Luc, 1 kb cFLIPpr-Luc and tk-Luc, as the control vector with tk-promoter.
Luciferase reporter activity was normalized based on β-gal activity; β-gal expression construct was cotransfected at a ratio of
1/4. A ratio of the specific luciferase reporter activity to luciferase activity driven by the empty tk-Luc vector in the mock control
culture is indicated. Error bars represent mean±S.D. from three independent experiments. (E) Effects of pharmacological
inhibitors of MEK-ERK (U0126, 10 μM), p38 MAPK (SB203580, 10 μM) and JNK (SP600125, 10 μM) on RSV-induced apoptosis in
LU1205 cells. Error bars represent mean± S.D. from three independent experiments.
1168 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
cancer cells are critical factors regulating initiation of death
signaling in cancer cells. Normal human melanocytes and
many human melanomas exhibit TRAIL-Receptor-2/DR5 on
their surface (Fig. 4A). TRAIL-R1/DR4 could also be detected on
cell surface of some human melanomas, however, at relatively
low levels [11,32]. For early radial growth phase (RGP) WM35
melanoma, levels of surface expression of DR5 are quite
similar to melanocytes. In vertical growth phase (VGP)
melanoma WM793, and in some metastatic melanomas
(LU1205, WM9 and LOX), surface expression of DR5 is higher
than in melanocytes (Fig. 4A). In contrast, both FEMX (Fig. 4A)
and HHMSX (data not shown) metastatic melanomas consist
of two cell subpopulations with low (18 MFI) and negligible
levels of DR5 on the cell surface. Finally, metastatic OM431
melanoma cells appear to have lost expression of DR5 on their
surface almost completely (Fig. 4A).
In general, most human melanomas are relatively resistant
to apoptosis induced by TRAIL alone. Furthermore, there was
no simple linear correlation between surface levels of DR5 and
apoptotic response to recombinant TRAIL (50 ng/ml) for the
melanoma lines tested, including LOX and LU1205 cells
(Figs. 4A, D). Evidently, additional components may be involv-
ed in regulating the death signaling pathway. Indeed, TRAIL in
the presence of an inhibitor of protein synthesis, cyclohex-
imide (CHX, 2 μg/ml), induced caspase-8/caspase-3 activation,
leading to PARP cleavage (Fig. 4B), and dramatically accelerated
TRAIL-mediated apoptotic signaling that in this case was
proportional to death receptor surface expression levels
(Figs. 4A and D). Caspase activation and death signaling was
suppressed by the universal inhibitor of caspases, zVAD-fmk.
Enhancing effects of CHX on TRAIL-induced death signaling
were probably due to translational suppression of short-lived
protein inhibitors of apoptosis, including cFLIP [33,34].
Apart from protein synthesis inhibitors, RSV was previously
implicated in sensitization to TRAIL-mediated apoptosis in
different cancer cell lines [16,17,35]. Based on its effects on
signaling pathways and expression of anti-apoptotic proteins
(Fig. 3), RSV dramatically increased sensitivity of DR5-positive
Fig. 4 –Surface expression of TRAIL-R2/DR5 and TRAIL-induced apoptosis in human melanomas. (A) Surface expression of
TRAIL-R2/DR5 levels in human melanocytes and melanoma cell lines was determined using PE-labeled mAb to DR5 and FACS
analysis. MFI (medium fluorescence intensity) and % positive cells are indicated; ns —nonspecific staining. (B) Western blot
analysis of caspase-8, caspase-3 and PARP cleavage 8 h after apoptotic stimulation. TRAIL (50 ng/ml), RSV (50 μM), zVAD-fmk
(5 μM) and their combinations were used for treatment of LU1205 human melanoma cells. (C) RSV (50 μM) suppressed
TRAIL-induced up-regulation of NF-κB p65-p50 DNA-binding activity in LOX melanoma cells. RSV was added into the media
30 min before TRAIL (50 ng/ml). EMSA was performed with nuclear extracts of control and treated cells using NF-κB- and
NF-Y-binding labeled probes. Positions of DNA-binding complexes are indicated. (D) Resveratrol (RSV) dramatically increased
sensitivity to TRAIL in human melanomas. Effects of TRAIL (50 ng/ml), RSV (50 μM), CHX (2 μg/ml) and their combinations on
apoptosis of human melanocytes, melanomas and the normal human fibroblasts TIG-3 were determined 48 h after treatment.
Error bars represent mean±S.D. from three independent experiments.
1169EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
metastatic melanomas, including LU1205 and LOX, to exogen-
ous recombinant TRAIL that was revealed by an enhanced
level of activation of the death signaling cascade after
combined treatment (Fig. 4B). We observed that the death
signaling pathway was highly active after either TRAIL+CHX or
TRAIL+RSV stimulation of the DR5 receptor. As early as 6-8 h
Fig. 5 –Synergistic interaction of TRAIL and RSV for induction of apoptosis in human metastatic melanomas LU1205 and LOX. A
role of cFLIP in resistance to TRAIL. (A) Effects of TRAIL (50 ng/ml), RSV (25 μM and 50 μM) and their combinations on apoptosis
of LU1205 and LOX human melanoma cells. Cells were stained by PI 48 h after treatment. Apoptosis levels were determined as
percentage of cells with hypodiploid content of DNA in the pre-G0/G1 region using flow cytometry. Results of typical
experiments (one of four) are presented. (B) Clonogenic survival assay of LU1205 and LOX cells 12 days after indicated
treatment. Error bars represent mean±S.D. from three independent experiments. (C) Suppression of cFLIP expression by
specific RNAi. Protein levels of cFLIP and β-actin were determined by Western blot analysis of LU1205 cells stably transfected by
the empty vector (puro) and FLIP-RNAi expression construct. (D) Apoptosis levels 48 h after treatment of control (puro),
TAM67-transfected LU1205 cells and cFLIP-RNAi transfected LU1205 cells with indicated stimuli: TRAIL (50 ng/ml), RSV (50 μM)
or their combination. Apoptosis levels were determined as the percentage of cells with hypodiploid content of DNA in the
pre-G0/G1 region using flow cytometry. Error bars represent mean± S.D. from three independent experiments. (E, F) LOX
melanoma cells were transfected by the empty vector or cFLIP expression construct in the presence of pEF-GFP. Apoptotic levels
were determined in GFP-positive cells 48 h after indicated treatment using PI staining and the flow cytometry.
1170 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
after treatment, protein processing of caspase-8, caspase-3
and cleavage of PARP, the classical target of caspase-3, were
easily detectable. RSV (50 μM) by itself demonstrated low to
moderate caspase activation in LU1205 cells (Fig. 4B). Determi-
nation of nuclear NF-κB DNA-binding activity by EMSA
revealed again modest negative effects of RSV on the basal
levels of nuclear NF-κB in LU1205 and LOX melanoma cells,
while a negative effect of RSV on TRAIL-induced nuclear NF-κB
p65-p50 activities was more pronounced (Fig. 4C and data not
shown). Importantly, RSV suppressed initial survival signals
induced by TRAIL via NF-κB activation and shifted the life-
death balance to favor TRAIL-R2-mediated death signaling.
The levels of TRAIL+RSV induced apoptotic death in
human melanomas were determined 48 h after treatment.
Combined treatment of DR5-positive metastatic melanomas,
LU1205 and LOX, with exogenous TRAIL (50 ng/ml) and RSV
(25–50 μM) resulted in an effective induction of apoptosis,
which was more pronounced in LOX cells with higher levels of
DR5 surface expression (Fig. 4D). WM9 cells displayed elevated
sensitivity to TRAIL alone and did not require any additional
stimulation. On the other hand, OM431 melanoma cells, which
were deficient in surface DR5 expression (Fig. 4A) but
possessed modest DR4 surface levels [11], exhibited TRAIL+
RSV mediated apoptosis at low levels (data not shown). FEMX
and HHMSX metastatic melanoma cells (Fig. 4D) also weakly
responded to combined treatment of TRAIL+RSV. Although
TIG-3 (DR5-positive normal human lung fibroblasts) modestly
responded to TRAIL alone, neither CHX nor RSV increased
TRAIL-induced apoptotic levels in these cells. Melanocytes
also developed only low levels of apoptosis after combined
treatment (Fig. 4D). This remarkable feature of normal human
cells allowed select TRAIL+RSV combination for specific
targeting of TRAIL-R-positive cancer cells.
As we observed in the present study, RSV treatment notably
inducedendogenous TRAIL surface expression in LOX, butnot in
LU1205 cells, and consequently initiated moderate TRAIL-
mediated apoptosis in LOX ce lls (Fig. 1). However, a combination
of exogenous recombinant TRAIL and RSV induced pronounced
apoptosis in both cell lines, which still was notably higher for
LOX cells, probably due to higher DR5 surface expression in these
cells (Fig. 5A). A clonogenic survival assay also demonstrated
very low survival of LOX cells when compared to LU1205 cells
12 days after combined treatment of TRAIL and RSV (Fig. 5B).
Effects of modulation of cFLIP protein levels on TRAIL-induced
apoptosis
Activation of the JNK-cJun pathway is involved in downreg-
ulation of cFLIP expression [11,13], which has been described
as one of the main negative regulators of the extrinsic apop-
totic pathway. Since inhibition of JNK activity modulates
numerous cell functions besides cFLIP regulation, we used
direct cFLIP suppression by specific RNAi to determine a role of
cFLIP in TRAIL+RSV induced apoptosis. Suppression of cFLIP
by specific RNAi (Fig. 5C) increased the levels of TRAIL-induced
apoptosis, but did not additionally affect TRAIL+RSV induced
apoptosis, suggesting that RSV and FLIP-RNAi affected the
same target (Fig. 5D). In contrast, TAM67, a dominant negative
cJun expression construct, substantially upregulated cFLIP
expression and protein levels [11]. LU1205 melanoma cells
with increased levels of cFLIP after transfecion with TAM67
were also more resistant to TRAIL+RSV induced apoptosis
(Fig. 5D). Mass cultures of both LU1205 and LOX cells trans-
fected with the cFLIP expression construct demonstrated up-
regulation of cFLIP expression and decreased levels of TRAIL+
RSV induced apoptosis (Figs. 6E, F and data not shown). Taken
together, these data further demonstrated a role for cJun-
dependent negative regulation of cFLIP expression, including
RSV-induced downregulation of cFLIP, for the sensitization
and upregulation of TRAIL-induced cell death in human
melanomas.
Increasing DR5 surface expression in metastatic melanoma
cells enhances TRAIL and RSV induced apoptosis
We attempted to further increase DR5 surface expression in
LU1205 cells in order to achieve effective killing of these
metastatic melanoma cells. We have recently described several
approaches to increase surface expression of DR5 in LU1205
human melanoma, including the use of sodium arsenite treat-
ment and γ-irradiation [11,19]. Treatment of LU1205 melanoma
cells by RSV (50 μM) alone had only minor effects on surface DR5
levels, while sodium arsenite in combination with RSV demon-
strated some additive effect, compared to sodium arsenite alone
(Fig. 6A). However, a combination of sodium arsenite and NS398
(50 μM), a COX-2 inhibitor, had the most pronounced effects on
up-regulating DR5 surface levels in LU1205 melanoma cells (Fig.
6B). The effect of sodium arsenite+NS398 on DR5 surface
expression was dependent on the upregulation of intracellular
protein levels of both DR5 and dynamin-2, an adaptor protein
critically involved in death receptor translocation from the Golgi
to the cell surface [19,36] (Fig. 6C).
Finally, an initial treatment of LU1205 cells with arsenite+
NS398 or with γ-irradiation (2.5–5 Gy) and subsequent treat-
ment with TRAIL+RSV caused strong upregulation of LU1205
cell death (Fig. 6D). Taken together, these results confirmed a
role of elevated DR5 surface expression to further accelerate
TRAIL+RSV induced apoptosis in LU1205 melanoma cells.
Upregulation of TRAIL-mediated apoptosis in highly resistant
HHMSX metastatic melanoma cells using the extrinsic
receptor-mediated pathway
HHMSX metastatic melanoma cells were highly resistant to the
death-receptor-mediated apoptosis. Mitochondrial phenotype
of these cells was evaluated using immunostaining. In normal
human fibroblasts and melanoma WM9 cells the mitochon-
drial staining consistently displayed a characteristic elongated
morphology was mainly perinuclear (Fig. 7A and data not
shown). In contrast, the mitochondrial staining of HHMSX
melanoma cells differed significantly from cell to cell and had
an inconsistent and variable morphology, indicating probably
an alteration of mitochondrial function in these cells (Fig. 7A).
Intracellular levels of BID-p22 and its cleaved form BID-p15
proteins, which were critically important for the transmission
of caspase-8-mediated signaling to mitochondria, were sub-
stantially decreased in HHMSX cells, further demonstrating a
low efficiency of the mitochondrial pathway (Fig. 7B).
Specific inhibitors of caspases also indicated that HHMSX
melanoma cells have a dysfunctional mitochondrial death
1171EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
pathway. Indeed, TRAIL+RSV induced apoptosis in several
human melanomas was suppressed by Ac-DEVD-CHO, a cas-
pase-3 inhibitor, and zVAD-fmk, a universal inhibitor of cas-
pases. Ac-IETD-CHO (5 μM), a caspase-8 inhibitor, and Ac-LEHD-
CHO (5 μM), a caspase-9 inhibitor, provided some protection
against TRAIL-mediated apoptosis in WM9 and LU1205 cells. In
contrast, Ac-LEHD-CHO was not effective in HHMSX cells,
revealing a deficiency in their mitochondrial pathway (Fig. 7C).
These data correlated well with the involvement of both caspase-
8 and caspase-9 mediated apoptotic pathways in response to
TRAIL in WM9 and LU1205 melanoma cell lines. However, it also
indicated that apoptotic death of melanoma cells, such as
HHMSX, with their suppressed mitochondrial pathway, could
be independent of caspase-9 activity.
We attempted to increase the levels of TRAIL-mediated
apoptosis in highly resistant HHMSX melanoma cells via
Fig. 6 –Upregulation of TRAIL and RSV induced apoptosis in LU1205 melanoma cells. (A) Combined effects of sodium arsenite
(As, 5 μM) and resveratrol (RSV, 50 μM) on DR5 surface expression in LU1205 cells. RSV was added into cell media 1 h after
sodium arsenite. Cells were stained 6 h after treatment with PE-labeled anti-human DR5 mAb and analyzed by the flow
cytometry; MFI was indicated (B) Combined effects of sodium arsenite (As, 5 μM) and NS398 (50 μM) on DR5 surface expression.
(C) Western blot analysis of total protein levels of DR5 and dynamin-2 6 h after indicated treatment of LU1205 cells.
(D) Acceleration of TRAIL-mediated apoptosis in LU1205 cells after pretreatment with sodium arsenite (5 μM) + NS398 (50 μM)
for 16 h, or after γ-irradiation (2.5–5 Gy). TRAIL (50 ng/ml), RSV (50 μM) and CHX (2 μg/ml) were added 16 h after an initial
treatment and remained in the media for the next 32 h. Error bars represent mean ± S.D. from three independent experiments.
1172 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
the extrinsic TRAIL-R2-mediatedpathway. Pretreatment with γ-
irradiation significantly upregulated DR5 surface levels in
HHMSX cells (Fig. 7D). The sequential treatment of HHMSX
cells by γ-irradiation, and 16 h after irradiation with TRAIL
(50 ng/ml)+ RSV (50μM) caused a substantialincrease in levels of
TRAIL-mediated apoptosis (Fig. 7E). Clonogenic survival assay
further demonstrated that combined treatment with TRAIL and
RSV of HHMSX melanoma cells was especially efficient follow-
ing γ-irradiation, while normal human fibroblasts TIG-3 were
affected substantially less under these conditions (data not
shown).
Hence, we observed an efficient TRAIL/DR5/caspase-8/cas-
pase-3-mediated death signaling in the presence of RSV, in
melanomas harboring defects in mitochondrial death pathway,
demonstrating a possibility for the upregulation of apoptotic
signaling in these cancer cells via the extrinsic pathway.
Discussion
Numerous investigations over the last two decades have att-
empted to restore apoptotic signaling pathways in cancer cells.
Understanding the role of NF-κB, STAT3 and AP1 in general
regulation of cell survival, via transcriptional control of genes
with pro-survival and pro-apoptotic functions, was an important
goal of these investigations [7,37,38]. The discovery of TRAIL and
Fig. 7 –HHMSX melanoma cells have dysfunctional mitochondrial apoptotic pathway, but undergo TRAIL-mediated apoptosis.
(A) Mitochondrial morphology of melanoma cell lines. WM9 and HHMSX metastatic melanomas were stained with
anti-Pyruvate dehydrogenase (PDH —green) to visualize mitochondria, and propidium iodide (PI —red) to observe the nucleus.
The mitochondrial and nuclear staining of HHMSX cells was aberrant, indicative of a highly transformed cell line. (B) Western
blot analysis of BID (p22 and p15) levels in control and TRAIL+RSV treated melanoma cells. (C) Induction of apoptosis in
melanoma lines 48 h after indicated treatment. TRAIL (50 ng/ml) and RSV (50 μM) were used. Caspase inhibitors: IETD, LEHD,
DEVD and zVAD (20 μM) have been added to the media 30 min before TRAIL+RSV. Cells were stained by PI 48 h after treatment.
Apoptosis levels were determined as the percentage of cells with hypodiploid content of DNA in the pre-G0/G1 region using
flow cytometry. Error bars represent mean±S.D. from three independent experiments. (D) Upregulation of surface DR5 levels in
HHMSX cells following γ-irradiation (16 h) or sequential treatment with irradiation and RSV (16 h after γ-irradiation for the next
16 h) and was determined by immunostaining with PE-labeled anti-human DR mAb using the flow cytometry; MFI is indicated.
(E) Acceleration of apoptosis in HHMSX cells with increased levels of DR5. Cell were not treated or γ-irradiated at 5 Gy; 16 h after
irradiation TRAIL (50 ng/ml), RSV (50 μM) or TRAIL+RSV were added and remained in the media for an additional 32 h. Cell
cycle-apoptosis analysis was performed using PI staining DNA and the FACS analysis. Results of a typical experiment are
indicated.
1173EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
detailed analysis of TRAIL-R1/R2-mediated death signaling in
normal and cancer cells led to the theory that TRAIL-R-mediated
pathway might beeffective for the induction of apoptosis [39,40].
One important advantage of TRA IL, compared to TNFαand FasL,
is its relatively low toxicity in vivo [41]. The safety and efficacy of
soluble TRAIL and anti-TRAIL-R1/R2 agonistic mAbs combined
with chemotherapy are currently undergoing evaluation in
several clinical trials [41]. Unfortunately, sensitivity to TRAIL is
not a universal feature of cancer cells. Most melanomas do not
respond to apoptotic stimulation induced by TRAIL alone and
require cotreatment with an additional sensitizing agent [42].
The internal strategy of genetic and epigenetic regulation
during progression of cancer, based on Darwinian selection, is to
suppress death signaling at all levels, including down-regu-
lation of transcription of death receptor genes, inhibition of
receptor's modification, suppression of translocation of death
receptors to the cell surface, elevated mutagenesis in the death
domains, the dramatic STAT3/NF-κB-dependent upregulation
of expression of apoptoticinhibitors and upregulation of surface
expression of decoy receptors, DcR1 and DcR2 [43–45]. Investiga-
tion of natural and pharmacological inhibitorsof STAT3 and NF-
κB represents a significant and novel tactics in the struggle
against cancer. In this respect, correlation of anticancer activity
of RSV and its capacity to suppress the activation of STAT3 and
NF-κB has provided insights into the regulation of apoptosis in
cancer cells [9,34,16,46]. Jang et al. [47] have demonstrated the
abilities of RSV to inhibit carcinogenesisat multiple stages, and
the systemic administration of RSV was shown to inhibit tumor
progression in different mouse cancer models [14].
The results of the present study highlighted a significance
of RSV-induced p53-p21WAF activation and RSV-induced
downregulation of cyclin D1 levels for RSV-induced cell cycle
arrest in LU1205 (Figs. 1 and 3). However, RSV has many addi-
tional targets within the cell, including general modulators of
transcription Sirtuins (class III histon deacetylases) [48,49],
transcription factors NF-κB and STAT3 [16,17] and many
enzymes of general cell metabolism [46]. RSV treatment of
several melanoma lines induced a relatively modest decrease
in the high basal NF-κB activity and NF-κB-dependent trans-
cription in these cells, while downregulation of STAT3 levels
and STAT3-dependent transcription was more pronounced
(Figs. 2 and 3). The well known control of Bcl-xL,Survivin, TRAIL
and Fas genes by STAT3 certainly demonstrated its integral
role as a regulator of apoptosis in melanomas [9]. An addi-
tional important target of RSV in melanoma cells was the
activation of the JNK-cJun pathway, which was involved in
regulating the expression and turnover of cFLIP [11,13] and the
upregulation of sensitivity to TRAIL. Interestingly, that strong
activation of MAPK p38-ATF2 by RSV in melanoma cells was
involved in the regulation of cell survival that was strongly
decreased after specific inhibition of this pathway (Fig. 3).
The results of our study demonstrated that RSV rarely
initiates notable upregulation of endogenous TRAIL expres-
sion on the cell surface or TRAIL-mediated cell suicide in
human melanomas, as most melanomas tested were rela-
tively resistant to either RSV or recombinant TRAIL alone.
Importantly, the combined treatment with RSV and TRAIL had
a profound synergistic effect on the induction of apoptosis in
certain metastatic melanomas. In addition, pretreatment of
melanoma cells by either γ-irradiation or sodium arsenite
(especially in combination with COX-2 inhibitor) that upregu-
lates DR5 surface levels [19] and subsequent treatment with
TRAIL+RSV has promising therapeutic potential, as evidenced
by the induction of apoptosis in the most resistant melanoma
lines. A role of COX-2 inhibitors in transcriptional upregula-
tion of dynamin-2 expression, which was important for death
receptor translocation, was described previously [50].
We should highlight that an alternative approach, which
used γ-irradiation and RSV treatment was effective only for
killing early stage human melanomas (such as WM35 and
WM793) and had only modest improvement in metastatic
melanomas (such as LOX). In contrast, this approach was very
effective for mouse SW1 metastatic melanoma cells, where
TRAIL was actively translocated to cell surface after RSV treat-
ment (unpublished obsevations). Mechanisms that control
TRAIL translocation from the intracellular pools to the cell
surface [51] are currently unknown and are the subject of con-
tinuing investigation. Monoubiquitination was shown to play
an important role in the protein translocation ofFas Ligand [52].
However, protein modifications of TRAIL linked with its
intracellular trafficking are still incompletely investigated.
Many cancer cells, including melanoma cells, have mito-
chondrial respiration defects and suppressed mitochondrial
function [53]. We observed a similar situation in some
metastatic melanomas investigated in our study. Despite in-
herent mitochondrial deficiencies, we demonstrated in the
present study that the extrinsic DR5-mediated death pathway
could still be upregulated in metastatic melanoma cells, using
pretreatment with γ-irradiation that was followed by com-
bined treatment of TRAIL and RSV. This finding provides a
potentially important rational approach to more efficacious
melanoma treatment.
Acknowledgments
We would like to thank Drs. A. Chan and S. Y. Fuchs for
discussion, Drs. M. Herlyn and Z. Ronai for melanoma cell
lines, Drs. R. Davis, W. S. El-Deiry, G. J. Gores, J. Hiscott, M.
Karin, J. J. Manfredi, M. E. Perer, S.-Y. Sun, A. N. Shajahan and
H. Wajant for plasmid constructs. This work was supported by
NIH Grants CA 49062, ES 11804, Superfund Grant P42 ES 10349,
and Environmental Center Grant P30 ES 09089.
REFERENCES
[1] P.H. Krammer, M. Kaminski, M. Kiessling, K. Gulow, No life
without death, Adv. Cancer Res. 97C (2007) 111–138.
[2] J.C. Reed, Drug insight: cancer therapy strategies based on
restoration of endogenous cell death mechanisms, Nature
clinical practice 3 (2006) 388–398.
[3] K.M. Debatin, P.H. Krammer, Death receptors in
chemotherapy and cancer, Oncogene 23 (2004) 2950–2966.
[4] V. Gray-Schopfer, C. Wellbrock, R. Marais, Melanoma biology
and new targeted therapy, Nature 445 (2007) 851–857.
[5] C. Perlis, M. Herlyn, Recent advances in melanoma biology,
Oncologist 9 (2004) 182–187.
[6] P. Hersey, L. Zhuang, X.D. Zhang, Current strategies in
overcoming resistance of cancer cells to apoptosis melanoma
as a model, International review of cytology 251 (2006)
131–158.
1174 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
[7] M. Karin, Nuclear factor-kappaB in cancer development and
progression, Nature 441 (2006) 431–436.
[8] J.E. Darnell Jr., Transcription factors as targets for cancer
therapy, Nat. Rev. Cancer 2 (2002) 740–749.
[9] V.N. Ivanov, A. Bhoumik, Z. Ronai, Death receptors and
melanoma resistance to apoptosis, Oncogene 22 (2003)
3152–3161.
[10] Y. Ueda, A. Richmond, NF-kappaB activation in melanoma,
Pigment cell research / sponsored by the European Society for
Pigment Cell Research and the International Pigment Cell
Society 19 (2006) 112–124.
[11] V.N. Ivanov, T.K. Hei, Sodium arsenite accelerates
TRAIL-mediated apoptosis in melanoma cells through
upregulation of TRAIL-R1/R2 surface levels and
downregulation of cFLIP expression, Exp. Cell Res. 312 (2006)
4120–4138.
[12] W. Li, X. Zhang, A.F. Olumi, MG-132 sensitizes TRAIL-resistant
prostate cancer cells by activating c-Fos/c-Jun heterodimers
and repressing c-FLIP(L), Cancer Res. 67 (2007) 2247–2255.
[13] L. Chang, H. Kamata, G. Solinas, J.L. Luo, S. Maeda, K.
Venuprasad, Y.C. Liu, M. Karin, The E3 ubiquitin ligase itch
couples JNK activation to TNFalpha–induced cell death by
inducing c-FLIP(L) turnover, Cell 124 (2006) 601–613.
[14] J.A. Baur, D.A. Sinclair, Therapeutic potential of resveratrol:
the in vivo evidence, Nat. Rev. Drug Discov. 5 (2006) 493–506.
[15] V.N. Ivanov, T.K. Hei, Arsenite sensitizes human melanomas
to apoptosis via tumor necrosis factor alpha-mediated
pathway, J. Biol. Chem. 279 (2004) 22747–22758.
[16] B.B. Aggarwal, A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S.
Shishodia, Y. Takada, Role of resveratrol in prevention and
therapy of cancer: preclinical and clinical studies, Anticancer
research 24 (2004) 2783–2840.
[17] A. Bhardwaj, G. Sethi, S. Vadhan-Raj, C. Bueso-Ramos, Y.
Takada, U. Gaur, A.S. Nair, S. Shishodia, B.B. Aggarwal,
Resveratrol inhibits proliferation, induces apoptosis, and
overcomes chemoresistance through down-regulation of
STAT3 and nuclear factor-kappaB-regulated antiapoptotic
and cell survival gene products in human multiple myeloma
cells, Blood 109 (2007) 2293–2302.
[18] A. Kotha, M. Sekharam, L. Cilenti, K. Siddiquee, A. Khaled, A.S.
Zervos, B. Carter, J. Turkson, R. Jove, Resveratrol inhibits Src
and Stat3 signaling and induces the apoptosis of malignant
cells containing activated Stat3 protein, Mol. Cancer Ther. 5
(2006) 621–629.
[19] V.N. Ivanov, H. Zhou, T.K. Hei, Sequential treatment by
ionizing radiation and sodium arsenite dramatically
accelerates TRAIL-mediated apoptosis of human melanoma
cells, Cancer Res. 67 (2007) 5397–5407.
[20] K. Satyamoorthy, E. DeJesus, A.J. Linnenbach, B. Kraj, D.L.
Kornreich, S. Rendle, D.E. Elder, M. Herlyn, Melanoma cell lines
from different stages of progression and their biological and
molecular analyses, Melanoma Res. 7 (Suppl 2) (1997) S35–S42.
[21] H. van Dam, S. Huguier, K. Kooistra, J. Baguet, E. Vial, A.J. van
der Eb, P. Herrlich, P. Angel, M. Castellazzi, Autocrine growth
and anchorage independence: two complementing
Jun-controlled genetic programs of cellular transformation,
Genes Dev. 12 (1998) 1227–1239.
[22] T.M. Baetu, H. Kwon, S. Sharma, N. Grandvaux, J. Hiscott,
Disruption of NF-kappaB signaling reveals a novel role for
NF-kappaB in the regulation of TNF-related
apoptosis-inducing ligand expression, J. Immunol. 167 (2001)
3164–3173.
[23] T. Bartke, D. Siegmund, N. Peters, M. Reichwein, F. Henkler, P.
Scheurich, H. Wajant, p53 upregulates cFLIP, inhibits
transcription of NF-kappaB-regulated genes and induces
caspase-8-independent cell death in DLD-1 cells, Oncogene
20 (2001) 571–580.
[24] M.S. Ricci, Z. Jin, M. Dews, D. Yu, A. Thomas-Tikhonenko, D.T.
Dicker, W.S. El-Deiry, Direct repression of FLIP expression by
c-myc is a major determinant of TRAIL sensitivity, Mol. Cell.
Biol. 24 (2004) 8541–8555.
[25] L. Resnick-Silverman, S. St Clair, M. Maurer, K. Zhao, J.J.
Manfredi, Identification of a novel class of genomic
DNA-binding sites suggests a mechanism for selectivity in
target gene activation by the tumor suppressor protein p53,
Genes Dev. 12 (1998) 2102–2107.
[26] A. Bakhtiarova, P. Taslimi, S.J. Elliman, P.A. Kosinski, B.
Hubbard, M. Kavana, D.M. Kemp, Resveratrol inhibits firefly
luciferase, Biochem. Biophys. Res. Commun. 351 (2006)
481–484.
[27] M.A. Partridge, S.X. Huang, E. Hernandez-Rosa, M.M.
Davidson, T.K. Hei, Arsenic induced mitochondrial DNA
damage and altered mitochondrial oxidative function:
implications for genotoxic mechanisms in mammalian cells,
Cancer Res. 67 (2007) 5239–5247.
[28] A. Brunet, A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J.
Anderson, K.C. Arden, J. Blenis, M.E. Greenberg, Akt promotes
cell survival by phosphorylating and inhibiting a Forkhead
transcription factor, Cell 96 (1999) 857–868.
[29] M. Selvakumaran, H.K. Lin, T. Miyashita, H.G. Wang, S.
Krajewski, J.C. Reed, B. Hoffman, D. Liebermann, Immediate
early up-regulation of bax expression by p53 but not TGF beta
1: a paradigm for distinct apoptotic pathways, Oncogene 9
(1994) 1791–1798.
[30] T. Gritsko, A. Williams, J. Turkson, S. Kaneko, T. Bowman, M.
Huang, S. Nam, I. Eweis, N. Diaz, D. Sullivan, S. Yoder, S.
Enkemann, S. Eschrich, J.H. Lee, C.A. Beam, J. Cheng, S.
Minton, C.A. Muro-Cacho, R. Jove, Persistent activation of
stat3 signaling induces survivin gene expression and confers
resistance to apoptosis in human breast cancer cells, Clin.
Cancer Res. 12 (2006) 11–19.
[31] D. Sinibaldi, W. Wharton, J. Turkson, T. Bowman, W.J. Pledger,
R. Jove, Induction of p21WAF1/CIP1 and cyclin D1 expression
by the Src oncoprotein in mouse fibroblasts: role of activated
STAT3 signaling, Oncogene 19 (2000) 5419–5427.
[32] C. Xiao, B.F. Yang, J.H. Song, H. Schulman, L. Li, C. Hao,
Inhibition of CaMKII-mediated c-FLIP expression sensitizes
malignant melanoma cells to TRAIL-induced apoptosis, Exp.
Cell Res. 304 (2005) 244–255.
[33] S. Kreuz, D. Siegmund, P. Scheurich, H. Wajant, NF-kappaB
inducers upregulate cFLIP, a cycloheximide-sensitive
inhibitor of death receptor signaling, Mol. Cell. Biol. 21 (2001)
3964–3973.
[34] S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis
pathways in anticancer chemotherapy, Oncogene 25 (2006)
4798–4811.
[35] S. Fulda, K.M. Debatin, Sensitization for tumor necrosis
factor-related apoptosis-inducing ligand-induced apoptosis
by the chemopreventive agent resveratrol, Cancer Res. 64
(2004) 337–346.
[36] V.N. Ivanov, Z. Ronai, T.K. Hei, Opposite roles of FAP-1 and
dynamin in the regulation of Fas (CD95) translocation to the
cell surface and susceptibility to Fas ligand-mediated
apoptosis, J. Biol. Chem. 281 (2006) 1840–1852.
[37] E. Shaulian, M. Karin, AP-1 as a regulator of cell life and death,
Nat. Cell Biol. 4 (2002) E131–E136.
[38] D.E. Levy, J.E. Darnell Jr., Stats: transcriptional control and
biological impact, Nat. Rev., Mol. Cell Biol. 3 (2002) 651–662.
[39] A. Ashkenazi, Targeting death and decoy receptors of the
tumour-necrosis factor superfamily, Nat. Rev. Cancer 2 (2002)
420–430.
[40] R. Takimoto, W.S. El-Deiry, Wild-type p53 transactivates the
KILLER/DR5 gene through an intronic sequence-specific
DNA-binding site, Oncogene 19 (2000) 1735–1743.
[41] U. Schaefer, O. Voloshanenko, D. Willen, H. Walczak, TRAIL: a
multifunctional cytokine, Front. Biosci. 12 (2007) 3813–3824.
[42] P. Hersey, X.D. Zhang, How melanoma cells evade
trail-induced apoptosis, Nat. Rev. Cancer 1 (2001) 142–150.
1175EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
[43] H. Wajant, CD95L/FasL and TRAIL in tumour surveillance and
cancer therapy, Cancer Treat. Res. 130 (2006) 141–165.
[44] K.W. Wagner, E.A. Punnoose, T. Januario, D.A. Lawrence, R.M.
Pitti, K. Lancaster, D. Lee, M. von Goetz, S.F. Yee, K. Totpal, L.
Huw, V. Katta, G. Cavet, S.G. Hymowitz, L. Amler, A.
Ashkenazi, Death-receptor O-glycosylation controls
tumor-cell sensitivity to the proapoptotic ligand
Apo2L/TRAIL, Nat. Med. 13 (2007) 1070–1077.
[45] A. Almasan, A. Ashkenazi, Apo2L/TRAIL: apoptosis signaling,
biology, and potential for cancer therapy, Cytokine Growth
Factor Rev 14 (2003) 337–348.
[46] S. Shankar, G. Singh, R.K. Srivastava, Chemoprevention by
resveratrol: molecular mechanisms and therapeutic
potential, Front. Biosci. 12 (2007) 4839–4854.
[47] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W.
Beecher, H.H. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G.
Mehta, R.C. Moon, J.M. Pezzuto, Cancer chemopreventive
activity of resveratrol, a natural product derived from grapes,
Science 275 (1997) 218–220.
[48] J.A. Baur, K.J. Pearson, N.L. Price, H.A. Jamieson, C. Lerin, A.
Kalra, V.V. Prabhu, J.S. Allard, G. Lopez-Lluch, K. Lewis, P.J.
Pistell, S. Poosala, K.G. Becker, O. Boss, D. Gwinn, M. Wang, S.
Ramaswamy, K.W. Fishbein, R.G. Spencer, E.G. Lakatta, D. Le
Couteur, R.J. Shaw, P. Navas, P. Puigserver, D.K. Ingram, R. de
Cabo, D.A. Sinclair, Resveratrol improves health and survival
of mice on a high-calorie diet, Nature 444 (2006) 337–342.
[49] M. Kaeberlein, T. McDonagh, B. Heltweg, J. Hixon, E.A.
Westman, S.D. Caldwell, A. Napper, R. Curtis, P.S. DiStefano, S.
Fields, A. Bedalov, B.K. Kennedy, Substrate-specific activation
of sirtuins by resveratrol, J. Biol. Chem. 280 (2005) 17038–17045.
[50] Z. Zhang, R.N. DuBois, Detection of differentially expressed
genes in human colon carcinoma cells treated with a
selective COX-2 inhibitor, Oncogene 20 (2001) 4450–4456.
[51] M.A. Cassatella, V. Huber, F. Calzetti, D. Margotto, N.
Tamassia, G. Peri, A. Mantovani, L. Rivoltini, C. Tecchio,
Interferon-activated neutrophils store a TNF-related
apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular
pool that is readily mobilizable following exposure to
proinflammatory mediators, J. Leukoc. Biol. 79 (2006) 123–132.
[52] E. Zuccato, E.J. Blott, O. Holt, S. Sigismund, M. Shaw, G. Bossi,
G.M. Griffiths, Sorting of Fas ligand to secretory lysosomes is
regulated by mono-ubiquitylation and phosphorylation,
J. Cell Sci. 120 (2007) 191–199.
[53] H. Pelicano, R.H. Xu, M. Du, L. Feng, R. Sasaki, J.S. Carew, Y. Hu,
L. Ramdas, L. Hu, M.J. Keating, W. Zhang, W. Plunkett, P.
Huang, Mitochondrial respiration defects in cancer cells
cause activation of Akt survival pathway through a
redox-mediated mechanism, J. Cell Biol. 175 (2006) 913–923.
1176 EXPERIMENTAL CELL RESEARCH 314 (2008) 1163–1176
