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Caspase-mediated Cleavage of

-Catenin Precedes
Drug-induced Apoptosis in Resistant Cancer Cells
*
□
S
Received for publication, January 13, 2009, and in revised form, March 16, 2009 Published, JBC Papers in Press, March 16, 2009, DOI 10.1074/jbc.M900248200
Subramanian Senthivinayagam
‡
, Prajna Mishra
‡
, Suresh Kanna Paramasivam
‡
, Srinivas Yallapragada
§
,
Malay Chatterjee
¶
, Lucas Wong
储
, Ajay Rana**
‡‡
, and Basabi Rana
‡
**
‡‡1
From the
‡
Department of Medicine, Division of Gastroenterology, Hepatology & Nutrition, and the **Department of Pharmacology
and Experimental Therapeutics, Loyola University, Chicago, Maywood, Illinois 60153, the
‡‡
Hines Veterans Affairs Medical Center,
Hines, Illinois 60141, the
§
Department of Internal Medicine, The Texas A&M University System-Health Science Center, College of
Medicine, Temple, Texas 76504, the
¶
Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University,
Kolkata, India 700032, and the
储
Division of Hematology/Oncology, Scott & White Hospital, Temple, Texas 76504
A delicate balance between cell death and survival pathways
maintains normal physiology, which is altered in many cancers,
shifting the balance toward increased survival. Several studies have
established a close connection between the Wnt/

-catenin path-
way and tumorigenesis, aberrant activation of which might con-
tribute toward increased cancer cell growth and survival. Extensive
research is underway to identify therapeutic agents that can induce
apoptosis specifically in cancer cells with minimal collateral dam-
age to normal cells. Although tumor necrosis factor-related apo-
ptosis-inducing ligand (TRAIL) can induce apoptosis specifically in
tumor cells, many cancer cells develop resistance, which can be
overcome by combinatorial treatment with other agents: for exam-
ple, peroxisome proliferator-activated receptor
␥
(PPAR
␥
) ligands.
To identify the molecular target mediating combinatorial drug-
induced apoptosis, we focused on

-catenin, a protein implicated
in oncogenesis. Our results show that co-treatment of TRAIL-re-
sistant cancer cells with TRAIL and the PPAR
␥
ligand troglitazone
leads to a reduction of

-catenin expression, coinciding with max-
imal apoptosis. Modulation of

-catenin levels via ectopic over-
expression or small interference RNA-mediated gene silencing
modulates drug-induced apoptosis, indicating involvement of

-catenin in regulating this pathway. More in-depth studies
indicated a post-translational mechanism, independent of gly-
cogen synthase kinase-3

activity regulating

-catenin expres-
sion following combinatorial drug treatment. Furthermore,
TRAIL- and troglitazone-induced apoptosis was preceded by a
cleavage of

-catenin, which was complete in a fully apoptotic
population, and was mediated by caspases-3 and -8. These
results demonstrate

-catenin as a promising new target of
drug-induced apoptosis, which can be targeted to sensitize apo-
ptosis-resistant cancer cells.
Apoptosis is a form of cell death that permits the removal of
damaged, senescent, or unwanted cells in multicellular orga-
nisms, without damage to the cellular microenvironment.
Alterations of cellular machinery that lead to inactivation or
evasion of apoptosis represents a major causative factor in the
development and progression of cancer. Therapeutic
approaches that can restore cancer cell apoptosis are expected
to provide an effective means of treating various forms of can-
cer. Induction of cancer cell apoptosis via TNF
2
-related apo-
ptosis-inducing ligand (TRAIL/apo2L) is an attractive novel
form of cancer therapy, because TRAIL targets transformed
cells with minimal damage to the normal cells (1, 2). TRAIL
belongs to the tumor necrosis factor
␣
(TNF-
␣
) superfamily of
cytokines, which include TNF
␣
and Fas ligand (FasL)/CD95L
all of which function via binding to their corresponding death
receptors (3) leading to activation of caspases (4). Stimulation
by TRAIL leads to its binding to death receptors DR4 and DR5,
resulting in activation of initiator caspases (caspases-8 and -10),
executioner caspase (caspase-3) (5, 6), and finally apoptosis via
extrinsic pathway. Activated caspase-8 can also amplify death
signal via an intrinsic pathway, through cleavage of proapopto-
tic BID resulting in its translocation to the mitochondria and
release of cytochrome cin the cytoplasm. Cytochrome cin the
presence of dATP and apoptotic protease-activating factor 1
activates caspase-9, which in turn activates caspase-3 further
leading to more apoptosis (7).
Results from preclinical studies with recombinant TRAIL
have demonstrated its significant anti-tumor activities, indicat-
ing the potential of utilizing TRAIL as an anticancer agent (8).
Despite this proapoptotic role of TRAIL in the transformed
cells, many cancer cells develop resistance toward TRAIL-in-
duced apoptosis (9, 10). One potential reason for this resistance
could be due to the presence of non-signaling decoy receptors
for TRAIL, DcR1, DcR2 (11), and osteoprotegerin (12). Sensi-
tivity of the tumor cells toward TRAIL-mediated apoptosis
have also been linked with the activity of various pro- and anti-
*This work was supported, in whole or in part, by National Institutes of Health
Grants CA121221 (to B. R.) and GM55835 (to A. R.). This work was also sup-
ported by Veterans Affairs Merit and VISN17 awards (to B. R.), the Susan Komen
Breast Cancer Foundation, and a Veterans Affairs Merit award (to A. R.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S5.
1
To whom correspondence should be addressed: Bldg. 101, Rm. 2729, Loyola
University Chicago, 2160 S. First Ave., Maywood, IL 60153. Tel.: 708-216-
5583; Fax: 708-216-6596; E-mail: brana@lumc.edu.
2
The abbreviations used are: TNF
␣
, tumor necrosis factor
␣
; APC, adenoma-
tous polyposis coli; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride;
FasL, Fas ligand; FL, full-length; GSK3

, glycogen synthase kinase 3

; KO,
knockout; MEFs, mouse embryonic fibroblasts; PARP, poly(ADP-ribose) po-
lymerase; PPAR
␥
, peroxisome proliferator-activated receptor
␥
; siRNA,
small interference RNA; TCF/LEF, T cell factor/lymphoid enhancer factor;
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TZD, tro-
glitazone; WT, wild type; ELISA, enzyme-linked immunosorbent assay;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Z, benzyloxycar-
bonyl; fmk, fluoromethyl ketone.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13577–13588, May 15, 2009
Printed in the U.S.A.
MAY 15, 2009•VOLUME 284 •NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13577
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apoptotic proteins (10, 13) as well as loss of caspase-8 activity
(14). Identification of drugs or agents that can overcome TRAIL
resistance and sensitize cancer cells toward TRAIL-induced
apoptosis is thus critically important for targeting TRAIL-resis-
tant cancer cells. In earlier studies, a combination of TRAIL
with radiation and some chemotherapeutic agents (9, 15) have
provided limited success, because this combination also
resulted in an increase in systemic toxicity.
Peroxisome proliferator-activated receptor
␥
(PPAR
␥
)
belongs to the nuclear receptor superfamily, which is involved
in regulating various cellular processes including proliferation
and apoptosis (16). Multiple artificial ligands of PPAR
␥
have
been reported so far, which belong to the thiazolidinedione
family of insulin sensitizers and include troglitazone (TZD),
ciglitazone, pioglitazone, and rosiglitazone. Our earlier studies
have demonstrated that TZD can induce cell cycle arrest in
proliferating liver cells, via targeting cell cycle regulators (17).
At high doses these ligands can inhibit tumor growth in vivo via
regulating cellular proliferation and apoptosis (18, 19). More
recent studies have also demonstrated that PPAR
␥
ligands can
sensitize various cancer cells toward TRAIL-induced apoptosis
(13, 20–24) thus raising the possibility of utilizing the TRAIL-
PPAR
␥
ligand combination for treating cancer cells. However,
the detailed mechanism how this drug combination increases
TRAIL sensitivity in TRAIL-resistant cancer cells is still
unclear. Although one study demonstrates the cell cycle regu-
lator cyclin D3 as a target of TRAIL-PPAR
␥
agonist-mediated
apoptosis (21), others suggest the anti-apoptotic protein cellu-
lar FLICE inhibitory protein as the potential target (22–24).
Identification of molecular effectors, which can be targeted to
increase TRAIL sensitivity in resistant cancer cells, is an impor-
tant first step toward the development of effective therapeutic
approaches.
In an attempt to identify additional targets contributing
toward TRAIL resistance in the cancer cells, we focused on

-catenin due to a close link of

-catenin activation with cancer
pathways (25, 26). Recent studies have also established a con-
nection of

-catenin in mediating cell survival (27, 28).

-Cate-
nin is a multifunctional protein, the expression of which is
tightly regulated in normal cells and aberrant activation of
which can lead to tumorigenesis (25). In the conventional path-
way of

-catenin degradation, the serine threonine kinase gly-
cogen synthase kinase 3

(GSK3

) in the presence of axin and
functionally active adenomatous polyposis coli (APC) phos-
phorylates specific N-terminal residues of

-catenin and tar-
gets it toward degradation (29). Mutations of either APC or

-catenin itself (30, 31) or activation of the Wnt signaling path-
way (25) can lead to inhibition of

-catenin degradation, result-
ing in increased cytoplasmic pools of

-catenin. Other path-
ways of

-catenin degradation have also been reported recently
(32–34). Stabilization of

-catenin results in its translocation
into the nucleus, interaction with transcription factors of the T
cell factor/lymphoid enhancer factor (TCF/LEF) family to acti-
vate target gene transcription (25). A significant percentage of
the human and mouse liver tumors harbor oncogenic muta-
tions of the

-catenin gene (30), (35), whereas inactivating
mutations of APC are responsible for

-catenin stabilization in
the colorectal adenocarcinomas (29, 37).
In the present study we determined whether combinatorial
treatment with TRAIL and TZD induces apoptosis in TRAIL-
resistant cancer cells via targeting

-catenin. Our studies indi-
cate that TRAIL- and TZD-mediated apoptosis is associated
with a dramatic reduction of full-length

-catenin expression,
coinciding with the degree of apoptosis. Ectopic overexpression
of

-catenin antagonizes this drug-induced apoptosis, whereas
small interference RNA (siRNA)-mediated knock-down of
endogenous

-catenin initiates spontaneous apoptosis in can-
cer cells. TRAIL-TZD-mediated attenuation of

-catenin
expression involved a GSK3

independent post-translational
mechanism. Reduction of full-length

-catenin expression was
also associated with a cleavage of C-terminal

-catenin medi-
ated by caspases-3 and -8. These results indicate the promising
possibility of targeting

-catenin as an effective means of over-
coming TRAIL resistance.
EXPERIMENTAL PROCEDURES
Reagents—Dulbecco’s modified Eagle’s medium/F-12, Dul-
becco’s modified Eagle’s medium, RPMI, McCoy’s 5A tissue
culture media, geneticin sulfate, and Lipofectamine 2000 were
purchased from Invitrogen; troglitazone, TRAIL, cyclohexi-
mide, Z-VAD-fmk, Z-DEVD-fmk, IEDT-fmk, MG-132, lacta-
cystin, and GSK3

inhibitor VIII (AR-A014418) were pur-
chased from Calbiochem; the ELISA
PLUS
kit and in situ Cell
Death Detection Kit (fluorescein) were purchased from Roche
Applied Sciences; and 4,6-diamidino-2-phenylindole dihydro-
chloride (DAPI) was from Vector Laboratories (Burlingame,
CA). The antibodies were obtained from the following sources:
poly(ADP-ribose) polymerase (PARP), caspase-3, cleaved
caspase-3, caspase-8, GSK3

, phospho-GSK3

Ser9
, Akt, and
phospho-glycogen synthase
Ser641
from Cell Signaling Technol-
ogy (Danvers, MA);

-catenin from Zymed Laboratories Inc.,
Invitrogen, and BD Biosciences (San Jose, CA); glycogen syn-
thase from Invitrogen; cyclin-D1 from Neomarkers (Fremont,
CA); Bcl-xL from Exalpha Biologicals Inc. (Watertown, MA);
GAPDH from Ambion Inc. (Austin, TX). The Huh-7 cells were
obtained from Dr. Robert E. Lanford (University of Texas
Health Science Center, San Antonio, TX) (38) and the RKO and
RKO-

-catenin cells were obtained from Dr. Wallace K. Mac-
Naughton and Hongying Wang (University of Calgary, Calgary,
Canada) (39). The LNCaP and SKOV3 cells were obtained com-
mercially from ATCC. The wild type (WT) and GSK3

knock-
out (⫺/⫺) mouse embryonic fibroblasts (MEFs) were obtained
from Dr. James R. Woodgett (University of Toronto, Toronto,
Canada) (40).
Cell Culture—Huh-7 and RKO cells were grown in Dulbec-
co’s modified Eagle’s/F-12 medium supplemented with 10%
fetal bovine serum and RKO-

-catenin cells were maintained
in a similar medium containing 500
g/ml Geneticin. LNCaP
cells were maintained in RPMI containing 10 mMHEPES, 4.5
g/liter glucose, and 10% fetal bovine serum and SKOV3 cells
were grown in modified McCoy’s 5A medium supplemented
with 10% fetal bovine serum. WT-GSK3

(⫹/⫹) and GSK3

knock-out (⫺/⫺) MEFs were maintained in Dulbecco’s modi-
fied Eagle’s medium containing 4.5 g/liter glucose, 4 mMgluta-
mine, and 10% fetal bovine serum. In TRAIL and TZD experi-
ments, cells were treated with 100 ng/ml TRAIL or 50
MTZD

-Catenin Cleavage in Drug-induced Apoptosis
13578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 20 •MAY 15, 2009
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(unless indicated otherwise) alone or in combination for vari-
ous lengths of time followed by either apoptosis assays or West-
ern blot analysis. In the studies with GSK3

inhibitor (AR-
A014418) or caspase inhibitors,
cells were pretreated for 1 h with 10
MAR-A014418 or 50
MZ-VAD-
fmk or 25
MDEVD-fmk or 25
M
IETD-fmk, respectively, followed by
TRAIL-TZD treatment.
Apoptosis Detection by Fluores-
cence Microscopy—This was per-
formed utilizing protocols described
earlier (41). Briefly, subconfluent
cells were plated in 2-well chamber
slides and were treated with 50
M
TZD and 100 ng/ml TRAIL either
alone or in combination. To deter-
mine the optimal drug concentra-
tions required to induce maximal
apoptosis in cancer cells, increasing
concentrations of TZD or TRAIL
were utilized in pilot experiments
and apoptosis assays were per-
formed. Following treatment with
TZD and TRAIL, cells were fixed
with 4% paraformaldehyde, perme-
abilized with phosphate-buffered
saline containing 0.1% Triton
X-100, and stained with DAPI.
DAPI-stained nuclei were analyzed
for apoptotic morphology by fluo-
rescence microscopy (Axiovert 200
inverted microscope, Zeiss). A min-
imum of 200 nuclei were analyzed
per treatment. The percentage of
apoptotic cells was calculated as the
number of cells with condensed/
fragmented nuclear morphology
divided by the total number of cells
analyzed. To determine the effect of
various agents on apoptotic mor-
phology, cells were also photo-
graphed by inverted phase-contrast
microscopy.
Apoptosis Detection by Cell Death
Detection ELISA—This assay was
performed utilizing the cell death
detection ELISA
PLUS
kit (Roche
Applied Sciences) following the
manufacturer’s specification as de-
scribed previously (42), with slight
modifications. This assay utilizes a
quantitative sandwich immunoas-
say principle to detect and quanti-
tate the mono- and oligonucleo-
somes specifically released in the
cytoplasm during apoptotic cell
death. Cells plated on 6-well plates
were treated with TZD or TRAIL, following which both adher-
ent and floating (apoptotic) populations were harvested. Cells
were lysed in Nonidet P-40 lysis buffer and the supernatant was
AHuh-7
DMSO
Cell Type
TZD
TRAIL
+
+
++
+
-
--
-
---
PARP
PARP (Cl)
GAPDH
Caspase 3 (Cl)
Caspase 3
GAPDH
0
10
20
30
40
123456789101112
Enrichm ent Factor
Lane
DMSO
TZD
TRAIL
Cell Type Huh-7
++++
+
---
-
---
Time
+
-
-+
-
-+
-
-
12hrs 24hrs 48hrs
+
+
-
+
+
-
+
-
-
+
-
-
+
-
-
E
BHuh-7
DMSO
Cell Type
TZD
TRAIL
+
+
++
+
-
--
-
---
GAPDH
β-catenin
DCell Type
Time
DMSO
TZD
TRAIL
Huh-7
4hrs 8hrs 12hrs 24hrs
++ ++
+
+
++
+
+++
+
++
+
-
-
-
-
-
-
-
--
-
-- - -
--
-- --
β-catenin
GAPDH
C
TRAIL+ TZD TRAIL TZD DMSO
TUNEL DAPI
FIGURE 1. Effect of combinatorial treatment with TRAIL and TZD on apoptosis and

-catenin expres-
sion in Huh-7 hepatocellular carcinoma cells. Aand B, Huh-7 cells were treated with 50
MTZD and 100
ng/ml TRAIL either alone or in combination for 24 h, following which cells were harvested and total
protein was extracted. Western blot analysis of the cell extracts was then performed with the indicated
antibodies specific for apoptosis (A) or with antibodies against

-catenin and GAPDH (as control) (B).
C, Huh-7 cells were treated as in A, and analyzed by TUNEL assay. The fluorescent signal, emitted by
fluorescein-labeled dUTP incorporated into fragmented DNA (green), and from DAPI (blue) were visualized
and photographed utilizing fluorescence microscopy (Axiovert 200 inverted microscope, Zeiss), inter-
faced with the camera (Axiocam). D, Huh-7 cells were treated with 50
MTZD or 100 ng/ml TRAIL either
alone or in combination for different periods of time. At the end of the incubation total protein was
extracted and Western blot analysis was performed with antibodies against

-catenin and GAPDH (as
control). E, Huh-7 cells were treated with the two agents for different periods of time. At the indicated
times cells were harvested and apoptosis assays were performed using the cell death detection ELISA
PLUS
kit. The data in each set represent the mean ⫾S.D. of two independent experiments.

-Catenin Cleavage in Drug-induced Apoptosis
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collected following centrifugation at 3000 ⫻gfor 10 min.
Nucleosomes were detected photometrically at 405 nm in an
ELISA plate reader (SpectraMax 190, Molecular Devices) by
measuring the peroxidase activity of 2,2⬘-azino-di(3-ethylben-
zthiazolinsulfonate) as substrate. The readings were expressed
as degree of apoptosis considering the corresponding untreated
control as 1.
Apoptosis Detection by TUNEL Assay—Terminal deoxy-
nucleotidyl transferase (TdT)-mediated deoxyuridine triphos-
phate (dUTP) nick end-labeling (TUNEL) assay was performed
utilizing the in situ Cell Death Detection Kit (fluorescein), from
Roche Applied Sciences according to the manufacturer’s
instructions. Briefly, Huh-7 cells plated in 2-well chamber
slides were treated with various agents, fixed in freshly prepared
4% paraformaldehyde, and then permeabilized in 0.1% Triton
X-100. Cells were then washed twice with phosphate-buffered
saline and subjected to the TUNEL reaction at 37 °C in a
humidified atmosphere in the dark for 60 min. At the end of the
incubation cells were counterstained with DAPI. The fluores-
cent signal, emitted by fluorescein-labeled dUTP incorporated
into fragmented DNA, was visualized by fluorescence micros-
copy (Axiovert 200 inverted microscope, Zeiss), interfaced with
a camera (Axiocam), and image analyzer software (Axiovision,
Zeiss).
Small Interference RNA (siRNA)—The

-catenin siRNA oligo-
nucleotides with the sequence (sense 5⬘-AGCUGAUAUU-
GAUGGACAG-3⬘) along with the
corresponding antisense oligonu-
cleotide were synthesized from Dhar-
macon (Lafayette, CO) as described
(43). A negative control siRNA (Santa
Cruz Biotechnology) was used as con-
trol siRNA. Caspase-8 siRNA was
obtained from Santa Cruz Biotech-
nology (Santa Cruz, CA) and
caspase-3 siRNA was obtained from
Cell Signaling Technology (Dan-
vers, MA). siRNA transfection was
performed using Lipofectamine
2000 as per the manufacturer’s
instructions. Briefly, subconfluent
Huh-7 cells were plated in 6-well
plates in regular growth medium.
The next day they were transfected
with either 100 nMcontrol siRNA or
the target protein siRNA for 24 h
followed by recovery in serum con-
taining medium. After 72 h of
siRNA transfection, the cells were
treated with either DMSO or a com-
bination of TZD and TRAIL for an
additional 24 h followed by apopto-
sis assays.
Western Blot Analysis—Western
blot analysis was performed follow-
ing treatment of the cells with vari-
ous agents and at different time
intervals following the procedures
described previously (34). Equal amounts of total protein were
fractionated by SDS-PAGE, transferred to polyvinylidene diflu-
oride membranes, followed by Western blotting with the anti-
bodies indicated. In the studies with cycloheximide, cells were
treated with (20
g/ml) cycloheximide in the presence or
absence of TRAIL-TZD for various lengths of time up to a max-
imum of 8 h.
RESULTS
TRAIL and TZD-mediated Apoptosis in Hepatocellular Car-
cinoma Cells Is Associated with a Reduction of

-Catenin
Expression—Earlier studies have reported that combinatorial
treatment of TRAIL-resistant cells with TRAIL and PPAR
␥
ligand (21) sensitizes them toward apoptosis. To identify a spe-
cific target mediating this apoptosis, we used the hepatocellular
carcinoma cell line Huh-7, because they show resistance to apo-
ptosis when treated with TRAIL alone (44). Western blot anal-
ysis indicated an activation of PARP and caspase-3 (indicated
by the presence of the corresponding cleaved fragments) fol-
lowing incubation with the TRAIL and TZD combination (Fig.
1A,fourth lane). Apoptosis assays designed under similar con-
ditions also showed an increase in Huh-7 cell apoptosis with the
TRAIL and TZD combination, compared with either agent
alone, as shown in Fig. 1Cand supplemental Fig. S1A. To gain
further insight into the effectors involved in mediating the
pathway of apoptosis following combinatorial drug treatment,
FIGURE 2. Effect of increasing concentrations of TRAIL or TZD on apoptosis and

-catenin expression in
Huh-7 hepatocellular carcinoma cells. Aand B, Huh-7 cells were treated with either DMSO or a combination
of 100 ng/ml TRAIL and increasing concentrations of TZD for 24 h. At the end of incubation, apoptotic cells were
analyzed either via Western blot analysis with the indicated antibodies (A) or counted via DAPI staining (B). The
data in each set represent the mean ⫾S.D. of two independent experiments. Cand D, Huh-7 cells were treated
with either DMSO or a combination of 50
MTZD and increasing concentrations of TRAIL. At the end of the
incubation, apoptotic cells were analyzed either via Western blot analysis (C) or counted via DAPI staining (D).
The data in each set represent the mean ⫾S.D. of two independent experiments.

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we estimated changes of

-catenin protein expression follow-
ing treatment with TRAIL and TZD. Treatment of Huh-7 cells
with the TRAIL and TZD combination resulted in a com-
plete loss of

-catenin expression (Fig. 1B,fourth lane), com-
pared with treatment with either drug alone, indicating a
possible involvement of

-catenin in this apoptotic pathway.
TRAIL and TZD Combination Decrease

-Catenin Expres-
sion and Induce Apoptosis in a Time- and Dose-dependent
Manner—To establish a correlation between the decrease of

-catenin expression and increase in apoptosis, time course
studies with the drug combination were designed next. These
indicated that a decrease in

-catenin expression is maximal at
24 h (Fig. 1D), which coincided with maximal apoptosis (Fig. 1E
and supplemental Fig. S1B). To determine the optimal concen-
tration of TRAIL and TZD required for maximal

-catenin
reduction, Western blot analyses were performed with Huh-7
cell extracts following treatment with increasing concentra-
tions of either TZD or TRAIL. The results indicated that
50–100
MTZD (Fig. 2A) and 50–100 ng/ml TRAIL (Fig. 2C)
were optimal for reducing

-cate-
nin expression maximally. This
drug concentration also correlated
with apoptosis, which was maxi-
mum with 50 –100
MTZD (Fig. 2B
and supplemental Fig. S2A), and 100
ng/ml TRAIL (Fig. 2Dand supple-
mental Fig. S2B). Based on these
results, the 50
MTZD and 100
ng/ml TRAIL combination was uti-
lized in all future studies. These
results indicated a close correlation
between down-regulation of

-cate-
nin expression and induction of
apoptosis.
TRAIL and TZD-induced Apop-
tosis Is Associated with Reduced

-Catenin Expression in Different
Cancer Cells—To determine any
correlation of decreased

-catenin
expression and TRAIL-TZD-in-
duced apoptosis in other cancer
cells, similar studies were per-
formed with TRAIL-resistant pros-
tate cancer (LNCaP), ovarian cancer
(SKOV3), and colon cancer (RKO)
cells. TRAIL-TZD combinatorial
treatment resulted in a significant
induction of apoptosis in LNCaP
cells, as indicated by the apoptosis
assays (Fig. 3Aand supplemental
Fig. S3A) and Western blot analysis
showing PARP and caspase-3 cleav-
age (Fig. 3B). Increased apoptosis
was also observed in SKOV3 (Fig.
3Cand supplemental Fig. S3B), and
RKO cells (Fig. 3Dand supplemen-
tal Fig. S3C). This drug combination
thus was effective in sensitizing var-
ious TRAIL-resistant cancer cells toward apoptosis. Western
blot analysis carried out following a similar combinatorial drug
treatment also showed a corresponding decrease of

-catenin
expression following co-incubation with TRAIL and TZD (Fig.
3, Eand F). These results indicated a correlation between
reduced

-catenin expression and increased apoptosis, and
suggested the possibility that decreasing

-catenin expres-
sion might increase drug-induced apoptosis in drug-resis-
tant cancer cells.
In an attempt to identify candidate downstream targets of

-catenin mediating this apoptotic response, we focused on the
following

-catenin target proteins: cyclin D1 (which mediates
cell growth) and Bcl-xL and AKT (which mediate cell survival)
(45–47). However, Western blot analysis of total protein
extracts from Huh-7 and LNCaP cells following treatment with
these drugs showed a significant decrease in the expression of
cyclin D1, AKT, and Bcl-xL with TZD alone (supplemental Fig.
S4, Aand B,second lane), and did not seem to correlate with the
apoptotic response (Figs. 1Aand 3B).
FIGURE 3. Effect of TRAIL-TZD combinatorial treatment on the apoptotic potential and

-catenin expres-
sion in various cancer cells. LNCaP prostate cancer cells (Aand B), SKOV3 ovarian cancer cells (C), and RKO
colon cancer cells (D) were treated with 50
MTZD or 100 ng/ml TRAIL either alone or in combination for 24 h.
Apoptosis was estimated via the cell death detection ELISA
PLUS
kit (A,C, and D) and Western blot analysis with
the indicated antibodies (B). The data in each set represent the mean ⫾S.D. of at least two independent
experiments. LNCaP (E) and SKOV3 (F) cells were treated as in A, total cell extracts were prepared and Western
blot analysis was performed with antibodies against

-catenin and GAPDH (as control).

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TRAIL and TZD-induced Apoptosis Involves

-Catenin—To
determine whether

-catenin was involved in providing
resistance to apoptosis, we utilized the RKO and RKO-

-
catenin cells, the latter stably overexpressing a mutant stable
form of

-catenin (39). Apoptosis assays showed an increase
in the apoptotic potential of the RKO cells following treat-
ment with TRAIL-TZD (Fig. 4Aand supplemental Fig. S5,
RKO panels), which was attenuated in the RKO-

-catenin

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cells (RKO-

-catenin panels). Western blot analysis showed
higher levels of

-catenin expression in the RKO-

-catenin
cells even after TRAIL-TZD treatment (Fig. 4B,RKO-

-cate-
nin panel) compared with lower levels in the RKO cells (RKO
panel). These results suggested that

-catenin overexpres-
sion can antagonize TRAIL-TZD-mediated apoptosis and
might contribute to TRAIL resistance. To establish the
involvement of

-catenin more conclusively, we designed
siRNA studies to knockdown endogenous

-catenin expres-
sion in the Huh-7 hepatocellular carcinoma cells. Treatment
with

-catenin siRNA resulted in a significant decrease in
endogenous

-catenin expression (Fig. 4C,lane 5), which
was unaffected by control siRNA (lane 3). Apoptosis assays
designed following

-catenin knockdown showed an
increase in basal apoptotic response in the absence of either
TRAIL or TZD (Fig. 4D,lane 5), which was increased further
following treatment with the TRAIL and TZD combination
(lane 6). In addition, a time course analysis carried out fol-
lowing

-catenin knockdown showed that at 24 h where

-catenin knockdown is partial (Fig. 4E, compare lanes 1 and
3), there is no increase in spontaneous apoptosis in the
absence of TRAIL and TZD (Fig. 4, E, compare lanes 1 and 3,
PARP cleaved panel, and F,lanes 1 and 3). However, at 48 h
when

-catenin knockdown is significant (Fig. 4E, compare
lanes 5 and 7,

-catenin panel), there is an increase in spon-
taneous apoptosis in the absence of drugs as evident from
PARP cleavage (Fig. 4E,lanes 5 and 7,PARP cleaved panel),
and apoptosis assays (Fig. 4F, compare lanes 5 and 7). Addi-
tion of TRAIL and TZD at 48 h produces a synergistic effect
on apoptosis (Fig. 4F,lane 8). These studies showed that
overexpression of

-catenin provides a survival advantage to
the cancer cells and knocking down

-catenin expression
sensitizes them toward apoptosis.
TRAIL-TZD Attenuates

-Catenin Expression at a Post-
translational Level Independent of GSK3

Activity—Because

-catenin expression can be regulated via different pathways,
studies were designed next to determine the mechanism by
which TRAIL-TZD stimulation might be regulating

-catenin
expression. To determine whether this regulation was at a
post-translational level, TRAIL-TZD studies were designed
following treatment with the protein synthesis inhibitor cyclo-
heximide. These studies showed a significant decrease in the
half-life of

-catenin protein following treatment with the
TRAIL-TZD combination compared with untreated controls
(Fig. 5A, compare lanes 3–7 with lanes 8–12). This suggested
that this drug combination regulated

-catenin expression at a
post-translational level. In the conventional pathway of

-cate-
nin degradation, GSK3

phosphorylates specific N-terminal
residues of

-catenin in the presence of functional APC and
Axin, which targets it toward proteasomal degradation (29).
However, our earlier studies showed that TZD alone can
degrade

-catenin via a novel GSK3

-APC and p53-independ-
ent pathway (34). To determine any involvement of GSK3

in
the TRAIL-TZD-mediated decrease of

-catenin expression,
cells were pretreated with a pharmacological inhibitor of
GSK3

(AR-A014418) prior to TRAIL-TZD treatment. Pre-
treatment with AR-A014418 resulted in a decrease in phospho-
rylation of glycogen synthase (downstream target of GSK3

)as
evident from the low phospho-glycogen synthase
Ser641
levels in
the absence of TRAIL-TZD (Fig. 5B, phospho-GS
Ser641
panel,
compare lanes 1 and 3), indicating that AR-A014418 inhibited
GSK3

activity. However, inhibition of GSK3

by AR-A014418
was unable to restore

-catenin expression following TRAIL-
TZD treatment (

-catenin panel, compare lanes 2 and 4). Sim-
ilar studies with other known GSK3

inhibitors including ken-
paullone and lithium chloride were also unable to restore

-catenin expression in the presence of TRAIL-TZD (data not
shown). In addition, treatment with TRAIL-TZD resulted in an
increase in GSK3

Ser9
phosphorylation (phospho-GSK3

Ser9
panel) indicating an inactivation of the kinase. This is also evi-
dent from a reduction of phospho-glycogen synthase
Ser641
lev-
els following TRAIL-TZD treatment (phospho-GS
Ser641
panel,
lane 2). To rule out the participation of GSK3

conclusively,
TRAIL-TZD treatments were carried out with control
(WT
⫹/⫹
) and GSK3

knock-out (KO
⫺/⫺
) MEFs (40). Time
course analysis of these cells showed that TRAIL-TZD treat-
ment was capable of reducing

-catenin expression in WT
⫹/⫹
and to a greater extent in the GSK3

KO
⫺/⫺
MEFs (Fig. 5C,
compare lanes 2 and 4, and lanes 6 and 8). In addition, pretreat-
ment with proteasomal inhibitors MG132 or lactacystin were
unable to restore the

-catenin full-length form following
TRAIL-TZD treatment, indicating no involvement of protea-
somes (Fig. 8C, compare lanes 2,4, and 6,

-catenin-FL- panel).
These results indicated that the TRAIL-TZD combination reg-
ulated

-catenin expression via a post-translational mecha-
nism, independent of the conventional degradation pathway
mediated by GSK3

.
The fact that GSK3

KO
⫺/⫺
MEFs showed a greater reduc-
tion of

-catenin when incubated with TRAIL-TZD indicated
the interesting possibility that inhibition of GSK3

expression
or activity might increase TRAIL-TZD-mediated apoptosis. In
fact, apoptosis assays showed that AR-A014418-mediated inhi-
bition of GSK3

activity produced synergistic effects on
TRAIL-TZD-induced apoptosis (Fig. 5, Dand E). These results
are consistent with earlier reports that showed a profound
increase in liver cell apoptosis in GSK3

knock-out mice (40).
FIGURE 4. TRAIL-TZD-induced apoptosis is mediated via down-regulation of

-catenin expression. A, RKO or RKO-

-catenin cells were treated with either
DMSO or the TRAIL (100 ng/ml) and TZD (50
M) combination for 24 h. At the end of the incubation cells were harvested and apoptosis assays were performed
using DAPI staining. The data in each set represent the mean ⫾S.D. of at least two independent experiments. B, RKO and RKO-

-catenin cells were treated as
in Afollowed by Western blot analysis with antibodies against

-catenin and GAPDH (as control). Cand D, Huh-7 cells were transfected with either none (lanes
1and 2) or control siRNA (lanes 3 and 4), or

-catenin siRNA (lanes 5 and 6) followed by treatment with the TRAIL-TZD combination for 24 h. At the end of the
incubation Western blot analysis was performed utilizing the antibodies indicated (C) or apoptosis assays were performed utilizing the cell death detection
ELISA
PLUS
kit (D). The data represent the mean ⫾S.D. of two independent experiments. Eand F, Huh-7 cells were transfected with control siRNA (lanes 1,2and
5,6)or

-catenin siRNA (lanes 3,4and 7,8)asinCfor the indicated periods of time, followed by treatment with DMSO or a combination of 50
MTZD and 100
ng/ml TRAIL for 24 h. At the end of the incubation Western blot analysis was performed utilizing the antibodies indicated (E) or apoptosis assays were
performed utilizing the cell death detection ELISA
PLUS
kit (F).

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TRAIL-TZD-induced Apoptosis Is Preceded by a Cleavage of

-Catenin—Earlier reports have shown that

-catenin can
undergo caspase-induced cleavage during cellular apoptosis
(48). Studies were designed next to determine whether the
TRAIL-TZD combinatorial treatment induced any cleavage of

-catenin protein. Western blot assays were performed with an
antibody created against the C-terminal 210 amino acid res-
idues of

-catenin (from BD Biosciences) as well as with the
one used in earlier studies from
Zymed Laboratories Inc., created
against the C-terminal 100 amino
acid residues of

-catenin. The anti-
body from BD Biosciences detected
the full-length (FL) form as well as a
cleaved

-catenin fragment (⬍97
kDa), the latter specifically follow-
ing TRAIL-TZD treatment (Fig. 6A,
lane four,

-catenin-cleaved BD
panel), whereas the Zymed Labora-
tories Inc. antibody only detected
the full-length form (

-catenin, FL-
Zymed panel). It was thus conceiv-
able that TRAIL-TZD-mediated
activation of caspases might lead to

-catenin cleavage, which contrib-
utes to the induction of apoptosis.
The BD antibody also detected the
full-length form following TRAIL-
TZD treatment (Fig. 6A,lane four,

-catenin-FL-BD panel), which
could be due to the presence of both
apoptotic and non-apoptotic cells in
this preparation. To confirm this,
floating cells (fully apoptotic) and
adherent cells (partially apoptotic)
were separately isolated after
TRAIL-TZD treatment and ana-
lyzed. Western blot analysis per-
formed with these cells indicated
the presence of both the cleaved and
noncleaved forms of

-catenin in the
partially apoptotic (adherent) popula-
tion (Fig. 6B,lane 2), and only the
cleaved form in the fully apoptotic
(floating) population (lane 3). TRAIL-
TZD-mediated cleavages of

-cate-
nin were also detected in LNCaP (Fig.
6C) and RKO cells (Fig. 6D), indicat-
ing a similar pathway operating in all
cancer cells. Interestingly, the degree
of

-catenin cleavage correlated with
the degree of apoptosis, with greater
apoptosis in cells showing more
cleavage (LNCaP, Figs. 3Aand 6C)
than those with less cleavage (Huh,
Figs. 1Eand 6A).
TRAIL-TZD-induced Apoptosis
and Cleavage of

-Catenin Is
Mediated via Caspases-3 and -8—To determine any involve-
ment of caspases in

-catenin cleavage, and to understand
whether cleavage of

-catenin occurred early or late during the
apoptotic process, detailed time course studies were designed fol-
lowing incubation of Huh cells with the TRAIL and TZD combi-
nation. The results indicated an early activation of caspase-8 start-
ing at 2 h (Fig. 7, lane 2,caspase-8 cleaved panel), followed by

-catenin cleavage starting at4h(lane 4,

-catenin cleaved panel)
FIGURE 5. TRAIL-TZD regulates

-catenin expression via a post-translational mechanism independent of
GSK3

.A, Huh-7 cells were treated with DMSO or the TRAIL-TZD combination in the presence of 20
g/ml
cycloheximide (CHX,lanes 3–12). Cells were harvested at 0, 2, 4, 6, and 8 h after treatment followed by Western
blot analysis with the indicated antibodies. The Huh-7 cells treated with either DMSO or the TRAIL-TZD com-
bination in the absence of cycloheximide were included in lanes 1 and 2, respectively, as positive controls to
show TRAIL-TZD effects on

-catenin. B, Huh-7 cells were treated with the TRAIL-TZD combination for 24 h in
the presence (⫹) or absence (⫺) of a pretreatment with the GSK3

inhibitor AR-A014418. At the end of the
incubation total cell extracts were prepared and Western blot analysis was performed with the indicated
antibodies. C, wild type (WT
⫹/⫹
) or GSK3

knock-out (KO
⫺/⫺
) MEFs were incubated with either DMSO or the
TRAIL-TZD combination. Total cell extracts were prepared at the indicated time points followed by Western
blot analysis with the antibodies shown. D, Huh-7 cells were treated as in Bfollowed by apoptosis assay utilizing
the cell death detection ELISA
PLUS
kit. The data represent the mean ⫾S.D. of two independent experiments.
E, phase-contrast microscopic pictures showing apoptotic morphology of Huh-7 cells following treatment with
either DMSO or the TRAIL-TZD combination in the presence (⫹) or absence (⫺) of AR-A014418.

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and caspase-3 cleavage starting at6h(lane 6,caspase-3 cleaved
panel). Although

-catenin cleavage was initiated following
caspase-8 activation and preceded caspase-3 cleavage, it was max-
imal at 24 h (lane 12), and correlated well with maximum
caspase-3 cleavage. This suggested that

-catenin might be
cleaved by both caspase-3 and -8 in this apoptotic cascade. In addi-
tion, this sequential and early cleavage pattern suggested that

-catenin cleavage might be a critical
step in the progression of apoptosis.
To confirm whether TRAIL-TZD-
induced cleavage of

-catenin
involved caspase activity, Western
blot analysis was performed with
cell extracts obtained following
pretreatment of the cells with the
pan-caspase inhibitor Z-VAD-fmk.
Pretreatment with caspase inhibitor
abolished TRAIL-TZD-induced
cleavage of

-catenin as shown in
Fig. 8A(

-catenin-Cl panel, com-
pare lanes 4 and 8), suggesting the
involvement of caspases in mediat-
ing this. Z-VAD-fmk pretreat-
ment also abolished TRAIL-TZD-
induced cleavage of caspase-3
(caspase 3-Cl panel), confirming the
efficacy of this inhibitor. Apoptosis
assays were designed next following
pretreatment with the pan-caspase
inhibitor Z-VAD-fmk, to determine
the role of caspases in mediating
this apoptotic pathway. The results
indicated a complete inhibition
of TRAIL-TZD-induced apoptosis
(Fig. 8B, compare lanes 2 and 4) fol-
lowing inhibition of caspase activity,
suggesting that caspases are involved in both

-catenin cleav-
age and induction of apoptosis. In addition, pretreatment with
two proteasomal inhibitors MG132 and lactacystin resulted in a
synergistic increase in caspase-3 cleavage (Fig. 8C, compare
lane 2 with 4and 6,caspase-3 cleaved panel) and

-catenin
cleavage (

-catenin cleaved panel), indicating again a correla-
tion between increased caspase activity and

-catenin cleavage.
Because our time course analysis indicated an activation of both
caspase-3 and -8 following incubation with TRAIL and TZD (Fig.
7), studies were designed next to identify the caspase(s) responsi-
ble for mediating

-catenin cleavage in this pathway. Western blot
analysis performed with cell extracts following pretreatment sep-
arately with inhibitors of caspase-8 (IETD-fmk) or caspase-3
(DEVD-fmk) resulted in an inhibition of

-catenin cleavage (Fig.
8D, compare lanes 2,6, and 8) as well as PARP and caspase-3
cleavage, suggesting the involvement of both of these caspases in
regulating

-catenin cleavage. To further confirm the partici-
pation of these two caspases in this, siRNA studies were de-
signed following knockdown of caspase-3 or -8 with their respec-
tive siRNA sequences. Western blot analysis showed a reduction
in the expression of the full-length forms of caspase-3 (Fig. 8E,
compare lane 1 with 3and 7,caspase-3FL panel) and caspase-8
(compare lane 1 with 5and 7,caspase-8 FL panel) following
knockdown with their respective siRNA oligonucleotides. TRAIL
and TZD studies designed following siRNA treatment showed a
decrease in

-catenin cleavage following knockdown of both
caspase-3 and caspase-8 (compare lane 2 with 4,6, and 8,

-cate-
nin-cleaved panel). This suggested that TRAIL-TZD-induced
cleavage of

-catenin involves caspase-3 as well as 8. Cleavage of
FIGURE 6. Effect of TRAIL-TZD on

-catenin cleavage. A, Huh-7 cells were treated with 50
MTZD or 100 ng/ml
TRAIL either alone or in combination for 24 h. Western blot analysis was performed with the cell extracts utilizing
antibodies against

-catenin from Zymed Laboratories Inc., recognizing the full-length (

-catenin-FL-Zymed panel),
or from BD Biosciences, recognizing the full-length (

-catenin-FL-BD panel) and cleaved forms (

-catenin-cleaved-BD
panel). The samples were also blotted with GAPDH antibody as control (GAPDH panel). B, Huh-7 cells were treated
with either DMSO or a combination of 50
MTZD and 100 ng/ml TRAIL for 24 h. At the end of the incubation,
adherent (non-apoptotic, lane 1), adherent (partially apoptotic, lane 2), and floating (fully apoptotic, lane 3) popula-
tions of cells were separately isolated and analyzed by Western blot analysis with antibodies against

-catenin (BD
Biosciences), PARP, cleaved caspase-3 or GAPDH (as control). LNCaP (C) and RKO (D) cells were treated as in A
followed by Western blot analysis with antibodies against

-catenin (from BD Biosciences) and GAPDH.
FIGURE 7. Time course of

-catenin and caspase cleavage during TRAIL-
TZD-induced apoptosis. Huh-7 cells were treated with either DMSO or a com-
bination of 50
MTZD and 100 ng/ml TRAIL for the indicated periods of time.
Western blot analysis was performed with the cell extracts utilizing the following
antibodies:

-catenin antibody (from BD Biosciences), and PARP and caspase-8
antibodies recognizing full-length and cleaved forms of the respective proteins
(panels 1,2, and 3,respectively), caspase-3 antibody recognizing only the cleaved
form (panel 4) and GAPDH antibody as control (panel 5).

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PARP and caspase-3 were also reduced by caspase-8 siRNA
(PARP-cleaved and caspase-3-cleaved panels), suggesting both of
them to be downstream of caspase-8 activation.
DISCUSSION
The fact that TRAIL can activate apoptosis specifically in
tumor cells with little or no affect on the normal cells raises
a great deal of enthusiasm toward
utilizing TRAIL as a target thera-
peutic agent for treating cancer.
However, this enthusiasm is seri-
ously challenged by increasing
reports that the majority of tumor
cells develop resistance via dif-
ferent mechanisms that evade
TRAIL-induced apoptosis (9, 10).
Intense research is currently
underway to develop drugs or drug
combinations that can overcome
TRAIL resistance and sensitize
cancer cells toward TRAIL-in-
duced apoptosis, without jeopardiz-
ing the tumor-specific pro-apopto-
tic properties of TRAIL. This
requires a thorough understanding
of the events and identification of
molecular targets that contribute to
tumor cell resistance. Development
of an effective drug combination
currently imposes the biggest chal-
lenge in designing the next genera-
tion of cancer drugs. Recent studies
with TRAIL in combination with
various nontoxic chemopreventive
agents (49, 50), or PPAR
␥
ligands
(13, 20–24), have provided promis-
ing results in promoting TRAIL-in-
duced apoptosis. Despite this pro-
gress, the mechanism by which this
combination therapy overcomes
TRAIL resistance is largely unclear
and needs to be addressed to further
increase the potency of this drug
combination. This will also help in
determining which cancer cells
might respond to this combinatorial
treatment.
The studies described here indi-
cated that combinatorial treatment
with TRAIL and TZD can sensitize
various TRAIL-resistant cancer
cells toward apoptosis, and identi-
fied

-catenin as a novel target of
this apoptosis pathway. This is evi-
dent from an extensive decrease in
full-length

-catenin expression,
coinciding well with maximal apo-
ptosis. This also suggested that
antagonizing

-catenin expression or activity might be a
critical event in promoting TRAIL sensitivity. This is con-
firmed by our studies showing that ectopic overexpression of

-catenin can antagonize TRAIL-TZD-mediated apoptosis
(Figs. 4Aand supplemental Fig. S5). Similarly, siRNA-medi-
ated knockdown of endogenous

-catenin increased the
basal level of apoptosis in the absence of TRAIL or TZD (Fig.
FIGURE 8. Effect of inhibition of caspase-3 and -8 on TRAIL-TZD-induced apoptosis and

-catenin cleavage.
A, Huh-7 cells were treated with the TRAIL and TZD combination for 24 h following a pretreatment in the absence (⫺)
or presence (⫹)of50
MZ-VAD-fmk. Western blot analyses of the cell extracts were performed with the indicated
antibodies. B, Huh-7 cells were treated as in A, followed by apoptosis assay utilizing the cell death detection
ELISA
PLUS
kit. The data represent the mean ⫾S.D. of two independent experiments. C, Huh-7 cells were treated with
either DMSO or a combination of TRAIL and TZD for 10 h following pretreatment with 10
MMG132 (lanes 3 and 4)
or 5
Mlactacystin (lanes 5 and 6) for 1 h. Western blot analysis was performed with the cell extracts utilizing the
antibodies indicated. D, Huh-7 cells were treated with the TRAIL and TZD combination for 24 h following pretreat-
ment in the absence (⫺) or presence (⫹)of50
MZ-VAD-fmk (lanes 3 and 4), 25
MDEVD-fmk (lanes 5 and 6), or 25
MIETD-fmk (lanes 7 and 8). Western blot analyses of cell extracts were performed with the indicated antibodies.
E, Huh-7 cells were transfected with either control-siRNA (lanes 1 and 2), caspase-3-siRNA (lanes 3 and 4), caspase-8-
siRNA (lanes 5 and 6), or caspase-3 and -8 siRNA (lanes 7 and 8), followed by treatment with TRAIL and TZD for 24 h.
Western blot analyses were then performed with the indicated antibodies.

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4, Dand F). In fact

-catenin overexpression has been shown
to be linked with pro-survival pathways in various cell types
(27, 28, 39).
To gain a mechanistic insight toward the pathway involved in
TRAIL-TZD-mediated modulation of

-catenin expression,
we designed studies with the protein synthesis inhibitor cyclo-
heximide. These studies indicated a reduction of

-catenin pro-
tein half-life following treatment with TRAIL-TZD, suggesting
regulation at a post-translational level. In the conventional deg-
radation pathway,

-catenin is phosphorylated at specific
N-terminal residues by GSK3

in the presence of functional
APC and Axin, and is then targeted toward ubiquitination-me-
diated proteasomal degradation (29). However, pretreatment
of the cells with AR-A014418, a pharmacological inhibitor of
GSK3

, was unable to restore

-catenin levels following
TRAIL-TZD treatment (Fig. 5B), whereas it inhibited glycogen
synthase Ser-641 phosphorylation, indicating inhibition of
GSK3

activity (Fig. 5B,phospho-GS
Ser641
panel). Studies with
other known GSK3

inhibitors (kenpaullone and lithium chlo-
ride) produced similar results (data not shown). In addition,
studies with GSK3

KO
⫺/⫺
MEFs showed a greater decrease of

-catenin expression following TRAIL-TZD stimulation com-
pared with the WT
⫹/⫹
MEFs. These results, combined with the
fact that TRAIL-TZD stimulation resulted in an increase
in GSK3

Ser9
phosphorylation (inhibition) suggested that
TRAIL-TZD-mediated degradation of

-catenin was inde-
pendent of GSK3

activity. In an attempt to identify the regu-
lator(s) modulating

-catenin expression during the onset of
apoptosis, we examined any potential role of caspases in cleav-
ing

-catenin. In fact, earlier studies have shown caspase (48,
51, 52) and Calpain (53) induced cleavage of

-catenin. Our
studies detected a TRAIL-TZD-induced cleavage of

-catenin
(Fig. 6, A,C, and D), and a complete loss of the full-length form
in the fully apoptotic population (Fig. 6B,lane 3). Inhibition of
caspase activation by a pan-caspase inhibitor (Z-VAD-fmk)
inhibited TRAIL-TZD-induced cleavage of

-catenin (Fig.
8A) as well as apoptosis (Fig. 8B), suggesting the involvement
of caspases in mediating both events. This also indicated that
cleavage of

-catenin is an important part of this apoptosis
pathway, and might be a mechanism explaining how
caspases induce apoptosis. Additional studies designed to
identify the caspases mediating this indicated the involve-
ment of caspases-3 and -8 (Fig. 8, Dand E) in mediating

-catenin cleavage.
The functional significance of

-catenin cleavage in these
studies is still unknown, and is unlikely to be to decrease the
transactivation potential of

-catenin as suggested earlier (48).
This is supported by two lines of evidence: (i) expression of

-catenin target proteins showed a significant decrease with
TZD alone in both Huh-7 (supplemental Fig. S4A) and LNCaP
cells (supplemental Fig. S4B), which was not associated with
apoptosis (Figs. 1, E, lanes 2,6, and 10, and A,lane 2); (ii) TZD
treatment alone resulted in a significant decrease in

-catenin/
TCF responsive reporter activity in our earlier studies (17), but
was unable to induce any apoptosis in those cells (data not
shown). It thus seemed that there is no correlation between the
decrease in

-catenin/TCF transcriptional activity and the
induction of apoptosis. Importantly, treatment with TZD alone
also reduces full-length

-catenin expression as shown in Figs.
1, B, and 3, Eand F(lane 2), which might contribute to the
reduced

-catenin/TCF transcriptional activity as reported
earlier (17). However, this decrease is not associated with
increased apoptosis, indicating the possibility that TRAIL-
TZD-induced cleavage of

-catenin is important for this apo-
ptosis. It is likely that TRAIL-TZD-mediated cleavage of

-catenin results in the disassembly of adherens junctions, as
was reported earlier with NO-donating aspirin (36), which con-
tributes toward apoptosis.
Results described here indicate the possibility that targeting

-catenin in TRAIL-resistant cancer cells might be an effective
means of increasing TRAIL sensitivity. Combination of TRAIL
with drugs that can antagonize the

-catenin pathway might
thus be effective in ameliorating TRAIL resistance in cancer
cells. Abnormal activation of the

-catenin pathway has been
linked with various tumorigenic pathways including those in
liver and colon (30, 31). It is important to note that this activa-
tion of the

-catenin pathway is mostly due to either inactivat-
ing mutations of APC or Axin, or stabilizing mutations of

-catenin itself, all of which result in evasion of GSK3

-medi-
ated degradation. Because TRAIL-TZD-mediated degradation
is independent of GSK3

pathway, this combinatorial therapy
might be effective in treating those tumors that have GSK3

degradation-resistant

-catenin activation. Identification of
the effectors and signaling pathways involved will provide
important insight toward increasing the potency of this novel
therapeutic approach.
Acknowledgments—We are grateful to Dr. Robert E. Lanford for pro-
viding the Huh-7 cells, Drs. Wallace K. MacNaughton and Hongying
Wang for the RKO and RKO-

-catenin cells, and Dr. James R.
Woodgett for the WT and GSK3

knock-out (⫺/⫺) MEFs.
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-Catenin Cleavage in Drug-induced Apoptosis
13588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 20 •MAY 15, 2009
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SUPPLEMENTAL FIGURE LEGENDS:
Supplemental Figure S1: Effect of TZD and TRAIL treatment on Huh-7 cell apoptosis: (A) Phase
Contrast microscopic pictures, showing apoptotic morphology of Huh-7 cells following treatment with
either DMSO or TRAIL-TZD combination for 24 hours. (B) Huh-7 cells were treated with DMSO or
50μM TZD and 100ng/ml TRAIL either alone or in combination for the indicated amounts of time. At the
end of incubation, cells were harvested and apoptosis assays were performed via DAPI staining. The data
in each set represent the mean + s.d. of two independent experiments.
Supplemental Figure S2: Effect of increasing concentrations of TZD or TRAIL on Huh-7 cell
apoptosis: (A & B) Huh-7 cells were treated with either DMSO or a combination of 100ng/ml TRAIL
and increasing concentrations of TZD (A) and DMSO or a combination of 50μM TZD and increasing
concentrations of TRAIL (B). At the end of incubation, cells were harvested and apoptosis assays were
performed using cell death detection ELISAPLUS kit. The data in each set represent the mean + s.d. of two
independent experiments.
Supplemental Figure S3: Effect of TRAIL-TZD combination on various cancer cell apoptosis: LNCaP
(A) SKOV3 (B) and RKO (C) cells were treated with TZD and TRAIL either alone or in combination for
24 hours, following which apoptosis assays were performed via DAPI staining. The data in each set
represent the mean + s.d. of two independent experiments.
Supplemental Figure S4: Effect of TRAIL-TZD combination on
β
-catenin downstream targets: (A)
Huh-7 and (B) LNCaP cells were treated with TZD and TRAIL either alone or in combination for 24
hours, following which Western Blot analyses were performed with the antibodies indicated.
Supplemental Figure S5: Effect of
β
-catenin overexpression on TRAIL-induced apoptosis: RKO or
RKO-β-catenin cells were treated with either DMSO or TRAIL-TZD combination for 24 hours. At the
end of incubation cells were harvested and apoptosis assays were performed via cell death detection
ELISAPLUS kit. The data in each set represent the mean + s.d. of at least two independent experiments.
ADMSO TZD+TRAIL
Supplementary Fig S1: Senthivinayagam et al
B
0
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123456789101112
% apoptotic
Lane
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+
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+
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Supplementary Fig S2: Senthivinayagam et al
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% a
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o
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tosis
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TRAIL
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Supplementary Fig S3: Senthivinayagam et al
A
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1234
% a
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o
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to s is
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% apoptotic
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Cell Type
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Huh-7
+++ +
+
-
-
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-
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--
Cyclin D1
GAPDH
Bcl-xL
AKT
Supplementary Fig S4: Senthivinayagam et al
B
Cyclin D1
AKT
GAPDH
+ +
DMSO
Cell Type
TZD
TRAIL
LNCaP
++ +
-
-
-
-
-
--
BclxL
Supplementary Fig S5: Senthivinayagam et al
0
1
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1234
Enrichment
Fcator
Lane
DMSO
TZD (50μM)
TRAIL (100ng/ml)
Cell Type
++-+
+
-+-
-
--
RKO RKO-β-catenin
+
Ajay Rana and Basabi Rana
Yallapragada, Malay Chatterjee, Lucas Wong,
Mishra, Suresh Kanna Paramasivam, Srinivas
Subramanian Senthivinayagam, Prajna
Resistant Cancer Cells
Precedes Drug-induced Apoptosis in
-CateninβCaspase-mediated Cleavage of
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M900248200 originally published online March 16, 2009
2009, 284:13577-13588.J. Biol. Chem.
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