A Human scFv Antibody against TRAIL Receptor 2 Induces
Autophagic Cell Death in Both TRAIL-Sensitive and
TRAIL-Resistant Cancer Cells
2Kyeong Sook Choi,
2and Yong-Sung Kim
1Department of Molecular Science and Technology, Ajou University;
Ajou University School of Medicine, Woncheon-dong, Yeongtong-gu, Suwon, Korea
2Department of Microbiology and
3Institute for Medical Sciences,
Tumor necrosis factor (TNF)–related apoptosis-inducing
ligand (TRAIL) induces apoptotic cell death in a variety of
tumor cells without significant cytotoxicity on normal cells.
However, many cancer cells with apoptotic defects are
resistant to treatment with TRAIL alone, limiting its potential
as an anticancer therapeutic. Here, we report on the tumor-
icidal activity of a human single-chain fragment variable,
HW1, which specifically binds to TRAIL receptor 2 (TR2)
without competing with TRAIL for the binding. HW1
treatment as a single agent induces autophagic cell death in
a variety of both TRAIL-sensitive and TRAIL-resistant cancer
cells, but exhibits much less cytotoxicity on normal cells. The
HW1-induced autophagic cell death was inhibited by an
autophagy inhibitor, 3-methyladenine, or by RNA interference
knockdown of Beclin-1 and Atg7. We also show that the HW1-
mediated autophagic cell death occurs predominantly via the
c-Jun NH2-terminal kinase pathway in a caspase-independent
manner. Analysis of the death-inducing signaling complex
induced by HW1 binding to TR2 exhibits the recruitment of
TNF receptor–associated death domain and TNF receptor–
associated factor 2, but not Fas-associated death domain,
caspase-8, or receptor-interacting protein, which is distinct
from that induced by TRAIL. Our results reveal a novel TR2-
mediated signaling pathway triggering autophagic cell death
and provides a new strategy for the elimination of cancer cells,
including TRAIL-resistant tumors, through nonapoptotic cell
death. [Cancer Res 2007;67(15):7327–34]
Tumor necrosis factor (TNF)–related apoptosis-inducing ligand
(TRAIL) and its associated receptors (TRAIL-R/TR) are attractive
targets for cancer therapy as they can kill tumor cells with little
cytotoxicity on normal cells in vitro and in vivo through the
p53-independent extrinsic apoptotic pathway (1–3). TR1 (TRAIL-
R1/DR4) and TR2 (TRAIL-R2/DR5) contain a cytoplasmic death
domain that triggers apoptosis upon TRAIL binding, whereas TR3
(TRAIL-R3/DcR1) and TR4 (TRAIL-R4/DcR2) lack such a functional
death domain (reviewed in ref. 2). TRAIL binding to TR1 or TR2
results in conformational changes of the receptor that promote the
formation of death-inducing signaling complexes (DISC), which
consists of receptors, Fas-associated death domains (FADD), and
activated caspase-8 and/or caspase-10 to trigger apoptosis
signaling (2). However, many highly malignant tumor cells (>50%)
even expressing TR1 and/or TR2 remain resistant to TRAIL-
induced apoptosis, the underlying mechanism of which has been
poorly understood and varies with the cellular context (4, 5).
TRAIL-resistant cells become sensitized to apoptotic cell death via
a combination of TRAIL with either chemotherapeutic agents or
irradiation (6–10). A number of agonistic monoclonal antibodies
(mAb) that specifically target TR1 or TR2 have been isolated to
induce apoptotic cell death in multiple tumor cell types, and some
of these also exhibit minimal cytotoxicity on normal cells (11–16).
However, all of the TR1- or TR2-specific mAbs reported thus far
have shown tumoricidal activities as a single agent in most TRAIL-
sensitive cells, but not in TRAIL-resistant cells (12, 13). Thus, the
isolation of agonistic mAbs that are cytotoxic to TRAIL-resistant
cells but not to normal cells is highly desirable for developing novel
When stimulated by either TRAIL or agonistic mAbs, TR1 and/or
cells predominantly through the sequential activation of caspases
(2, 12, 15). Depending on the external stimuli and specific cell types,
TR1 and/or TR2 can also transduce multiple cellular signaling
pathways in a caspase-independent manner, including c-Jun NH2-
terminal kinase (JNK) and p38, although the components of which
remain largely undefined (4, 7, 8, 14, 17). Furthermore, TRAIL can
also induce caspase-independent autophagic cell death in normal
epithelial cells (18) and in the breast cancer cell line MCF-10A (19),
implying that TR1 and/or TR2 are involved in autophagic cell death.
However, the specific roles of TR1 and/or TR2 in autophagic cell
death have not yet been defined. Autophagy is an intracellular
process in which double membrane–bound vesicles encapsulate
cytoplasmic proteins and organelles to degrade by fusion with
lysosomes in response to external stresses (reviewed in refs. 20, 21).
Recent studies have suggested that autophagy is another cell death
pathway that is morphologically distinct from apoptosis (22, 23).
Here, we have characterized the cell death–inducing properties
of an agonistic anti-TR2 single-chain fragment variable (scFv), HW1,
in multiple TRAIL-sensitive and TRAIL-resistant cancer cell types.
Unlikely with TRAIL and agonistic TR2-specific mAbs previously
reported, HW1 induced autophagic cell death as a single agent
in both TRAIL-sensitive and TRAIL-resistant cancer cells exhibiting
much less cytotoxicity on normal cells. We also found that
HW1-mediated autophagic cell death occurs predominantly via
Note: Supplementary data for this article are available at Cancer Research Online
K-J. Park, S-H. Lee, and T-I. Kim contributed equally to this work.
Requests for reprints: Yong-Sung Kim, Department of Molecular Science and
Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749,
Korea. Phone: 82-31-219-2662; Fax: 82-31-219-2394; E-mail: email@example.com or
Myung-Hee Kwon, Department of Microbiology, Ajou University School of Medicine,
San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea. Phone: 82-31-219-5074;
Fax: 82-31-219-5079; E-mail: firstname.lastname@example.org.
I2007 American Association for Cancer Research.
Cancer Res 2007; 67: (15). August 1, 2007
caspase-independent JNK activation. DISC analysis showed that
HW1 binding to TR2 induced the recruitment of TNF receptor
(TNFR)–associated death domain (TRADD) and TNFR-associated
factor 2 (TRAF2), but not FADD, caspase-8, or receptor-interacting
protein (RIP), showing that the components of HW1-induced
DISC are distinct from those of TRAIL-induced DISC. These
results provide a potential therapeutic strategy to kill not only
TRAIL-sensitive but also TRAIL-resistant cancer cells through
TR2-mediated autophagic cell death, which is distinct from
TRAIL-mediated apoptotic pathway.
Materials and Methods
Cell lines and reagents. Adherent human cancer cell lines, HCT116,
MCF-7, HepG2, DU-145, ZR75-1, and U87MGwere purchased from American
Type Culture Collection and grown in DMEM medium supplemented with
10% (v/v) FCS, 100 units/mL of penicillin, and 100 Ag/mL of streptomycin
(Life Technologies Invitrogen). Nonadherent human cancer cell lines, Molt-4
and HL-60, were from American Type Culture Collection and grown in RPMI
1640 supplementedwith 10% FCS, 100 units/mL of penicillin, and 100 Ag/mL
of streptomycin. Normal human hepatocytes and mammary epithelial cells
were purchased from Cambrex BioScience and were cultured under the
recommended conditions. Normal human astrocytes were prepared and
subcultured as described previously (9). Recombinant human TRAIL
(residues 114–281) without any tags were from KOMA Biotech. Soluble
TR2-Fc, TNFR1, and TNFR2 were from R&D Systems, and CD95 was from
Biovision. AlexaFluor633 and AlexaFluor488 labeling kits were from
Molecular Probes. Mouse anti-His6 IgG was from Sigma. The following
rabbit antibodies were used for Western blotting: anti–caspase-3 and anti–
caspase-8 were from Stressgen, Inc.; anti-poly(ADP-ribose) polymerase
(PARP) and anti-FADD were from Upstate Biotechnology; anti–phospho-
JNK, anti-JNK, anti–phospho-p38, anti-p38, anti-TRADD, anti-TRAF2, and
anti–h-actin were from Cell Signaling Technology; anti-TR2 was from Koma
Biotech; and anti-RIP was from BD Bioscience.
Biochemical characterizations. Previously, we isolated human scFv
HW14(281 amino acids, 30.9 kDa) against the extracellular domain of TR2
(TR2-ECD) from a yeast surface–displayed pseudoimmune scFv library (24).
The detailed experimental methods of expression and purification of HW1
(with COOH-terminal FLAG and 6? His tags), TRAIL-His (residues 114–281
with a COOH-terminal 6? His-tag), TRAIL-Flag (residues 114–281 with a
COOH-terminal Flag tag), TR2-ECD, TR1-ECD, TR3-ECD, and TR4-ECD
(25, 26), competitive ELISA (25), size exclusion chromatography (25), and
surface plasmon resonance (SPR; refs. 25, 26) are described in the
Specific binding of HW1 to cell surface–expressed TR2. Plasmids of
T010 and T30 encoding TR2DCD-YFP fusion protein (the cytosolic domain
deleted TR2 fused to yellow fluorescent proteins) and TR4DCD-YFP (the
cytosolic domain deleted TR4 fused to yellow fluorescent proteins),
respectively, were kindly provided by Prof. F.K. Chan (University of
Massachusetts). HCT116 cells were transfected with the respective plasmid
by electroporation (1,000 V and 1,500 AF) using a 0.1-cm diameter capillary
(Digital Biotech, Korea), seeded at a density of 5 ? 104cells/well in 24-well
plate over glass coverslips and grown for 30 h. The cells were then washed
once with PBS containing 2% FCS and then stained with either Alexa633-
labeled HW1 (5 Ag/mL) or TRAIL-His (5 Ag/mL) for 30 min at 4jC. The cells
were then washed thrice with 2% FCS and fixed using 4% paraformaldehyde
in PBS for 1 h at 4jC. Images were obtained using a LMS510 model laser
scanning confocal fluorescence microscope (LSM510; Carl Zeiss).
Cell viability assays. Cells seeded at a density of 1 ? 104cells/well in
96-well plates were cultured overnight and treated with HW1 or TRAIL in
the absence and presence of the following agents, as specified in the figure
legends: Z-VAD [benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl
ketone; Santa Cruz Biotechnology], SP600125 (Calbiochem), SB203580
(Sigma), and 3-methyladenine (3-MA; Sigma). Cell viability was analyzed
using a colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide]–based cell growth determination kit (Sigma; refs. 6, 26).
Transmission electron microscopy. Cells treated with TRAIL-His or
HW1, as specified in the figure legends, were prefixed in Karnovsky’s
solution (2% glutaraldehyde, 1% paraformaldehyde in 100 mmol/L of
sodium cacodylate buffer; pH 7.4) at room temperature for 2 h. The samples
were rinsed with cacodylate buffer, postfixed in 1% osmium tetroxide and
1.5% potassium ferrocyanide for 1 h, dehydrated through a graded series of
ethanols (50–100%), and then embedded in Poly Bed 812 resin (Pelco).
Ultrathin sections were cut on a Reichert Ultracut E microtome, double-
stained with uranyl acetate and then lead citrate, and viewed under an
electron microscope (EM 902A; Carl Zeiss).
protein–fused LC3 (GFP-LC3) localization, U87MG cells were transfected
with a plasmid encoding GFP-LC3 (27, 28) kindly provided by Prof. T.
Yoshimori (Osaka University) and stably expressing cells were selected with
changes of media containing 500 Ag/mL of G418. After treatment of the
stablecell linesexpressing GFP-LC3 withHW1 (25 Ag/mL,24 h), imageswere
obtained under a live fluorescence microscope (Axiovert 200M; Carl Zeiss).
The nucleuswas stainedwith 4¶,6-diamidino-2-phenylindole dihydrochloride
(DAPI; Vector Laboratories). The images were obtained using a LMS510
The fluorescence images taken and processed using identical settings are
representative of experiments done at least in duplicate.
Western blotting. Cells (3 ? 105cells/well) were seeded in six-well
plates, grown overnight, and then treated under the conditions specified in
the figure legends. The standard procedure for Western blotting was then
done as described previously (6, 9). The secondary anti-rabbit IgG
conjugated to horseradish peroxidase (Zymed Laboratories) was used for
developing by chemiluminescence (Amersham Pharmacia Biotech).
RNA interference. Small interfering RNA (siRNA) oligonucleotides were
synthesized at Bioneer Co. The mRNA sequences targeted by Beclin-1 and
Atg7 siRNAs were 5¶-CAGUUUGGCACAAUCAAUA-3¶ and 5¶-GGAGUCA-
CAGCUCUUCCUU-3¶, respectively (22). An unrelated siRNA with a sequence
of 5¶-AGACACACGCACUCGUCUC-3¶ was employed as a control. HCT116 or
U87MG cells were transfected with 1 Amol/L of siRNA by electroporation in
a 0.1-cm diameter capillary (Digital Biotech). Transfected cells were
incubated for 48 h at a density of 3 ? 105cells/well in a six-well plate for
Western blotting using anti-Beclin 1 (Novus Biologicals) or anti-Atg7
antibody (Novus Biologicals). Twenty-four hours posttransfection, the cells
(1 ? 104cells/well in 96-well plate) were treated with 200 ng/mL of TRAIL
for 2 h or 25 Ag/mL of HW1 for 30 h prior to MTT assay.
Immunoprecipitations. Cells (1 ? 107cells/time point) were left
untreated or treated with 2 Ag/mL of Flag-tagged TRAIL (29) or 25 Ag/mL
of Flag-tagged HW1 for the indicated time periods (see figure legends). The
cells were collected and lysed for 30 min on ice in 1 mL of a lysis buffer
[20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 10% (v/v) glycerol, 1% (v/v)
Triton X-100, and 2 mmol/L EDTA] with protease inhibitor cocktail (Roche).
After removingcelldebrisby centrifugingat15,000?g for 15minat 4jC,the
cell lysates (800 AL, 15 mg protein) were precipitated with 80 AL of EZview
Red anti-Flag M2 Affinity Gel (Sigma) overnight at 4jC. The complexes
were subsequently washed five times with TBS buffer [50 mmol/L Tris,
150 mmol/L NaCl (pH 7.4)], suspended in 20 AL of 2? SDS sample buffer
[125 mmol/L (pH 6.8), 10% (w/v) SDS, 20% (v/v) glycerol, 14.4 mmol/L 2-ME,
and 0.0002% (w/v) BPB], and subjected to SDS-PAGE and Western blotting.
HW1 specifically binds to TR2 without competition with
TRAIL for the binding. Human scFv HW1 recently isolated
against TR2-ECD from a pseudoimmune scFv library (24) was
solubly expressed and purified without intermolecular disulfide
bonds from bacterial supernatants with >98% purity (Fig. 1A). HW1
existed in a monomeric form (f30.9 kDa) in solution at
concentrations of up to 500 Ag/mL (f16 Amol/L), as determined
4The HW1 sequence data has been deposited into the Genbank under accession
Cancer Res 2007; 67: (15). August 1, 2007
by size exclusion chromatography (Fig. 1B). SPR analyses showed
that HW1 bound to TR2-ECD with association and dissociation
rate constants of 2.33 F 0.02 ? 104(mol/L)?1s?1and 4.71 F 0.05 ?
10?3s?1, respectively, resulting in a KDvalue of 202 F 6 nmol/L
(Fig. 1C). However, HW1 bound to neither the homologous ECDs of
TR1, TR3, and TR4, nor the TNF family death receptors of TNFR1,
TNFR2, and CD95 (Supplementary Fig. S1A), which was further
confirmed by ELISA (data not shown). Thus, HW1 specifically
binds to TR2 without cross-reactivity with the other death
receptors. Notably, competitive ELISA showed that HW1 did not
compete with TRAIL-His for the binding to TR2-ECD (Fig. 1D),
suggesting that it recognizes epitopes which do not overlap with
the TRAIL-binding sites on TR2-ECD.
The specific binding activity of HW1 for cell surface–expressed
TR2 was also analyzed by confocal fluorescence microscopy in
HCT116 cells transiently transfected with a plasmid encoding
TR2DCD-YFP using TR4DCD-YFP as a control (Fig. 1E; ref. 30).
The staining of cells expressing TR2DCD-YFP with Alexa633-
labeled HW1 at 4jC revealed a discrete colocalization pattern of
HW1 and TR2 along the plasma membrane, similar to the
staining with Alexa633-labeled TRAIL-His. However, HW1 did not
bind to cells expressing TR4DCD-YFP (Fig. 1E), which colocalized
with Alexa633-labeled TRAIL-His on the plasma membrane
(Supplementary Fig. S1B). Upon incubation at 37jC for 5 min,
the colocalized fluorescence signal from the HW1-TR2 complexes
exhibited strong punctuated foci (Supplementary Fig. S1C).
These observations indicate that HW1 specifically binds to TR2
to form oligomeric HW1-TR2 complexes on the cell surface.
Flow cytometry analysis of Alexa488-labeled HW1 binding to
endogenous TR2 on HCT116 cells, which overexpressed TR2 on
the cell membrane following pretreatment with sulforaphane (6),
showed a much more enhanced binding activity in the
sulforaphane-treated cells compared with untreated cells, further
indicative of the specific binding activity of HW1 for TR2
(Supplementary Fig. S2A).
HW1 induces cell death in multiple TRAIL-sensitive and
TRAIL-resistant tumor cells with much less cytotoxicity on
normal cells. Tumoricidal activities of HW1 were evaluated using
multiple human cancer cells with different sensitivities to TRAIL.
These included HCT116 (colorectal carcinoma), HL-60 (monocyte
leukemia), Du145 (prostate cancer), and MCF-7 (breast cancer) for
TRAIL-sensitive cells and HepG2 (hepatoma), U87MG (glioma),
Molt-4 (T cell leukemia), and ZR-75-1 (breast cancer) for
TRAIL-resistant cells (4). The cells were incubated with
various concentrations of TRAIL-His (0.008–0.5 Ag/mL) and HW1
Figure 1. Biochemical characterizations of human scFv HW1. A, reducing
(+DTT) and nonreducing (?DTT) SDS-PAGE analyses of the purified HW1.
B, size exclusion elution profile of HW1 injected at 500 Ag/mL (f16 Amol/L)
and monitored at 280 nm. C, kinetic interactions of HW1 with TR2-ECD
determined by SPR analyses. SPR sensograms were obtained from injections of
serially diluted HW1 at 1.5, 0.75, 0.38, 0.19, and 0.09 Amol/L (from top to
bottom) over a TR2-ECD immobilized surface. D, no competition between HW1
and TRAIL-His for the binding to TR2. ELISA plates coated with TR2-ECD
were incubated with the indicated concentrations of HW1 in the absence or
presence of 20 Ag/mL of TRAIL-His. Points, relative bound fractions of HW1 to
TR2-ECD from experiments done in triplicate; bars, SD. E, specific binding
activity of HW1 for cell surface–expressed TR2. Alexa633-labeled HW1
(5 Ag/mL) was incubated for 30 min at 4jC with HCT116 cells transiently
transfected with TR2DCD-YFP (top) and TR4DCD-YFP (bottom), respectively,
and then visualized by confocal fluorescence microscopy. Nuclei were costained
with DAPI. Magnification, ?630.
Figure 2. HW1 alone induces cell death in both TRAIL-sensitive and
TRAIL-resistant tumor cells with much less cytotoxicity on normal cells. A, cell
killing activities of TRAIL-His (a and b, top) and HW1 (a and b, bottom) in
various TRAIL-sensitive (a) and TRAIL-resistant tumor cells (b). Cells were
incubated with the indicated concentrations of TRAIL-His for 8 h (a) or 30 h (b)
and HW1 for 30 h. c and d, cytotoxicity assay of non-tagged TRAIL and HW1
in normal human hepatocytes (c) and mammary epithelial cells (d), which were
incubated with the indicated concentrations of non-tagged TRAIL or HW1 for
30 h. B, inhibition of cell death induced by TRAIL-His (filled columns) or HW1
(open columns) by the soluble competitor TR2-Fc. HCT116 cells were treated
with 0.1 Ag/mL of TRAIL-His for 8 h or 5 Ag/mL of HW1 for 30 h in the presence of
the indicated concentrations of TR2-Fc. C, no effects of cross-linked HW1 on
the cell death activity in HCT116 and HepG2 cells. Cells were incubated for
30 h with the indicated concentrations of HW1, which were untreated or
cross-linked by preincubation (4jC for 1 h) with a secondary cross-linking agent
of mouse anti-His6IgG (10 Ag/mL). A to C, cell viability was determined by
MTT assay. Points, percentages of viable cells compared with untreated control
cells from experiments done at least in triplicate; bars, SD.
TR2-Mediated Autophagic Cell Death
Cancer Res 2007; 67: (15). August 1, 2007
(0.008–25 Ag/mL) for the indicated periods specified in the figure
legends. Cell viability was determined by MTT assay (6, 26). All of
the known TRAIL-sensitive cells were susceptible to both TRAIL-
His– and HW1-mediated cell deaths in a dose-dependent manner
(Fig. 2A, a). DU145 cells were most susceptible to HW1, exhibiting
an EC50 (50% effective concentration) of f100 ng/mL. As
expected, the known TRAIL-resistant cells did not show significant
cell death (<12%) by TRAIL-His treatments of up to 0.5 Ag/mL for
30 h (Fig. 2A, b). Strikingly, however, HW1 exhibited cell-killing
activity for all of these TRAIL-resistant cells in proportion to the
concentration starting from 12 h at >1 Ag/mL (Fig. 2A, b). Cells
dying because of HW1 treatment became rounded and were
detached from the culture plates. The sensitivity to HW1-induced
cell death varied among the cells, with U87MG displaying the
highest susceptibility (EC50? 1.5 Ag/mL).
Next, we determined the cytotoxicity of HW1 on normal cells
using human hepatocytes, mammary epithelial cells, and astrocytes
in comparison with non-tagged TRAIL (residues 114–281). Tagged
TRAIL with NH2-terminal 6? His exogenous sequences have
previously exhibited cytotoxicity on normal human hepatocytes
(31), whereas non-tagged TRAIL and some anti-TR2 mAbs have
not (3, 14, 15). HW1 treatment exhibited very low levels of cyto-
toxicity (V20%) at doses <5 Ag/mL for 30 h incubation in normal
human hepatocytes and mammary epithelial cells, like the non-
tagged TRAIL (Fig. 2A, c and d). Treatment with 25 Ag/mL of HW1
for 30 h induced f40% cell death in both normal human cells
(Fig. 2A, c and d). However, the cytotoxicity of HW1 for the normal
cells showed far lower levels than for the cancer cells tested under
the same conditions (Fig. 2A). For normal human astrocytes, HW1
did not show any cytotoxicity even up to 40 Ag/mL for 48 h incu-
bation (data not shown). Thus, HW1 exhibited much less cyto-
Whether HW1 induced cell death through specific binding to TR2
was assessed using a soluble competitor of TR2-Fc–conjugated
protein (TR2-ECD fused to the constant region of the immuno-
globulin) for cell surface–expressed TR2. Cell death in HCT116 cells
mediated by either HW1 or TRAIL-His was gradually inhibited with
increased concentrations of TR2-Fc (Fig. 2B). Furthermore, HW1
showed enhanced cell death activity for HCT116 cells with TR2
overexpressed by pretreatment with sulforaphane, compared with
the untreated control cells (Supplementary Fig. S2B). These results
showed that HW1 specifically transduces cell death signaling
Some bivalent TR2-specific IgG mAbs induced apoptotic cell
death as a single agent (11, 14, 15), but their respective monovalent
Fab forms could not (16, 32), indicating that the oligomerization of
TR2 is required to recruit and activate subsequent molecules
involved in cell death, like other TNFRs (2). However, monovalent
scFv HW1 induced cell death in the absence of secondary cross-
linking agents. Even cross-linking of 6? His-tagged-HW1 using
mouse anti-His6IgG did not augment the cell death activity in
HCT116 and HepG2 cells examined (Fig. 2C). In contrast, the cross-
linking of TRAIL-His under the same conditions showed signifi-
cantly enhanced killing activity in HCT116 cells (data not shown),
consistent with previous results (1, 32).
HW1 induces autophagy in both TRAIL-sensitive and TRAIL-
resistant tumor cells. The tumoricidal activity of HW1, even
for TRAIL-resistant cells, prompted us to characterize the
associated cell death morphology by transmission electron
microscopy. Compared with untreated cells exhibiting normal
nuclear and cytoplasmic morphology (Fig. 3A, top), TRAIL-sensitive
HCT116 and Du145 cells treated with TRAIL-His showed typical
characteristics of apoptotic cell deaths, such as nuclear shrinkage,
chromatin condensation, and membrane blebbing (Fig. 3A, bottom;
refs. 20, 21). However, HW1-treated cells exhibited numerous
membrane-bound vesicles occupying the major cytoplasmic space
(>90%), which frequently contained electron-dense materials of
cytoplasmic fragments and organelles, such as entire or damaged
Figure 3. HW1 induces autophagy in both
TRAIL-sensitive and TRAIL-resistant
cancer cells. A, representative
transmission electron microscopy images
of TRAIL-sensitive HCT116 and DU145
cells and TRAIL-resistant U87MG and
HepG2 cells, which were left untreated as
controls (top), or treated with either
25 Ag/mL of HW1 for 20 h (middle) or
0.2 Ag/mL of TRAIL-His for 5 h (bottom).
Autophagic vesicles (black arrows); bars,
2 Am. B, representative high-magnification
images of multiple- (left) or double
autophagosomes (white arrows), and
autophagosome induction stage
(white arrowhead) and autophagosome
fusion with autophagic vacuoles (black
arrowhead; right); bars, 0.2 Am.
C, HW1-induced LC3 accumulation in
autophagic vacuoles. Phase contrast
(left) and fluorescence images (right) of
U87MG cells stably expressing GFP-LC3,
which were left untreated (top) or treated
with 25 Ag/mL of HW1 for 24 h (bottom).
Cancer Res 2007; 67: (15). August 1, 2007
mitochondria, in both TRAIL-sensitive and TRAIL-resistant cells
(Fig. 3A, middle). These are typical autophagic vesicle structures
associated with autophagy (20, 21). Higher magnification of
images clearly revealed multiple or double membrane–bound
autophagosomes with fragmented cellular organelles and debris
inside (Fig. 3B, left and middle; refs. 19–21). Simultaneously, auto-
hagosome induction stages where membrane-bound vacuoles
surround mitochondria and autophagosome fusion with autopha-
gic vacuoles were also observed (Fig. 3B, right). The dying cells by
HW1 treatment also exhibited common characteristics of apop-
tosis and autophagy, such as shrunk nuclei, partial chromatin
condensation, and membrane blebbing (Fig. 3A, middle; refs.
Another distinctive hallmark of autophagy is the translocation of
LC3 from the cytosol to the autophagic vacuoles (27, 28). When
TRAIL-resistant U87MG cells stably expressing GFP-LC3 were incu-
bated with HW1, the accumulation of GFP-LC3 was observed both
as shown by confocal fluorescence microscopy, compared with
accumulates in acidic vesicles (20, 21), they exhibited numerous
punctuate staining patterns, compared with untreated cells exhibit-
ing only very weak diffuse staining (Supplementary Fig. S3A and B).
This supports the cytosolic accumulation of autophagic vacuoles.
To determine whether autophagy was responsible for the HW1-
induced cell death, the effect of 3-MA, an autophagy inhibitor (21),
on the HW1-induced cell death was evaluated. Pretreatment of
HCT116 and U87MG cells with 3-MA (50 Amol/L) significantly
abolished the cytosolic accumulation of autophagic vacuoles
monitored by lysotracker red staining (Supplementary Fig. S3A
and B) and completely suppressed the HW1-induced cell death at
125 Amol/L (Fig. 5B), such as the TRAIL-mediated autophagic cell
death of epithelial cancer cells (19). Furthermore, long-term
clonogenic assays showed that inhibiting the HW1-induced
autophagic cell death of HCT116 cells by 3-MA rescued the
colony-forming ability of the cells (Supplementary Fig. S4).
Several autophagy-related (Atg) genes, including Beclin-1 and
Atg7, have been known to be associated with the autophagic
pathway (20–22). To determine whether Beclin-1 and/or Atg7
were involved in HW1-induced cell death, we reduced the
expression of Beclin-1 and Atg7 by RNA interference in TRAIL-
sensitive HCT116 and TRAIL-resistant U87MG cells before HW1
treatment (Fig. 4). The significantly suppressed expression of the
two respective genes by siRNA caused a dramatic inhibition of
the HW1-induced cell death in both HCT116 and U87MG cells,
but exhibited negligible effects on TRAIL-induced cytotoxicity in
HCT116 cells, compared with cells untreated or treated with the
unrelated siRNA (Fig. 4). This result suggested that Atg7 and
Beclin-1 are closely associated with the HW1-induced autophagic
cell death, consistent with typical autophagic cell death (20–22,
33). Taken together, we concluded that HW1 induces autophagic
cell death not only in TRAIL-sensitive cancer cells, but also in
TRAIL-resistant cancer cells.
HW1-induced autophagic cell death occurs mainly through
the JNK pathway. To elucidate the mechanisms underlying HW1-
induced cell death, activation of some participants involved in cell
death were monitored by Western blotting in TRAIL-sensitive
HCT116 and TRAIL-resistant U87MG cells. Caspase-3, caspase-8,
and PARP were significantly cleaved from their pro-forms in
HCT116 cells treated with TRAIL-His for 2 h, but was very slightly
cleaved in both HCT116 and U87MG cells treated with HW1 for the
various time periods (Fig. 5A). Instead, compared with TRAIL-His–
treated or untreated control cells, HW1-treated cells exhibited
significant activations of both JNK and p38 by at least 3-fold
starting from 12 h, which closely paralleled the time course of cell
death in both cells (Fig. 5A). We then examined further whether
caspases, JNK, or p38 plays a critical role in HW1-induced
autophagic cell death, using their specific inhibitors. The pan-
caspase inhibitor Z-VAD (10 Amol/L) completely blocked the
cleavage of PARP by caspase-3 and cell death in HCT116 cells
treated with TRAIL-His (Fig. 5B–D). However, for HW1-treated
HCT116 and U87MG cells, Z-VAD did not attenuate JNK
activations at all and only slightly reduced the cell death
(Fig. 5B–D), indicating that HW1-mediated JNK activation and
cell death occur independently of caspase activations. The p38
inhibitor SB203580 (10 Amol/L) efficiently inhibited HW1-mediated
p38 activation, but not JNK activation, resulting in only a slight
reduction in the HW1-induced cell death (Fig. 5B and D). Hence,
even though p38 was significantly activated in HW1-treated cells
(Fig. 5A), its activation was not essential for HW1-induced cell
death. Strikingly, the JNK inhibitor SP600125 (10 Amol/L), which
Figure 4. Inhibition of the HW1-mediated autophagic cell death by siRNAs
against Beclin-1 (A) and Atg7 (B) in TRAIL-sensitive HCT116 cells (left) and
TRAIL-resistant U87MG cells (right). After transfection with Beclin-1, Atg7, or
unrelated (con) siRNA, the cells were cultured for 48 h and then subjected to
Western blotting using anti–Beclin-1 or anti-Atg7 antibodies. Cell viability was
determined by MTT assay for siRNA-transfected cells cultured for 24 h and then
further incubated with 200 ng/mL of TRAIL-His for 2 h or 25 Ag/mL of HW1
for 30 h. Untransfected cells (Control) were also treated with TRAIL or HW1
under the same conditions. Columns, percentages of viable cells compared with
untreated control cells from experiments done in triplicate; bars, SD.
TR2-Mediated Autophagic Cell Death
Cancer Res 2007; 67: (15). August 1, 2007
efficiently inhibited JNK activations, completely suppressed HW1-
mediated cell death in both TRAIL-sensitive and TRAIL-resistant
cells (Fig. 5B and D), indicating an essential requirement of JNK
activation in HW1-mediated cell death. Furthermore, blocking the
cell death of HCT116 cells induced by HW1 with the JNK inhibitor
SP600125 efficiently rescued the colony-forming capability of the
cells (Supplementary Fig. S4). 3-MA (50 Amol/L) did not inhibit
JNK activations, but completely blocked the HW1-stimulated cell
death at 125 Amol/L (Fig. 5B and D), suggesting that it attenuates
autophagic cell death by operating downstream of JNK activation.
The HCT116 and U87MG cells dying because of HW1 in the
presence of Z-VAD or SB203580, but not with SP600125 or 3-MA,
exhibited numerous punctuate lysotracker red stainings (Supple-
mentary Fig. S3), indicative of the accumulation of autophagic
vacuoles, and thus, autophagic cell death. The inhibitors against
JNK, p38, and autophagy (3-MA) did not affect the TRAIL-induced
cell death at all in HCT116 cells (Fig. 5B), whereas Z-VAD almost
blocked it (Fig. 5B and C). Taken together, the results showed that
the specific inhibitors of JNK and autophagy (3-MA), but not of
caspases and p38, effectively protected both TRAIL-sensitive and
TRAIL-resistant tumor cells from the HW1-induced autophagic cell
death, consistent with the Z-VAD–mediated autophagic cell death
of L929 fibroblastic cells (22).
To further understand the role of JNK in HW1-induced cell
death, we transiently transfected HCT116 and U87MG cells with
wild-type JNK2 (wtJNK2) or dominant-negative JNK2 mutant
(dnJNK2) to modulate JNK activity. The expression of these two
respective proteins in both cells was confirmed by Western
blotting using anti-JNK antibody (Supplementary Fig. S5). The
ectopic expression of dnJNK2 significantly inhibited the HW1-
induced cell death in both cells, whereas that of wtJNK2 showed
the opposite effect of slightly enhancing the HW1-mediated cell
death, compared with control cells transfected with an empty
vector and then treated with HW1 (Supplementary Fig. S5). This
result, together with that of the JNK inhibitor, indicated that JNK
activation is required for the HW1-induced autophagic cell death.
Figure 5. HW1-induced autophagic cell death occurs predominantly via JNK activation in a caspase-independent manner in both TRAIL-sensitive HCT116 and
TRAIL-resistant U87MG cells. A, HW1 treatment slightly activates caspases, but significantly activates JNK and p38 in both HCT116 and U87MG cells. Cells were
left untreated (Control), or treated with either 0.2 Ag/mL of TRAIL-His for 2 h or with 25 Ag/mL of HW1 for the indicated periods, and then whole cell lysates were
prepared and subjected to Western blotting. B, effects of various inhibitors on the cell death of HCT116 and U87MG cells treated with either HW1 or TRAIL-His.
Cells pretreated for 1 h with Z-VAD (10 Amol/L), SB203580 (10 Amol/L), SP600125 (10 Amol/L), or 3-MA (50 and 125 Amol/L) were further incubated with either
0.2 Ag/mL of TRAIL-His in HCT116 for 5 h and U87MG cells for 30 h or 25 Ag/mL of HW1 in both cells for 30 h. C, no significant effects of Z-VAD on the cell
death of HCT116 cells and U87MG cells induced by varying concentrations of HW1. Cells pretreated with 10 Amol/L of Z-VAD for 1 h were further incubated with
the indicated concentrations of TRAIL-His (8 h) or HW1 (30 h) in HCT116 cells and HW1 (30 h) in U87MG cells. Cell viability was determined by MTT assay;
percentages of viable cells compared with untreated control cells (columns, B; and points, C) from experiments done at least in triplicate; bars, SD. D, effects of various
inhibitors on the activations of JNK and p38, and cleavage of PARP in HCT116 treated with TRAIL or HW1 and U87MG cells treated with HW1. Cells pretreated
for 1 h with either Z-VAD (10 Amol/L), SB203580 (10 Amol/L), SP600125 (10 Amol/L), or 3-MA (50 Amol/L) were further incubated with 25 Ag/mL of HW1 for 30 h or
0.2 Ag/mL of TRAIL-His for 5 h. A and D, the phosphorylated forms of JNK (P-JNK) and p38 (P-p38), and pro-forms (black arrowhead) and cleaved forms (white
arrowhead) of caspase-8, caspase-3, and PARP. The h-actin protein levels were included as a control for protein loading.
Cancer Res 2007; 67: (15). August 1, 2007
TRADD and TRAF2, but not FADD, caspase-8, or RIP, are
recruited to the HW1-TR2 signaling complex. To elucidate the
molecular components involved in HW1 signaling through TR2,
we analyzed DISC associated with HW1-TR2 complex by
immunoprecipitation after Flag-tagged HW1 treatment of
HCT116 and U87MG cells, compared with Flag-tagged TRAIL
treatment (Fig. 6). The TR2-associated DISC was coimmunopre-
cipitated from the cell lysates using anti-Flag antibody and
analyzed by Western blotting. TRAIL stimulated a DISC assembly
containing TR2, FADD, and caspase-8, but not RIP, TRADD, or
TRAF2 in HCT116 cells, consistent with the previous results (29).
However, HW1 induced a DISC composed of TR2, TRADD, and
TRAF2, whereas FADD, caspase-8, and RIP were not detected in
the TR2-associated complex in both HCT116 and U87MG cells
(Fig. 6). This result suggested that HW1 binding may lead to the
recruitment of TRADD into the intracellular death domain of
TR2, and subsequent association of TRADD with TRAF2, which
seems to trigger subsequent signaling for the JNK activation (34).
Taken together, our data suggested that HW1 triggers autophagic
cell death by forming a distinct DISC from that of TRAIL.
TRAIL or agonistic anti-TR2 mAbs induce apoptotic cell death
in various cancer cells, but was not sufficient as a single agent to
kill TRAIL-resistant tumor cells, which can be sensitized to cell
death by combined therapy with chemotherapeutics (6–12). We
showed here that HW1 alone can induce autophagic cell death in
both TRAIL-sensitive and TRAIL-resistant cancer cells in vitro
with much less cytotoxicity to normal cells. The tumoricidal
activity of TR2-specific HW1 was blocked by the soluble TR2-Fc
and augmented by TR2 overexpression on the cell surface,
suggesting that TR2-mediated extrinsic signaling is sufficient to
kill various tumors, including TRAIL-resistant cells, through the
distinct cell death pathway of autophagy from apoptosis. This is
indirectly supported by the TRAIL-mediated autophagic cell
death of normal epithelial cells (18, 19). Similar to typical
autophagy (20–22), two key autophagy genes, Beclin-1 and Atg7,
seem to be involved in HW1-induced autophagic cell death.
Although TRAIL induced apoptotic cell death in TRAIL-sensitive
cells via the caspase-dependent pathway, HW1 triggered auto-
phagic cell death in both TRAIL-sensitive and TRAIL-resistant
tumors predominantly through the JNK pathway in a caspase-
independent manner. Caspase-independent JNK activation has
been reported to contribute to autophagic cell death induced by
TNF-a (33) and chemicals (22, 35), which, together with our
results, indicates a molecular link between JNK activation and
autophagic cell death. DISC analysis revealed that TRADD and
TRAF2 were recruited to the HW1-TR2 complex, but not FADD
and caspase-8, which are essential for TRAIL-induced apoptotic
cell death. Thus, it is most likely that TRAF2 mediated HW1-
induced JNK activation, eventually triggering autophagy and
subsequent cell death (22, 33). Even though the detailed
mechanism of how HW1-triggered JNK activation mediates
autophagic cell death is yet to be determined, our study suggests
that induction of autophagic cell death through the JNK pathway
might be one of the strategies to overcome TRAIL-resistance of
In contrast to previous reports showing that at least the
bivalency of TR2-specific mAbs is required to trigger cell death (12,
14, 16, 32), monovalent HW1 alone induced cell death without
enhanced tumoricidal activity through cross-linking, raising the
question of how it directly activates TR2 to transmit the cell death
signal. Similar to TNFRs (2), preligand assembled oligomeric
complexes of TR2 through lateral interactions of TR2-ECD have
been observed on the cell surface (26, 30). The observed punctuate
colocalization signals upon HW1 binding to TR2 (Fig. 1E;
Supplementary Fig. S1C) indicates the formation of oligomeric
HW1-TR2 complexes on the cell membrane. It is not clear,
however, whether HW1 binds to preassembled homomeric TR2
complexes or to monomeric TR2, and then immediately induces
TR2 clustering. The latter might be feasible due to the different
binding sites of HW1 from those of TRAIL on TR2. In either case,
DISC analysis indicated that HW1 binding to TR2 may induce
different conformations of the TR2 intracellular death domain,
which recruits distinct adaptor molecules of TRADD and TRAF2
from those induced by TRAIL. This distinct DISC assembly seems
to explain the differences in the eventual outcome between HW1
and TRAIL upon binding to the same target TR2. For example, in
contrast with HW1-induced autophagic cell death, TRAIL-resistant
U87MG cells were sensitized to apoptotic cell death by the
combination of TRAIL and the chemotherapeutic agent rottlerin,
which was mediated by caspase activations without any activa-
tions of JNK and p38 (9).
The specific role of autophagy in programmed cell death,
whether it is a survival mechanism or another cell death pathway,
has been controversial. However, accumulating evidence suggests
that autophagy represents another cell death pathway in certain
circumstances, which occurs in the absence or presence of
apoptosis (20–23). Most TRAIL-resistant cancer cells have apopto-
tic defects. For example, antiapoptotic molecules of FLICE-like
inhibitory proteins and Bcl-2 family are overexpressed in TRAIL-
resistant cells (5). Thus, similar to HW1-induced autophagic cell
death, targeting nonapoptotic cell death might be an alternative
strategy to apoptotic cancer therapy for various cancers, particu-
larly for TRAIL-resistant tumors (21, 23). Although we need to
further characterize the tumoricidal activity of HW1 in vivo, our
study shows that TR2-specific HW1 can induce autophagic cell
death in various cancer cell types, including TRAIL-resistant
Figure 6. HW1 induces a different DISC from that of TRAIL in TRAIL-sensitive
HCT116 (left) and TRAIL-resistant U87MG (right) cells. Cells were treated
with 2 Ag/mL of Flag-tagged TRAIL for 30 min (only for HCT116 cells) or
25 Ag/mL of Flag-tagged HW1 for 30 or 360 min, and lysed. The TR2-associated
DISC was coimmunoprecipitated from the lysates using anti-Flag antibody
and analyzed by Western blotting for the presence of TR2, FADD, caspase-8,
TRADD, RIP, and/or TRAF2. The addition of beads alone to unstimulated
cell lysates (Control) was used to control for nonspecific interactions.
TR2-Mediated Autophagic Cell Death
Cancer Res 2007; 67: (15). August 1, 2007
tumors, providing a novel strategy of cancer therapy through
nonapoptotic cell death.
Received 12/27/2006; revised 5/18/2007; accepted 5/31/2007.
Grant support: National R&D Program for Cancer Control, Ministry of Health and
Welfare 0520110-1 (Y-S. Kim); Korea Research Foundation 205-2004-D00068 (Y-S. Kim)
and KRF-2005-204-E00034 (M-H. Kwon); and the ‘‘GRRC’’ Project of Gyeonggi
Provincial Government (M-H. Kwon and Y-S. Kim), Republic of Korea.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Profs. Francis K-M. Chan (University of Massachusetts Medical School,
Department of Pathology, Worcester, MA) and T. Yoshimori (Osaka University,
Department of Cell Regulation, Suita, Osaka, Japan) for providing us with T010 and
T30 plasmids and GFP-LC3 plasmid, respectively.
Cancer Res 2007; 67: (15). August 1, 2007
1. Walczak H, Miller RE, Ariail K, et al. Tumoricidal
activity of tumor necrosis factor-related apoptosis-
inducing ligand in vivo. Nat Med 1999;5:157–63.
2. Ashkenazi A. Targeting death and decoy receptors of
the tumour-necrosis factor superfamily. Nat Rev Cancer
3. Ashkenazi A, Pai RC, Fong S, et al. Safety and
antitumor activity of recombinant soluble Apo2 ligand.
J Clin Invest 1999;104:155–62.
4. Di Pietro R, Zauli G. Emerging non-apoptotic
functions of tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL)/Apo2L. J Cell Physiol 2004;201:
5. Zhang L, Fang B. Mechanisms of resistance to TRAIL-
induced apoptosis in cancer. Cancer Gene Ther 2005;12:
6. Kim H, Kim EH, Eom YW, et al. Sulforaphane sensi-
tizes tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL)-resistant hepatoma cells to TRAIL-
induced apoptosis through reactive oxygen species-
mediated up-regulation of DR5. Cancer Res 2006;66:
7. Muhlenbeck F, Haas E, Schwenzer R, et al. TRAIL/
Apo2L activates c-Jun NH2-terminal kinase (JNK) via
caspase-dependent and caspase-independent pathways.
J Biol Chem 1998;273:33091–8.
8. Sah NK, Munshi A, Kurland JF, McDonnell TJ, Su B,
Meyn RE. Translation inhibitors sensitize prostate
cancer cells to apoptosis induced by tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) by
activating c-Jun N-terminal kinase. J Biol Chem 2003;
9. Kim EH, Kim SU, Choi KS. Rottlerin sensitizes glioma
cells to TRAIL-induced apoptosis by inhibition of Cdc2
and the subsequent downregulation of survivin and
XIAP. Oncogene 2005;24:838–49.
10. Martin S, Phillips DC, Szekely-Szucs K, Elghazi L,
Desmots F, Houghton JA. Cyclooxygenase-2 inhibition
sensitizes human colon carcinoma cells to TRAIL-
induced apoptosis through clustering of DR5 and
concentrating death-inducing signaling complex com-
ponents into ceramide-enriched caveolae. Cancer Res
11. Griffith TS, Rauch CT, Smolak PJ, et al. Functional
analysis of TRAIL receptors using monoclonal anti-
bodies. J Immunol 1999;162:2597–605.
12. Georgakis GV, Li Y, Humphreys R, et al. Activity of
selective fully human agonistic antibodies to the
TRAIL death receptors TRAIL-R1 and TRAIL-R2 in
primary and cultured lymphoma cells: induction of
apoptosis and enhancement of doxorubicin- and
bortezomib-induced cell death. Br J Haematol 2005;
13. Pukac L, Kanakaraj P, Humphreys R, et al. HGS-ETR1,
a fully human TRAIL-receptor 1 monoclonal antibody,
induces cell death in multiple tumour types in vitro and
in vivo. Br J Cancer 2005;92:1430–41.
14. Guo Y, Chen C, Zheng Y, et al. A novel anti-human
DR5 monoclonal antibody with tumoricidal activity
induces caspase-dependent and caspase-independent
cell death. J Biol Chem 2005;280:41940–52.
15. Ichikawa K, Liu W, Zhao L, et al. Tumoricidal activity
of a novel anti-human DR5 monoclonal antibody
without hepatocyte cytotoxicity. Nat Med 2001;7:954–60.
16. Motoki K, Mori E, Matsumoto A, et al. Enhanced
apoptosis and tumor regression induced by a direct
agonist antibody to tumor necrosis factor-related
apoptosis-inducing ligand receptor 2. Clin Cancer Res
17. Lin Y, Devin A, Cook A, et al. The death domain
kinase RIP is essential for TRAIL (Apo2L)-induced
activation of InB kinase and c-Jun N-terminal kinase.
Mol Cell Biol 2000;20:6638–45.
18. Thorburn J, Moore F, Rao A, et al. Selective
inactivation of a Fas-associated death domain protein
(FADD)-dependent apoptosis and autophagy pathway
in immortal epithelial cells. Mol Biol Cell 2005;16:
19. Mills KR, Reginato M, Debnath J, Queenan B,
Brugge JS. Tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL) is required for induction of
autophagy during lumen formation in vitro. Proc Natl
Acad Sci U S A 2004;101:3438–43.
20. Kroemer G, Jaattela M. Lysosomes and autophagy in
cell death control. Nat Rev Cancer 2005;5:886–97.
21. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of
autophagy in cancer development and response to
therapy. Nat Rev Cancer 2005;5:726–34.
22. Yu L, Alva A, Su H, et al. Regulation of an ATG7-
beclin 1 program of autophagic cell death by caspase-8.
23. Tsujimoto Y, Shimizu S. Another way to die:
autophagic programmed cell death. Cell Death Differ
2005;12 Suppl 2:1528–34.
24. Lee HW, Lee SH, Park KJ, Kim JS, Kwon MH, Kim YS.
Construction and characterization of a pseudo-immune
human antibody library using yeast surface display.
Biochem Biophys Res Commun 2006;346:896–903.
25. Kim YR, Kim JS, Lee SH, et al. Heavy and light chain
variable single domains of an anti-DNA binding
antibody hydrolyze both double- and single-stranded
DNAs without sequence specificity. J Biol Chem 2006;
26. Lee HW, Lee SH, Lee HW, Ryu YW, Kwon MH, Kim YS.
Homomeric and heteromeric interactions of the extra-
cellular domains of death receptors and death decoy
receptors. Biochem Biophys Res Commun 2005;330:
27. Kabeya Y, Mizushima N, Yamamoto A, Oshitani-
Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and
GATE16 localize to autophagosomal membrane
depending on form-II formation. J Cell Sci 2004;117:
28. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a
mammalian homologue of yeast Apg8p, is localized in
autophagosome membranes after processing. EMBO
29. Varfolomeev E, Maecker H, Sharp D, et al. Molecular
determinants of kinase pathway activation by Apo2
ligand/tumor necrosis factor-related apoptosis-inducing
ligand. J Biol Chem 2005;280:40599–608.
30. Clancy L, Mruk K, Archer K, et al. Preligand assembly
domain-mediated ligand-independent association be-
tween TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-
induced apoptosis. Proc Natl Acad Sci U S A 2005;102:
31. Jo M, Kim TH, Seol DW, et al. Apoptosis induced
in normal human hepatocytes by tumor necrosis
factor-related apoptosis-inducing ligand. Nat Med
32. Wajant H, Moosmayer D, Wuest T, et al. Differential
activation of TRAIL-R1 and -2 by soluble and membrane
TRAIL allows selective surface antigen-directed activa-
tion of TRAIL-R2 by a soluble TRAIL derivative.
33. Jia G, Cheng G, Gangahar DM, Agrawal DK. Insulin-
like growth factor-1 and TNF-a regulate autophagy
through c-jun N-terminal kinase and Akt pathways in
human atherosclerotic vascular smooth cells. Immunol
Cell Biol 2006;84:448–54.
34. Jin Z, El-Deiry WS. Distinct signaling pathways in
TRAIL- versus tumor necrosis factor-induced apoptosis.
Mol Cell Biol 2006;26:8136–48.
35. Borsello T, Croquelois K, Hornung JP, Clarke PG.
N-methyl-D-aspartate-triggered neuronal death in orga-
notypic hippocampal cultures is endocytic, autophagic
and mediated by the c-Jun N-terminal kinase pathway.
Eur J Neurosci 2003;18:473–85.
A human scFv antibody against TRAIL receptor 2 induces autophagic cell death in both
TRAIL-sensitive and –resistant cancer cells
Kyung-Jin Park1,4, Seung-Hyun Lee1,4, Tae-In Kim2,4, Hae-Won Lee1, Chang-Han Lee1, Eun-Hee
Kim1,3, Ji-Young Jang2, Kyeong Sook Choi1,3, Myung-Hee Kwon2, and Yong-Sung Kim1
1Dept. of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu,
Suwon 443-749, Korea; 2Dept. of Microbiology and 3Institute for Medical Sciences, Ajou University
School of Medicine, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea.
Legends for Supplementary Figures.
Supplementary Figures S1, S2, S3, S4, and S5.
Protein expression and purification. The previously isolated human scFv HW1 (281 amino acids,
30.9 kDa) against the extracellular domain of TR2 (TR2-ECD) from a yeast surface-displayed pseudo-
immune scFv library (1) was subcloned into a bacterial expression plasmid resulting in a pKJ1-HW1
plasmid. The plasmid encodes N-terminal PelB signal peptide for targeting protein expression to the
periplasm under the control of T7 promoter and C-terminal FLAG and 6×His tags. The constructs
were confirmed by sequencing and transformed into E. coli BL21 (DE3) pLysE cells (Novagen).
The detailed expression and purification procedures of HW1, TRAIL-His (residues 114-281 with a C-
terminal 6×His-tag), TRAIL-Flag (residues 114-281 with a C-terminal Flag tag), TR2-ECD-His, TR1-
ECD-His, TR3-ECD-His, and TR4-ECD-His were performed essentially as described before (2, 3).
The purified proteins were sterilized by filtration using a cellulose acetate membrane filter (0.2 µm)
(Nalgene Co.) before usages in cell assays. The protein concentrations were determined using the
Bio-Rad protein assay.
Competitive ELISA. ELISA plates (Nunc, Invitrogen Ltd.) were coated with 10 μg/mL of TR2-
ECD overnight at 4oC and blocked with 2% (w/v) bovine serum albumin (Sigma). After incubations
with varying concentrations of HW1 (0.001-30 µg/mL) in the absence or presence of 20 µg/mL
TRAIL-His, the bound HW1 was detected with anti-Flag M2 mouse mAb (Sigma) and alkaline
phosphatase-conjugated goat anti-mouse mAb (Sigma). Absorbance was read at 405 nm in a
VersaMax microplate reader (Molecular devices, Crawley, UK).
Size exclusion chromatography. Size exclusion chromatography analysis of HW1 was performed,
essentially as described before (2). The injection concentration ranged between 5 and 20 µmol/L of
proteins in a volume of 20 µL. A set of molecular weight standard marker (Sigma) ranging from
13.7 to 66 kDa was used.
Surface plasmon resonance (SPR). Kinetic measurements of HW1 interactions with the antigens,
TR2-ECD, TR1-ECD, TR3-ECD, TR4-ECD, CD95, TNFR1 and TNFR2, were performed at 25oC
using a Biacore 2000 SPR biosensor (Pharmacia, Sweden), as described before (2, 3). After
immobilization of the antigens onto the carboxymethylated dextran surface of a CM5 sensor chip at a
level of about 1000 response units, 30 μL of HW1 (0.05-4 μmol/L) in PBS (137 mmol/L NaCl, 2.7
mmol/L KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4, pH 7.4) was injected into the flow cell at a
flow rate of 30 μL/min. The dissociation and association rate constants, and the dissociation
equilibrium constant (KD) values, were determined by the 1:1 binding model using the BIAevaluation
software provided by the manufacturer.
Flow cytometric analysis of HW1 binding to cell surface-expressed TR2. For the flow cytometry
analysis of HW1 binding to endogenously expressed TR2, HCT116 cells were left untreated or
pretreated with 10 µmol/L sulforaphane for 1 hour to overexpress TR2 on the cell surface (4) and then
labeled with Alexa488-labeled-HW1 (5 µg/mL) for 30 min at 4oC. After washing (3×) with 1 mL
ice-cold PBS, cells were analyzed using a Becton Dickinson FACSCaliburTM. Goat anti-human TR2
(R&D Systems, 1:400) and then FITC-conjugated anti-goat antibody (Pierce, 1:500) were used as a
positive control for TR2 binding. An irrelevant PE-conjugated anti-goat Ig (Pierce, 1:500) was used
as a negative control.
Clonogenic assay. Clonogenic assay was performed according to the procedure described by
Sinicrope et al. (5). HCT 116 cells were seeded at a concentration of 2 × 104 cells/ml in a 24-well
plate and grown for 24 hours. The cells were pretreated with 3-MA (125 µmol/L), SP600125 (10
µmol/L), or Z-VAD (10 µmol/L) for 1 hour and then incubated with medium alone, HW1 (25 µg/mL)
for 30 hours, TRAIL (200 ng/mL) for 90 min. After washing away all the agents, a known number
of cells were re-seeded in 6-well plate and incubate for 7 days to allow colony development. Plates
were stained with crystal violet containing 2% ethanol and taken photography. The colony numbers
in each well were also counted in the whole area of each well.
Transient transfection of wtJNK2 and dnJNK2. The plasmids of pcDNA3-JNK2 and pSR2-
dnJNK2 encoding wild type JNK2 (wtJNK2) and dominant negative JNK2 (dnJNK2) (6), respectively,
was kindly provided by Prof Choi KY (Yonsei University, Korea). Transient transfections with an
empty plasmid of pcDNA3.1 (Invitrogen), pcDNA3-JNK2, or pSR2-dnJNK2 were carried out using
Lipofectamine 2000 reagent (Invitrogen) in HCT116 and U87MG cells, according to the
manufacturer’s instructions. Twenty-four hour posttransfection, the cells were subjected to Western
blotting using anti-JNK antibody or further incubated with HW1 (25 µg/mL) for 30 hours prior to
1. Lee HW, Lee SH, Park KJ, Kim JS, Kwon MH, Kim YS. Construction and characterization
of a pseudo-immune human antibody library using yeast surface display. Biochem Biophys Res
2. Kim YR, Kim JS, Lee SH, et al. Heavy and light chain variable single domains of an anti-
DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence
specificity. J Biol Chem 2006;281:15287-95.
3. Lee HW, Lee SH, Lee HW, Ryu YW, Kwon MH, Kim YS. Homomeric and heteromeric
interactions of the extracellular domains of death receptors and death decoy receptors. Biochem
Biophys Res Commun 2005;330:1205-12.
4. Kim H, Kim EH, Eom YW, et al. Sulforaphane sensitizes tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through
reactive oxygen species-mediated up-regulation of DR5. Cancer Res 2006;66:1740-50.
5. Sinicrope FA, Penington RC, Tang XM. Tumor necrosis factor-related apoptosis-inducing
ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA
14-1, in human colon cancer cells. Clin Cancer Res 2004;10:8284-92.
6. Kim TI, Jin SH, Kang EH, Shin SK, Choi KY, Kim WH. The role of mitogen-activated
protein kinases and their relationship with NF-kappaB and PPARgamma in indomethacin-Induced
apoptosis of colon cancer cells. Ann N Y Acad Sci 2002;973:241-5.
Legends for Supplementary Figures
Supplementary Figure S1. Specific binding activity of HW1 for TR2. A, SPR determination of
the binding activity of HW1, injected at 125 µg/mL (~4 µmol/L), for the death receptors of TR1-ECD,
TR3-ECD, TR4-ECD, TNFR1, TNFR2, and CD95. HW1 at 4 µmol/L exhibited very weak binding
to TR4-ECD and TR1-ECD. However, in the concentration ranges of 0.1-1.5 µmol/L, HW1 bound
to TR2 (Fig. 1C), but did not show any binding activities for the other death receptors (data not
shown), suggesting that HW1 does not have significant cross-reactivity in those concentrations. The
highest concentration of HW1 used in the cell death assay was 25 µg/mL (~0.81 µmol/L). B, the
binding activity of TRAIL-His to cell-surface expressed TR2 and TR4. Alexa633-labeled TRAIL-
His (5 µg/mL) was incubated for 30 min at 4oC with HCT116 cells transiently transfected with
TR2 ΔCD-YFP (top panel) and TR4 ΔCD-YFP (bottom panel), respectively, and then visualized by
confocal fluorescence microscopy. Nuclei were costained with DAPI. C, oligomeric complexes of
HW1-TR2 observed on the plasma membrane. After staining of HCT116 cells expressing TR2ΔCD-
YFP with Alexa633-labeled HW1 (5 µg/mL) at 4oC, samples were incubated at 37oC for 5 min and
then visualized by confocal fluorescence microscopy. In B and C, Magnification, 630×.
Supplementary Figure S2. Enhanced binding and cell death-inducing activities of HW1 in HCT116
cells overexpressing TR2. A, specific binding activity of HW1 for TR2 overexpressed in HCT116
cells by pretreatment with sulforaphane, monitored by flow cytometry. Binding of TR2-specific
mAb (top panel) as a positive control or HW1 (bottom panel) to endogenously expressed TR2 on the
cell surface of HCT116 cells. HCT116 cells were left untreated or pretreated with 10 μmol/L
sulforaphane for 1 hour and then labeled with either goat anti-human TR2 followed by FITC-
conjugated anti-goat antibody or Alexa488-labeled HW1 (5 µg/mL). B, enhanced cell death-
inducing activity of HW1 for HCT116 cells overexpressing TR2 by pretreatment with sulforaphane.
Cells were left untreated or pretreated with 10 µmol/L sulforaphane for 1 hour to up-regulate cell
surface expression of TR2 and then incubated with the indicated concentrations of TRAIL-His for 5
hours or HW1 for 30 hours.
Supplementary Figure S3. Accumulation of lysotracker-red in autophagic vacuoles in TRAIL-
sensitive HCT116 (A) and TRAIL-resistant U87MG cells (B), which were treated with HW1 in the
absence or presence of various inhibitors. Cells were pretreated for 1 hour with either Z-VAD (10
µmol/L), SB203580 (10 µmol/L), SP600125 (10 µmol/L), or 3-MA (50 µmol/L) and then further
incubated with 25 µg/mL HW1 for 20 hours. After the cells were stained with 500 nmol/L
lysotracker-red DND-99 (Molecular probes) for 30 min at 37oC, images were obtained by confocal
fluorescence microscopy. Nuclei were costained with DAPI. Magnification, 630×. All
experiments were repeated at least twice with essentially same results showing the representative
Supplementary Figure S4. Blocking the cell death of HCT116 cells induced by TRAIL with Z-
VAD (A, B) or by HW1 with either 3-MA or SP600125 (C, D), respectively, efficiently rescued the
colony forming capability of the cells. HCT 116 cells were pretreated with 3-MA (125 µmol/L),
SP600125 (10 µmol/L), or Z-VAD (10 µmol/L) for 1 hour and then incubated with medium alone
(control), HW1 (25 µg/mL) for 30 hours, or TRAIL-His (200 ng/mL) for 90 min. After washing
away all the agents, cells were cultured for 7 days to allow colony development. Representative plate
images stained with crystal were shown (A, C). The colony numbers in each well were also counted
in the whole area of each well (B, D). Errors bars indicate the standard deviations for at least
Supplementary Figure S5. Effect of the ectopic expression of wtJNK2 and dnJNK2 on the HW1-
induced cell death in TRAIL-sensitive HCT116 (A) and TRAIL-resistant U87MG (B) cells. Twenty-
four hour posttransfection, the cells were subjected to Western blotting using anti-JNK antibody for
the analyses of expression of wtJNK2 and dnJNK2 or further incubated with 25 µg/mL HW1 for 30
hours prior to MTT assay. Transfected cells with the empty vector of pcDNA3.1 were also left
untreated or treated with HW1 under the same conditions. Cell viability was presented as percentage
of viable cells compared with HW1-untreated, pcDNA3.1-transfected cells.
Page 20 Download full-text