Group Title: BMC Cancer
Title: A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB
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 Material Information
Title: A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB
Physical Description: Archival
Language: English
Creator: Dai, Yao
Liu, Meilan
Tang, Wenhua
Li, Yongming
Lian, Jiqin
Lawrence, Theodore
Xu, Liang
Publisher: BMC Cancer
Publication Date: 2009
Abstract: BACKGROUND:Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.METHODS:The potential of Smac-mimetics to bind XIAP or cIAP-1 was examined by pull-down assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth assay. Silencing of XIAP or cIAP-1 was achieved by transient transfection of short hairpin RNA. Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular fractionation, real time RT-PCR and reporter assay.RESULTS:SH122, but not its inactive analog, binds to XIAP and cIAP-1. SH122 significantly sensitized prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP and cIAP-1 sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH122 to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for IAP-inhibitor-mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by suppressing NF-kappaB target gene expression.CONCLUSION:These results demonstrate that SH122 sensitizes human prostate cancer cells to TRAIL-induced apoptosis by mimicking Smac and blocking both IAPs and NF-kappaB. Modulating IAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate cancer with constitutively active NF-kappaB signaling.
General Note: Periodical Abbreviation: BMC Cancer
General Note: Start page 392
General Note: M3: 10.1186/1471-2407-9-392
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Bibliographic ID: UF00099901
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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BMC Cancer BioMed C

Research article

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced
apoptosis via modulating both lAPs and NF-kappaB
Yao Dait1,4, Meilan Liut2, Wenhua Tang', Yongming Lil, Jiqin Lian1,
Theodore S Lawrencel,3 and Liang Xu*1,3

Address: 'Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA, 2Department of Pathology, University of
Michigan, Ann Arbor, MI 48109, USA, 3Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA and 4Current address
: Department of Radiation Oncology, University of Florida Health Science Center, Gainesville, FL 32610
Email: Yao Dai; Meilan Liu; Wenhua Tang;
Yongming Li; Jiqin Lian; Theodore S Lawrence; Liang Xu*
* Corresponding author tEqual contributors

Published: 6 November 2009
BMC Cancer 2009, 9:392 doi:10.1 186/1471-2407-9-392

Received: 30 October 2008
Accepted: 6 November 2009

This article is available from:
2009 Dai et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a
promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both
X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative
regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule
mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced
apoptosis in prostate cancer cells.
Methods: The potential of Smac-mimetics to bind XIAP or clAP-1I was examined by pull-down
assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth
assay. Silencing of XIAP or clAP- I was achieved by transient transfection of short hairpin RNA.
Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot
analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular
fractionation, real time RT-PCR and reporter assay.
Results: SH 122, but not its inactive analog, binds to XIAP and clAP- I1. SH 122 significantly sensitized
prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced
apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP
and clAP- I sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH 122
to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for lAP-inhibitor-
mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by
preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by
suppressing NF-kappaB target gene expression.
Conclusion: These results demonstrate that SH 122 sensitizes human prostate cancer cells to
TRAIL-induced apoptosis by mimicking Smac and blocking both lAPs and NF-kappaB. Modulating
lAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate
cancer with constitutively active NF-kappaB signaling.

Page 1 of 15
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Primary or acquired resistance of prostate cancer to cur-
rent treatment protocols has been associated with apopto-
sis-resistance in cancer cells, leading to therapy failure
[1,2]. Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) is a member of the TNF family that is in
clinical trials for the treatment of prostate cancer, either
alone or in combination with other treatments [3]. TRAIL
selectively induces apoptosis in prostate cancer cells com-
pared to normal prostate epithelial cells [4]. The relative
resistance of normal cells to TRAIL has been explained by
the lower expression levels of functional death receptors
relative to cancer cells [5,6]. Hence, TRAIL exerts a selec-
tive antitumor activity without eliciting systemic toxicity
in multiple preclinical models, and is considered to be a
prime candidate for prostate cancer therapy [3].

Mechanistically, TRAIL triggers apoptosis via binding to
its functional death receptors DR4 and DR5, and activat-
ing both death receptor (extrinsic) and mitochondria
(intrinsic) apoptosis pathways [7]. Ligation of DR4/DR5
by TRAIL results in caspase-8 activation and directly
cleaves downstream effector caspases [8]. Signals originat-
ing from death receptors can be linked to mitochondria
via Bid, which causes mitochondrial cytochrome c release
and caspase-9 activation. The mitochondrial pathway is
engaged by the release of multiple pro-apoptotic factors
from mitochondria into the cytosol, such as cytochrome
c, Smac and apoptosis inducing factor (AIF). These factors
execute cells through apoptosis in either a caspase-
dependent or independent manner [9].

Despite the fact that TRAIL selectively induces apoptosis
in cancer cells, TRAIL-resistance has been observed in a
substantial number of cancers, including prostate cancer
[10]. It is widely accepted that the inhibitor of apoptosis
proteins (IAP) function as a key negative regulator in
TRAIL resistance [11,12]. Mounting evidence confirms
that XIAP, the most potent anti-apoptotic protein among
IAPs, is responsible for primary or acquired TRAIL resist-
ance in tumor cells [13-16]. Overexpression of XIAP
increases resistance to TRAIL-induced apoptosis, while
downregulation of XIAP restores responsiveness to TRAIL
[17,18]. At the transcriptional level, almost all IAP pro-
teins are driven by the upstream transcription factor NF-
kappa B (NF-KB), which can be stimulated by multiple
stimuli, including TRAIL [19]. TRAIL-induced NF-KB acti-
vation attenuates apoptosis, predominantly by upregulat-
ing various anti-apoptotic proteins, including IAPs
[20,21]. Therefore, NF-KB functions as an upstream regu-
lator of IAPs and negatively regulates TRAIL signaling. The
role of NF-KB in the anti-apoptotic process has been stud-
ied in prostate cancer cells both in vitro and in vivo. In
prostate cancer cell lines, there seems to be an inverse cor-
relation between androgen receptor (AR) status and con-

stitutive NF-KB activity [22]. Thus it is tempting to
speculate that the constitutive activation of NF-KB may
contribute to prostate cancer cell survival and treatment
resistance following androgen ablation [22].

Smac functions as an endogenous IAP-antagonist [23].
Upon stimulation by TRAIL, Smac is released from mito-
chondria into the cytosol [24]. The released Smac interacts
with XIAP through the N-terminal four conserved amino
acid residues (AVPI) that bind to the baculoviral IAP
repeat 3 (BIR3) domain of XIAP and eliminates the inhib-
itory effect of XIAP on caspase activation [25-27]. Due to
the impressive pro-apoptotic role of Smac, synthetic small
molecule Smac-mimicking compounds (Smac-mimetics)
are being developed to sensitize apoptosis-resistant cancer
cells to various apoptotic stimuli [3,28]. Smac-mimetic
IAP-antagonists induce TNFa-dependent apoptosis in sev-
eral transformed cell lines [29-31], and small molecule
Smac-mimetics successfully sensitize TRAIL-induced
apoptosis by blocking functions of IAPs in multiple can-
cer cells [11,16,32,33]. These studies provide a solid foun-
dation for our assertion that mimicking Smac may
represent a promising strategy for restoring defective
apoptosis signaling triggered by TRAIL in prostate cancer

Based on the high-resolution experimental 3D structure of
Smac in complex with the XIAP BIR3 domain, a group of
potent non-peptidic Smac-mimetics, called SH-com-
pounds, were designed that mimic the tetra-peptide at the
N-terminal of the Smac protein [34,35]. These cell-perme-
able compounds show at least 20-fold more potential
than the natural Smac peptide to bind to the XIAP BIR3
domain in a cell-free system [34-36]. In the current study,
we evaluated the sensitizing potential of one of these
compounds, SH122, on TRAIL-mediated cell death in sev-
eral human prostate cancer cell lines. We also investigated
potential molecular targets and the mechanism of action
involved in SH 122-mediated TRAIL sensitization.

Cell Culture and Reagents
Human prostate cancer DU145 and LNCaP cells were
obtained from the American Type Culture Collection
(Manassas, VA). Androgen-independent prostate cancer
cell line CL1 derived from its androgen-dependent cell
line LNCaP was kindly provided by Dr. Arie Belldegrun
(University of California, Los Angeles). Cells were rou-
tinely maintained in Dulbecco's Modified Eagle Medium
(DMEM, Gibco) with 10% fetal bovine serum (FBS) and
2 mM L-glutamine. Cultures were maintained in a humid-
ified incubator at 37C with 5% CO2. Small molecule
Smac-mimetic SH122 as well as its inactive analogs
SH123 and SH110 were kindly provided by Dr.
Shaomeng Wang, University of Michigan. TRAIL was pur-

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BMC Cancer 2009, 9:392

chased from Cell Sciences (Canton, MA). Antibodies
against XIAP and cIAP-1 were purchased from BD Bio-
sciences (San Jose, CA). Antibodies against PARP, RelA
and IKBa were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Other antibodies include: anti-caspase-
3 (BioVision, Mountain View, CA), anti-caspase-8 (Calbi-
ochem, San Diego, CA), anti-caspase-9 (Novus Biologi-
cals, Littleton, CO), anti-Bid (Cell Signaling Technology,
Beverly, MA), and anti-3-actin (Sigma, MO). Chemicals
were from Sigma unless otherwise indicated.

Cytotoxicity Assay
To detect the survival of cells after treatment, a standard
cell growth assay was performed using the CCK-8 detec-
tion kit (Dojindo Molecular Technologies, Gaithersburg,
MD) following the manufacturer's instructions. Absorb-
ance was detected at 450 nm and 650 nm respectively,
using a microplate reader (TECAN ULTRA, Research Tri-
angle Park, NC). Cell viability (%) was normalized by
dividing normalized absorbance of treated samples by
that of the untreated control [37]. Inhibitory concentra-
tion 50% (ICso0) was calculated by GraphPad Prism 5.0
(San Diego, CA).

Pull-down Assay
Cells (1.0 x 107) were disrupted in a lysis buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate), with freshly added protease inhib-
itor cocktail (Roche). Cell lysates were homogenized by
passing through a 27-1/2G syringe needle (BD). After cen-
trifugation at 10,000 x g for 15 min at 4C, the superna-
tant was harvested and incubated with biotin-labeled
Smac-mimetic compounds with or without non-labeled
compounds, for 1 hour at 40C. Lysates were incubated
with pre-cleared Streptavidin-Agarose beads (Invitrogen)
by gently rotating for 2 hours at 4 o C. The beads were col-
lected and washed with washing buffer (Roche) and
eluted in 60 il of loading buffer (Bio-Rad, Hercules, CA).
After boiling for 5 min, the eluents were analyzed by
immunoblotting to detect proteins that interacted with
the compounds [38].

Apoptosis Assay
For apoptosis analysis by flow cytometry, DU145 cells
were treated with SH 122 and TRAIL, alone or in combina-
tion, with SH110 used as a negative control. Cells were
harvested by trypsinization and washed twice with ice-
cold PBS. After centrifuge, cells were stained with 100 1il
Annexin V-FITC diluted in binding buffer (10 mM
HEPES,100 mM NaCl, 10 mM KC1, 1 mM MgCl2, 1.8 mM
CaC12) containing propidium iodide (50 ig/ml). Cells
were incubated for 15 min at room temperature before
analysis by flow cytometry with FACScan (BD) using a
488-nm laser line. Data were analyzed using WinMDI 2.8

software (Purdue University Cytometry Laboratories) as
described previously [38].

shRNA Transfection
The shRNA specific for XIAP in plasmid pRS-shXIAP29
was purchased from OriGene (Rockville, MD). The
shRNA specific for clAP-1 in plasmid pGB-shcIAP-1 was
purchased from BioVision (Mountain View, CA). shRNA
plasmids (2.0 rig) were transfected into DU145 cells using
LipofectAMINE 2000 (Invitrogen, Carlsbad, CA), follow-
ing the manufacturer's instructions. Forty-eight hours
after transfection, cells were treated with Smac-mimetic
compounds and/or TRAIL and processed for cytotoxicity
analysis. Knockdown effect was detected by Western blot.

Western Blot Analysis
Whole cell proteins were isolated by RIPA buffer (50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40,
0.25% Sodium deoxycholate and 1 mM EDTA) with
freshly added protease inhibitor cocktail (Roche). Whole
cell lysates were clarified by centrifugation at 10,000 x g
for 10 minutes at 40 C. Total protein concentrations were
determined by BCA Protein Assay (Pierce, Rockford, IL).
Equal amounts of proteins were loaded to pre-cast 4-20%
SDS-PAGE gels (Invitrogen). Proteins resolved on gels
were transferred to PVDF membranes (Bio-Rad). After
electro-transfer, membranes were blocked with 5 % nonfat
milk in TBS-T buffer (20 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween 20), and probed with the desired pri-
mary antibodies. Blots were then incubated with horse-
radish-conjugated secondary antibodies and detected
with the SuperSignal West Pico chemiluminescence sub-
strate (Pierce), and exposed to an X-ray film (Kodak,
Rochester, NY). Intensity of the desired bands was ana-
lyzed using TotalLab software (Nonlinear Dynamics, Dur-
ham, NC).

Cytosol and Nuclei Fractionation
To detect subcellular redistribution of NF-KB proteins,
cytoplasmic and nuclear fractions were prepared accord-
ing to the method reported [39] with modifications.
Briefly, treated cells (5 x 106 cells) were resuspended in
200 Al of hypotonic buffer (10 mM HEPES, 5 mM KC1, 1.5
mM MgCl2, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 1 mM dithiothreitol (DTT), and protease inhibi-
tors), mixed well, and incubated with constant rotation at
4C for 15 min. Nonidet P-40 (10%) was then added to
reach a final concentration of 0.5%. Cytosolic extract was
cleared by centrifugation at 12,000 rpm for 1 min. The
pellets were washed once with hypotonic buffer, and
resolved in nucleus extraction buffer (20 mM HEPES, 50
mM KC1, 300 mM NaCl, 1 mM PMSF, 1 mM DTT, and
protease inhibitors), with constant rotation at 40 C for 45
min. Nuclear extracts were harvested after centrifuging for
10 min at 12,000 rpm. Subcellular proteins were quanti-

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BMC Cancer 2009, 9:392

fled by BCA assay before being employed to Western blot

Quantitative Real-time PCR (qRT-PCR)
qRT-PCR was performed to determine the NF-KB target
gene expression level [40]. Total RNA was extracted from
DU 145 cells using TRIzol (Invitrogen). Reverse transcrip-
tion reaction with 1 jig of total RNA in 100 il was carried
out following the instructions of the TaqMan Reverse
Transcription Kit (Applied Biosystems, Foster City, CA).
For quantitative PCR, 1 jil of gene primers with SYBR
Green (Applied Biosystems) in 20 jil of reaction volume
was applied [41]. Primers were designed as: TNF, forward,
5'TCTITAGCACTCCITGGCAAAAC3'; Actin (as an inter-
nal control): forward, 5'ATGCAGAAGGAGATCACTGC3',
primers were commercially obtained from Applied Bio-
systems. All reactions with TaqMan PCR Master Mix
(Applied Biosystems) were performed on the Mastercycler
realplex2 S (Eppendorf, Westbury, NY). Target gene mRNA
levels were normalized to actin mRNA according to the
following formula: [2^-(Cr target Cr Actin)] x 100%,
where CT is the threshold cycle. Fold increase was calcu-
lated by dividing the normalized target gene expression of
the treated sample by that of the untreated control

NF-KB Reporter Assay
DU145 cells were seeded into a 48-well plate 24 h before
transfection. For each well, cells were transiently cotrans-
fected with 0.1 jig of a NF-KB reporter construct (pNF-KB)
or a control reporter plasmid (pControl) (Panomics Inc.,
Fremont, CA), together with 0.06 jig of P-galactosidase
reporter vector (Promega, Madison, WI), which was used
to normalize NF-KB reporter gene activity, using Lipo-
fectAMINE 2000 (Invitrogen). Twenty-four hours after
transfection, cells were treated with TNF-a or TRAIL for 0
h, 1 h, 2 h, 4 h and 6 h. To determine the effect of SH122
on TRAIL-induced NF-KB activation, cells were pretreated
with SH 122 or SH 123 for 1 h followed by TRAIL stimula-
tion for 4 h. Cell lysates were prepared using Reporter
Lysis Buffer (Promega). For luciferase and P-galactosidase
assays, the samples were measured on a microplate lumi-
nometer using the Bright-Glo luciferase assay kit and P-
galactosidase enzyme assay kit (Promega), respectively,
according to the manufacturer's instructions. Fold of acti-
vation was calculated for each treated sample by dividing
normalized luciferase activity with that of the untreated

Statistical Analysis
Two-tailed Student's t-test was employed, using GraphPad
Prism 5.0 software (San Diego, CA). A threshold of P <
0.05 was defined as statistically significant.

Validation of interactions between Smac-mimetic
compound SH122 and lAPs
Based on 3-D rational design and computational mode-
ling, SH 122 (Figure 1A) was synthesized and developed as
a non-peptide small molecule lAP-antagonist that is
20~30-fold more potent than the N-terminal tetrapeptide



SH122: R= -NHCH3
SH123: R= -OH


-SH22BL 0 20 20
SH122BL 0 20 20
SH122 0 0 200

XIAP -- -

clAP-1 -


SH122BL 0 1 1 2.5 2.5 0 0

SH122 0 0 10 0 25 0 0

SH123BL 0 0 0 0 0 2.5 2.5

SH123 0 0 0 0


0 25

clAP-1 -p

Figure I
Smac-mimetic compound interacted with XIAP and
clAP-I in human prostate cancer cells. A. Structure of
the Smac-mimetic compound SH 122, and its inactive control
compounds SH 123 and SH I 10. B. Pull-down assay. Biotin-
labeled SH 122 (SH I 22BL, 20 jiM) was incubated with whole
cell lysates of DU 145 cells with or without 10-fold excess of
non-labeled SH 122, followed by incubation with precleared
Streptavidin agarose beads. Eluted beads were employed to
Western blot analysis with anti-XIAP or clAP- I antibody.
Data shown represent one of at least three independent
experiments. SHI22BL, Biotin-labeled SH122. C. CLI cell
lysate was incubated with I jiM and 2.5 jiM of SH 122BL, or
2.5 jiM of SH-123BL, with or without 10-fold excess of their
unlabeled forms. SH 123BL, Biotin-labeled SH 123.

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BMC Cancer 2009, 9:392

of Smac in binding to IAPs, while inactive analogs SH123
and SH110 were shown to be 200-fold less potent [35]. To
verify the specific binding of SH122 to IAPs, we employed
a pull-down assay with biotin-labeled SH122 (SH 122BL)
in human prostate cancer cells. As shown in Figure 1 B,
both XIAP and clAP-1 were pulled down by SH122 in
DU145 cells. Furthermore, pre-incubation with a 10-fold
excess of non-biotin-labeled SH122 resulted in a more
than 90% block of the binding of SH122BL to XIAP/clAP-
1, suggesting that the binding was specific. In another
androgen-independent prostate cancer CL1 cell line, 1 gM
of SH 122 was sufficient to pull down both XIAP and clAP-
1 (Figure IC), and as with DU145, the binding effect was
blocked by a 10-fold excess of non-labeled SH122. By
contrast, the negative control compound SH123BL
showed almost no binding to either XIAP or clAP-1, as
evidenced in the previous report [38]. These results dem-
onstrate that Smac-mimetic SH122 potently and specifi-
cally interacts with XIAP and clAP-1 in human prostate
cancer cells.

SH 122 sensitizes TRAIL-induced prostate cancer cell
growth inhibition
To detect the combination effects of a Smac-mimetic and
TRAIL, cytotoxicity was tested after concurrent treatment
with TRAIL and SH122. In DU145 cells, while TRAIL
alone had a minor effect on decreasing cell viability, 5 gM
and 10 gM of SH122 showed a 100- and 600-fold sensiti-
zation, respectively, compared to TRAIL alone (Figure
2A). By contrast, the negative control compound SH110
produced no sensitization. Similar results were observed
in two other prostate cancer cell lines. As shown in Figure
2B, SH122, but not SH110, potentiated TRAIL-induced
cell growth inhibition in LNCaP cells. In CL1 cells that
were derived from LNCaP, SH122 showed dose-depend-
ent effects on TRAIL sensitization, although the concen-
tration used was approximately 10-fold less than that for
LNCaP cells (Figure 2C). Notably, CL1 seemed to be more
sensitive to TRAIL compared with LNCaP, as shown by
IC5s, suggesting that ablation of the androgen-receptor
may result in upregulation of TRAIL receptor expression.
These data demonstrate that the Smac-mimetic com-
pound SH 122 potentiates TRAIL-mediated cytotoxicity in
both TRAIL-resistant and TRAIL-sensitive prostate cancer
cell lines.

SH122 enhances TRAIL-induced apoptosis
To determine whether apoptosis was involved in TRAIL-
induced cell death, we examined apoptosis induced by
TRAIL in combination with SH122, with Annexin V-FITC
and PI staining by flow cytometry analysis. As shown in
Figure 3, SH122 increased TRAIL-induced apoptosis in a
dose-dependent manner. Even at a lower concentration
(2.5 gM), SH122 enhanced TRAIL-induced total apopto-
sis as compared with TRAIL and SH122 alone (Figure 3).

In contrast, TRAIL alone (50 ng/ml) moderately induced
apoptosis, and SH122 alone showed a minor effect on
apoptosis, with a slightly increased apoptotic cell popula-
tion compared with the control even at the highest con-
centration (Figure 3).

Both death receptor and mitochondrial pathways are
involved in SH 122-sensitized, TRAIL-induced apoptosis
Based on TRAIL-induced apoptotic signaling, we exam-
ined the expression of caspases treated with SH122 and
TRAIL. Caspase-8 was cleaved into fragments (p43/41 and
pl18) as early as 4 h after exposure to TRAIL alone, while
cleavage of caspase-8 became more intense with increased
concentrations of SH122, especially at 6 h after treatment
(Figure 4A). A similar tendency was observed for caspase-
3. Activation of caspase-3 was further confirmed by
poly(ADP-Ribose) polymerase (PARP) cleavage, a typical
feature of caspase-dependent apoptosis. PARP was
cleaved by TRAIL alone at 4 h (Figure 4A), while in com-
bination with SH122, cleaved PARP survived up to 8 h.
These results suggest that SH 122 enhances TRAIL-induced
apoptosis by activating caspases and promoting PARP

To determine the combination effect of TRAIL and SH 122
on the mitochondrial pathway, caspase-9 and Bid activa-
tion were examined. As shown in Figure 4B, Bid levels did
not change at 4 h or 6 h, while at 8 h, Bid levels decreased
after combination treatment, but not from treatment by
TRAIL alone. Furthermore, caspase-9 cleavage could be
detected as early as 4 h by either TRAIL alone or in combi-
nation with SH122, as evidenced by the appearance of its
cleaved forms, p35 and pl7. Interestingly, both pro- and
cleaved caspase-9 were diminished at 8 h, indicating that
caspase-9 activation was an early event with Bid cleavage
(Figure 4B). These data suggest that the mitochondria
pathway is also involved in TRAIL-induced, SH 122-medi-
ated apoptosis. Taken together, these in vitro apoptosis
data reveal that SH122 potentiates TRAIL-induced apop-
tosis by activating both the extrinsic death receptor path-
way and the intrinsic mitochondrial pathway, typically
through activation of the caspase cascade. More impor-
tantly, SH-122 combined with TRAIL induced a longer-
lasting apoptosis than TRAIL alone (8 h vs. 4 h), showing
that SH-122 enhanced the effect of TRAIL treatment.

Downregulation of XIAP and clAP-I sensitizes TRAIL-
induced cell death
It is well established that IAPs (especially XIAP) are
responsible for blocking TRAIL-induced apoptosis by
inhibiting caspase functions [ 11,14,43]. To investigate the
potential link between IAPs and TRAIL-resistance, short-
hairpin RNA (shRNA) of XIAP or clAP-1 was transfected
into DU145 cells, and cytotoxicity was examined. As
shown in Figure 5, XIAP or clAP-1 knockdown shifted the

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BMC Cancer 2009, 9:392

IC50 (ng/ml)
-m- DMSO >300
-0- SH110(10 pM) >300
-A- SH122(2.5 M) 72.9
-*- SH122(5 M) 2.4
--- SH122(10 iM) 0.5

TRAIL conc.(ng/ml)

IC50 (ng/mi)
-0- DMSO 292.1
-B- SH110 20um >300
-a- SH122 5um 137.6
-V- SH122 10um 52.3
-*- SH122 20um 8.5

TRAIL conc.(ng/ml)

IC50 (ng/ml)
.5 p.M) 19.5
ipM) 17.9

1 10 100 1000
TRAIL conc.(ng/ml)

Figure 2
SH122 promoted TRAIL-induced cell death in prostate cancer cell line DU145 (A), LNCaP (B) and CLI (C).
Cells (5,000 cells/well in 96-well plates) were treated with different concentrations of TRAIL and SH 122, alone or in combina-
tion, with SH I 10 as a negative control. After incubation for 96 h, cells were stained with Cell Counting kit-8 reagent. The opti-
cal density of each sample was measured. Data were normalized as described in Materials and Methods. Data were presented as
mean SD (n = 3).

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BMC Cancer 2009, 9:392




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Figure 3
SH 122 enhanced TRAIL-induced apoptosis. DU 145 cells were seeded into 6-well plates at a concentration of 2 x I 0/ml,
and exposed by 2.5, 5 and 10 iM of SH 122, with or without TRAIL (50 ng/ml). Eighteen hours after incubation, cells were har-
vested and processed for Annexin V-FITC and PI staining by flow cytometry. Numbers represented total apoptosis (Annexin V
positive cell population). Data represented one of three independent experiments.

cytotoxicity curve to the left, i.e., sensitized the cells to
TRAIL. Based on IC50, a more than 300-fold sensitization
was observed in the cells transfected with XIAP shRNA as
compared with the vector control (Figure 5A and 5B).
Similarly, in clAP-1-shRNA transfected cells, an approxi-
mately 100-fold sensitization was achieved as compared
with that of the vector control (Figure 5C and 5D). These
results demonstrate that knockdown of XIAP and clAP-1
effectively sensitizes the cells to TRAIL, indicating that
both XIAP and clAP-1 are involved in TRAIL-resistance,
and that silencing XIAP and/or c-IAPl can overcome such
resistance in prostate cancer cells.

Downregulation of XIAP attenuates sensitization potential
of SH122 on TRAIL-induced cell death
Earlier studies have shown that both XIAP and clAP-1 are
specific targets of SH122 (Figure 1B and IC), and knock-
down of XIAP/clAP-1 resulted in significant TRAIL-sensi-
tization (Figure 5). To evaluate the role of XIAP in SH122-
mediated TRAIL sensitization, we treated XIAP knock-
down cells with SH122 and TRAIL. In vector shRNA

(shControl)-transfected cells, negative compound SH 110
showed a moderate sensitizing effect, while SH122 dra-
matically synergized TRAIL-induced cell death in a dose-
dependent manner. Even at the lowest concentration (0.1
riM), SH 122 was 22-fold more potent in sensitizing TRAIL
than a high concentration of SH 110 (10 riM) (Figure 6A).
However, in XIAP shRNA-transfected cells, the sensitizing
potential of SH122 was 102l104-fold less potent than
shown by the shControl cells (Figure 6B). These results
demonstrate that silencing XIAP dramatically attenuates
SH122-mediated sensitization of TRAIL-induced cytotox-
icity, suggesting that XIAP is an important target of IAP-
inhibitors involved in TRAIL sensitization.

SHI22 inhibits TRAIL-induced NF-KB activation
Because NF-KB activation is known to play a crucial role in
inhibiting apoptosis [19,21], we thought it was important
to evaluate the effect of SH122 on NF-KB activation
induced by TRAIL. First, we treated cells with TRAIL alone
at the concentration that achieved apoptosis, to create
optimal conditions for NF-KB activation in our system.

Page 7 of 15
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10 (pM)

BMC Cancer 2009, 9:392



4 hours

TRAIL (ng/ml) -- -- -- 300 300 300 300 300
SH122 (plM) -- 10 20 -- 10 20 10 20
zVAD (WM) -- -- -- -- -- 2 2

A Death receptor pathway



Procaspase-3, r





B Mitochondria pathway

BID--__ ep.

Procaspase-9- __ - _


- -e -

6 hours

-- 10

300 300 300 300 300
-- 10 20 10 20
- -- 2 2

- ~

e -

8 hours

- -- 300 300 300 300
10 20 -- 10 20 10
-- -- -- -- -- 2


eflOf 40 ma- I OMWewa"

- r ~ -


a mm a - -~

I. I.


Figure 4
SH 122 potentiated TRAIL-induced apoptosis by activating both the death-receptor pathway (A) and mito-
chondrial pathway (B). DU 145 cells were treated with 10 and 20 gtM of SH 122, in the presence or absence of 300 ng/ml of
TRAIL, with or without pretreatment with zVAD (2 gtM), for 4, 6, and 8 h, respectively. Whole cell lysates (30 gtg) were sub-
jected to Western blot analysis. Membranes were probed with antibodies against caspase-8, caspase-3, caspase-9, PARP and
Bid. Actin was shown as a loading control.

TRAIL induced IKBa degradation by 60% at 40 min post-
treatment, and concomitantly, nuclear RelA expression
increased over 3-fold at 40 min and lasted for 3 h of treat-
ment (Figure 7A). This revealed that TRAIL induced NF-KB
activation via the classic pathway by degrading cytosolic
IKBa and promoting RelA nuclear translocation. It is
worth noting that at 120 min post treatment, cleaved
PARP was observed in the nuclei extracts, suggesting a
quick apoptosis induced by TRAIL along with NF-KB acti-
vation, also reflecting a balance of cell death and cell sur-
vival triggered by the same stimuli (Figure 7A). TRAIL
consistently induced multiple NF-KB target genes expres-
sion. For the four target genes examined, TNF and IL8

expression reached their peak at 2 h post treatment, while
ICAM-1 and BIRC4 expression continued to increase dur-
ing treatment (Figure 7B).

Next, we pretreated DU145 cells with varying concentra-
tions of SH122 or SH123 to evaluate the effect of the IAP-
antagonist on TRAIL-induced NF-KB activation. Pre-treat-
ment of SH122 potently suppressed IKBa degradation
and RelA translocation in a dose-dependent manner (Fig-
ure 7C). At the same time point, SH122 was consistently
shown to suppress expression of all three NF-KB target
genes by 30-80% (P < 0.01 vs. control) even at a lower
concentration (Figure 7D). In comparison, even high con-

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BMC Cancer 2009, 9:392

TRAIL (ng/ml)

Vector clAP-1

clAP-1---m -


D 120-
- 100-

> 80-


J 40-

0 20-


IC50 (ng/ml)
A shVector >300
shclAP-1 3.8

U ----
0.001 0.01

0.1 1 10
TRAIL (ng/ml)

Figure 5
Downregulation of XIAP or clAP-I sensitized TRAIL-induced cell death. DU 145 cells were transfected with shRNA
of XIAP (shXIAP) (A, B) or clAP- I (shclAP- I1) (C, D), or the vector control (shVector), and knockdown effect was measured
48 h after transfection (A, C). Transfected cells were treated with TRAIL, and cytotoxicity was determined by CCK-8 detec-
tion kit (B, D). Normalized data were presented as mean SD (n = 3).

centrations of the negative compound SH 123 altered nei-
ther NF-KB protein redistribution (Figure 7C) nor NF-KB
target genes expression (Figure 7D).

To further validate that SH-122 inhibits NF-KB pathway,
we analyzed the effects of SH122 on NF-KB activation
induced by TRAIL in DU145 cells using luciferase-based
NF-KB reporter assay. We first examined the time-course
of TRAIL-induced NF-KB activation by NF-KB reporter
assay. 4 h TRAIL treatment resulted in ~5-fold NF-KB acti-
vation, and at 6 h over 9-fold activation was observed
(Figure 7E), but at this time point the cells started to show
signs of cell death. Therefore, we selected the 4 h TRAIL
treatment in our NF-KB reporter assay. As shown in Figure
7F, SH122 inhibited TRAIL-induced NF-KB activation by
>60% at doses of 5 and 10 uM, whereas the control com-
pound SH 123 had no such effect. Similar results were also
observed in DU145 cells stimulated with TNFa (data not
shown). These results demonstrate that SH122 exhibits a

promising effect of blocking the TRAIL-induced NF-KB

In this study, we found that the small molecule Smac-
mimetic SH 122 potently sensitized TRAIL-induced apop-
tosis in multiple human prostate cancer cell lines. We also
found that although downregulation of either XIAP or
clAP-1 sensitized TRAIL-mediated cytotoxicity, XIAP
knockdown attenuated SH 122-mediated TRAIL sensitiza-
tion. Our results demonstrate that IAPs are valid molecu-
lar targets for modulating TRAIL sensitivity in prostate
cancer cells, and show that blocking IAPs achieves
improved efficacy and overcomes resistance to TRAIL. In
addition, our results demonstrate that NF-KB is involved
in regulating sensitivity of prostate cancer cells to TRAIL,
and a Smac-mimetic can augment TRAIL-induced apopto-
sis by blocking both IAPs and NF-KB (Figure 8).

Page 9 of 15
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Vector XIAP



100 1000

BMC Cancer 2009, 9:392


Sensitization (Fold)

DM SO 1.0
SH110 (10 pM) 8.8X102
SH122 (0.1 piM) 2.1X104
SH122 (1 pIM) 3.4X105
SH122 (10 pM) 1.2X106

TRAIL (ng/ml)



Sensitization (Fold)
DMSO 1.0
SH110 (10 pM) 1.9
SH122 (0.1 IM) 12.0
SH122 (1 piM) 41.3
SH122 (10 uM) 143.9

0.001 0.01 0.1 1 10 100
TRAIL (ng/ml)

Figure 6
Downregulation of XIAP attenuated sensitization effect of SH 122 on TRAIL-induced cell death. Cells transfected
with either XIAP shRNA (A) or the vector control shRNA (B) were treated with serial diluted TRAIL, alone or in combination
with 0.1, I and 10 iM of SH 122, respectively, with 10 iM of SH I 10 as a negative control. Cytotoxicity was determined by
CCK-8 detection kit. Normalized data were presented as mean SD (n = 3). Fold of sensitization was calculated by dividing
ICs0 of the compound-treated group by that of DMSO control.

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BMC Cancer 2009, 9:392

BMC Cancer 2009, 9:392


35 0

0 20 40 60 120 180 (min)

|KBa -
1.0 1.0 0.4 0.9 1.0 1.2 Fold CE
Actin (IKBalAclIn)

RelA- ]-ill llllglillll -
1.0 1.3 3.2 3.1 3.4 3.3 Fold NE

p85 -0'


SH122 SH123 MG132

Con Con 1 5 10 10 10 (liM)
IhBa -I W- i

1.0 0.3 0.4 0.9 1.3 0.4 0.8 Fold A CEt

RelA -I1 - ]
1.0 2.8 3.2 3.0 0.9 4.0 0.2 Fold NE
PARP O- eM- aft -ag n so .M (Re lAPARP)

-*-B IRC4

0 20 40 60 120 180 min

10 TNF


4 IL8
, 3

a 2


Con Con 1 5 10 10 10 (gM)

SH122 SH123 MG132


Cl pControl
- pNF-kB

= pControl
- pNF-kB

0 1 2 4 6
Time after TRAIL treatment (hours)

Con Con Sum 10um 10um

SH122 SH123

Figure 7 (see legend on next page)

Page 11 of 15
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Figure 7 (see previous page)
SH 122 suppressed TRAIL-induced NF-KB activation. A. DU 145 cells were treated with 300 ng/ml of TRAIL for the
indicated time. Cytosol and nuclei subcompartments were fractionated for detection of IKBa and RelA, with Actin and PARP
used as markers of cytosolic and nuclear extracts, respectively. Relative expression of IKBa and RelA was shown by dividing
band intensity by that of Actin and PARP, respectively. CE, cytosolic extract; NE, nuclear extract. B. After treatment with 300
ng/ml of TRAIL, cells were harvested for quantitative RT-PCR detect NF-KB target genes. Fold increase of gene expression was
calculated by dividing the normalized gene expression activity by that of the untreated control. C. Cells were pre-treated with
desired compounds for I h, and challenged with TRAIL (300 ng/ml) for 40 min. Cytosol and nuclei subcellular compartments
were fractionated for detection of IKBa and RelA, respectively. MG 132 was used as a positive control for blocking NF-KB. D.
After treatment as described in C, cells were harvested for quantitative RT-PCR to detect three NF-KB target genes. E, Time-
course of TRAIL-induced NF-KB activation examined by NF-KB luciferase reporter assay. DU145 cells were transiently co-
transfected with pNF-KB or pControl together with P-galactosidase plasmid, and then treated with TRAIL for the indicated
time. Luciferase and P-galactosidase activities were measured as described in Materials and Methods. F, SH 122 inhibited TRAIL-
induced NF-KB activation in NF-KB luciferase reporter assay. Transfected DU 145 cells were pretreated with SH 122 or SH 123
for I h followed by TRAIL treatment for 4 h. Fold of NF-KB activation was calculated by dividing the normalized luciferase
activity by that of the untreated control. Representative results of at least two independent experiments. Columns, mean; bars,
SD (n = 3). Con, DMSO vehicle control. ** P < 0.01; P < 0.05, Student's t-test (n = 3).

Smac-mimetic IAP-antagonists sensitize TRAIL-induced
apoptosis by blocking XIAP function in multiple tumor
models, including breast cancer [32], multiple myeloma
[16], glioblastoma [11] and ovarian cancer [33]. Embelin,
a natural XIAP inhibitor [44], sensitized TRAIL-induced
apoptosis in pancreatic cancer cells [45]. These findings
provide a strong rationale for using Smac-mimetics to
achieve TRAIL-sensitization by functional inhibition of
the overexpressed XIAP. To our knowledge, only a limited
number of studies have reported on the combination of
small molecule Smac-mimetic candidates with TRAIL in
prostate cancer therapy. Interestingly, in prostate cancer
cell lines, no strict correlation has been observed between
XIAP expression and TRAIL responsiveness [3]. This dis-
crepancy indicates that XIAP is not the only determinant
of TRAIL-resistance in prostate cancer. Nevertheless,
blocking XIAP function by transient overexpression of
Smac achieved a promising enhanced efficacy in combi-
nation with TRAIL in prostate cancer cell lines [46], indi-
cating that XIAP is the predominant target for TRAIL
sensitivity. In our system, we found that the lAP-antago-
nist alone did not induce apoptosis [35], but potently sen-
sitized TRAIL-induced cytotoxicity in TRAIL-resistant
DU145 and LNCaP cells, as well as in relatively TRAIL-
sensitive CL1 cells. Our finding, consistent with other
reports [17,47,48], suggests that the lAP-antagonist exerts
a potent sensitization effect independent of cell respon-
siveness to TRAIL, and provides an attractive strategy for
using lAP-antagonists in combination treatment.

In addition to IAPs, NF-KB is another well documented
pro-survival factor that is involved in mediating resistance
to TRAIL-induced apoptosis in tumor cells [49]. It has
been reported that constitutively active NF-KB signaling
leads to TRAIL-resistance by upregulating XIAP in multi-
ple human cancer cells [50], and in certain tumor cell

types, NF-KB is the primary cause for TRAIL resistance
[10]. Moreover, there is mounting evidence that clAP-1
physically interacts with adaptor proteins in TNFa/TRAIL-
mediated NF-KB signaling [51-53], suggesting that lAPs
serve as "bridging" molecules between the apoptosis path-
way and NF-KB pathway triggered by TRAIL (Figure 8).
Thus it is reasonable to postulate that the pro-apoptotic
lAP-antagonists may modulate NF-xKB. Indeed, several
recent studies reveal that Smac-mimetics (lAP-antago-
nists) can induce TNFa-dependent apoptosis in Smac-
sensitive cell lines by degrading clAPs and regulating NF-
KB signaling [29-31]. These findings indicate that in cell
lines that are sensitive to both Smac and TNFa, an IAP-
antagonist itself is sufficient to induce cell death through
autocrine TNFa signaling and caspase-8-dependent apop-
tosis [31,53].

Apart from these findings, our study shows that Smac-
mimetics as a single agent induce neither cell death nor
NF-KB activation in androgen-independent prostate can-
cer cells, suggesting that both apoptosis and NF-KB failed
to be activated by the Smac-mimetic alone in chemo- or
radioresistant cells with constitutively active NF-KB signal-
ing. The mechanism underlying such a discrepancy in dif-
ferent cell types remains to be investigated. However, our
data provide the first evidence that a potent Smac-mimetic
lAP-antagonist directly blocks TRAIL-induced NF-KB acti-
vation in prostate cancer cells. In fact, at the concentration
that effectively suppressed NF-KB activation, SH 122 sensi-
tized the effect of TRAIL several-hundred-fold, suggesting
that blocking NF-KB by a Smac-mimetic is sufficient for
TRAIL sensitization. Additionally, SH122-mediated inhi-
bition of IKBa degradation reflects an effect at the level of
the IKBa kinase (IKK) complex that is in agreement with
others' observations in different systems [11,54], indicat-
ing activation of the canonical NF-KB pathway. Further-

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BMC Cancer 2009, 9:392



I ^^ LclAP-1J

PARP cleavage

Figure 8
Working model of Smac-mimetic lAP-antagonist sensitizing TRAIL-induced apoptosis by suppressing NF-KB.
TRAIL triggers apoptosis via both the death-receptor (DR4/DR5) and mitochondrial pathways, by activating initiator caspase-8
and -9, and effector caspase-3. Furthermore, both Bid and PARP are cleaved by caspases, which are typical predictors of
TRAIL-mediated apoptosis. A Smac-mimetic effectively blocks IAP (XIAP/clAP-1) function and facilitates caspase activation.
Simultaneously, the Smac-mimetic suppresses TRAIL-induced classical NF-KB activation by preventing IKBUc degradation and
RelA nuclear translocation. Blockade of NF-KB XIAP signaling by small molecule Smac-mimetic abolishes counteraction of
pro-survival factors on TRAIL-mediated apoptosis.

more, SH122 suppresses XIAP mRNA expression driven
by NF-xB, demonstrating that TRAIL-mediated sensitiza-
tion by small molecule Smac-mimetic is associated with
functional inhibition of both XIAP and NF-xB, especially
in androgen-independent prostate cancer. Future work
will focus on evaluating the response to TRAIL and the
lAP-antagonists in androgen-dependent (AD) LNCaP
cells and their androgen-independent (AI) derivative CL1
cells. This isogenic (for hormone dependence) cell model
should permit us to determine if NF-KB plays an essential
role in the transition from AD to AI prostate cancer and to
discover if overcoming resistance to TRAIL-induced apop-
tosis can be achieved by down-modulating the NF-KB -
IAP signaling pathway.

Resistance to chemo- or radiotherapy, which is often asso-
ciated with recurrence after prior androgen deprivation
therapy in human prostate cancer, remains a severe clini-
cal problem [55]. TRAIL is currently being evaluated in

Phase I/II clinical trials, alone or in combination with
other therapies, for the treatment of prostate cancer
[3,10]. Our study indicates that blockade of IAPs by a
small molecule Smac-mimetic promotes TRAIL-induced
apoptosis in prostate cancer cells via modulating both the
apoptosis pathway and NF-KB pathway. As IAPs are key
molecular targets for the development of cancer cell-selec-
tive therapeutics, our findings reveal a potential mecha-
nism for a Smac-mimetic IAP-antagonist on TRAIL-
mediated signaling, and suggest that modulating IAPs
may contribute to enhanced TRAIL efficacy, especially in
androgen-independent prostate cancer with high levels of
IAPs and constitutively active NF-KB signaling. Therefore,
small molecule Smac-mimetics that specifically target
IAPs may yield a potential therapeutic benefit with TRAIL-
based therapy in chemo- or radioresistant prostate cancer.

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BMC Cancer 2009, 9:392

Competing interests
LX is a co-inventor of the Smac-mimetic compounds
involved in the study. All other authors declare no com-
peting interests.

Authors' contributions
YD and ML contributed equally to this study. YD partici-
pated in the design of the study, performed NF-KB assay
and drafted the manuscript; ML performed the cell cul-
ture, MTT assay, apoptosis assay, transfection and Western
Blot, WT helped with the Western Blot analysis; YL and JL
carried out NF-KB luciferase reporter assay; TSL partici-
pated in the project design and in revising the manuscript;
LX supervised the study, experimental design, data analy-
sis, and revision of the manuscript. All authors read and
approved the final manuscript.

We thank Dr. Arie Belldegrun at University of California Los Angeles for
kindly providing the androgen-independent prostate cancer cell line CL I;
Dr. Susan Harris for help with the manuscript; Drs. Shaomeng Wang and
Haiying Sun at University of Michigan for providing the Smac-mimetic com-
pounds; and the University of Michigan Comprehensive Cancer Center
(UMCCC) Flow Cytometry Core for flow cytometry analysis.

Grant support: This study was supported in part by Department of
Defense Prostate Cancer Research Program W81XWH-06-1-0010 (to L.
X.), NIH grants CA121830, CA128220 and CAl 34655 (to L. X.), and by
NIH through the University of Michigan's Cancer Center Support Grant (5
P30 CA46592).

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