DECOY RECEPTOR 3 EXPRESSION IN ASPC-1 HUMAN PANCREATIC ADENOCARCINOMA CELLS VIA THE PHOSPHATIDYLINOSITOL 3-KINASE-, AKT-, AND NF- B-DEPENDENT PATHWAY1

 
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The Journal of Immunology

Decoy Receptor 3 Expression in AsPC-1 Human Pancreatic
Adenocarcinoma Cells via the Phosphatidylinositol 3-Kinase-,
Akt-, and NF-␬B-Dependent Pathway1

Pei-Hsuan Chen and Chia-Ron Yang2
Many cancers develop different means of escaping destruction by the immune system, such as resistance to Fas ligand
(FasL)-Fas interaction-mediated apoptotic signals. Decoy receptor 3 (DcR3), a soluble receptor for FasL, is highly expressed in
cancer cells and plays a significant role in immune suppression and tumor progression. However, how DcR3 expression is
modulated is unclear. In this study, immunoprecipitation and ELISA using human pancreatic cancer cells showed the presence of
high levels of DcR3 protein in AsPC-1 cells, but not in PANC-1 cells. Treatment with herbimycin A (a tyrosine kinase inhibitor),
LY294002 or wortmannin (PI3K inhibitors), pyrrolidine dithiocarbamate (an NF-␬B inhibitor), or AG1024 (an insulin-like growth
factor-1 inhibitor) significantly reduced endogenous DcR3 levels in AsPC-1 cells. Furthermore, transfection of AsPC-1 cells with
Akt or I␬B␣ dominant-negative plasmids also markedly reduced DcR3 levels. In contrast, 48-h transfection of PANC-1 cells
with a constitutively active Akt induced DcR3 expression. Flow cytometry assays indicated that apoptosis was not seen in
AsPC-1 cells incubated with soluble FasL or membrane-bound FasL, but was seen when DcR3 small interfering RNA-transfected
AsPC-1 cells underwent the same treatment. In addition, PANC-1 cell incubation with conditioned medium from AsPC-1 cells
transfected with dominant-negative Akt or I␬B␣ plasmids or DcR3 small interfering RNA showed increased soluble FasL-
mediated apoptosis compared with the control group. Our results show that insulin-like growth factor-1-induced activation of the
PI3K/Akt/NF-␬B signaling pathway is involved in the modulation of endogenous DcR3 expression in AsPC-1 cells, and that
reducing endogenous DcR3 levels increases FasL-induced apoptosis of human pancreatic cancer cells. The Journal of Immunol-
ogy, 2008, 181: 8441– 8449.

D         ecoy receptor 3 (DcR3)3 is a member of the TNFR su-                               Evidence is accumulating that DcR3 plays a significant role
          perfamily. DcR3 cDNA encodes a 300-aa protein con-                             in immune suppression and tumor progression. DcR3 induces
          taining the four tandem cysteine-rich repeats character-                       dendritic cell apoptosis (6), modulates the differentiation of
istic of the TNFR superfamily and lacking a transmembrane                                dendritic cells and macrophages and impairs macrophage func-
sequence (1). DcR3 is therefore regarded as a secreted molecule.                         tion (7–9), regulates T cell/B cell activation, prevents T cell/
Previous studies have identified three ligands that interact with                        macrophage infiltration in the kidney (10), and inhibits T cell
DcR3, namely, Fas ligand (FasL), lymphotoxin-like, exhibits in-                          chemotaxis (11). Moreover, several reports link DcR3 with im-
ducible expression, and competes with HSV glycoprotein D for                             mune disease, e.g., DcR3 increases T cell activation in systemic
HVEM, a receptor expressed by T lymphocytes, and TL1A (1–3).                             lupus erythematosus (12), osteoclast formation (9, 13), and ad-
DcR3 is believed to block the cellular effects caused by the binding                     hesion molecule expression on endothelial cells (14), and over-
of these ligands to their membrane-bound cognate receptors by                            expression of DcR3 is seen in EBV- or human T-lymphotropic
blocking ligand/receptor binding, as shown for the binding of                            virus type 1-associated lymphomas (15). In addition, an asso-
FasL to Fas and TL1A to death receptor 3 (1, 3, 4). It has recently                      ciation between DcR3 expression and tumor progression is well
been reported that, in addition to binding to these ligands, DcR3                        documented (16, 17). Elevated serum concentrations of DcR3
may bind to, and cross-link, proteoglycans to induce monocyte                            have been detected in patients with various malignant cancers,
adhesion (5).                                                                            e.g., cancers of the esophagus, stomach, glioma, lung, colon,
                                                                                         rectum, and pancreas (1, 4, 16, 18, 19). In tumorigenesis, DcR3
                                                                                         not only helps tumor cells to escape immune surveillance by
School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan      neutralizing FasL- or lymphotoxin-like, exhibits inducible ex-
Received for publication. Accepted for publication.
                                                                                         pression, and competes with HSV glycoprotein D for HVEM, a
                                                                                         receptor expressed by T lymphocyte-mediated cell death (1, 2),
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance        down-regulating MHC-II expression by tumor-associated mac-
with 18 U.S.C. Section 1734 solely to indicate this fact.                                rophages (20), and inducing immune suppression, as described
1
 This work was supported by the National Science Council of Taiwan (NSC96-2320-          above, but also contributes to the development of a microenvi-
B-002-034; NSC97-2320-B-002-019-MY3).
                                                                                         ronment suitable for tumor growth, e.g., by inducing angiogen-
2
 Address correspondence and reprint requests to Dr. Chia-Ron Yang, School of Phar-       esis (21). DcR3 is therefore a critical factor in tumor
macy, College of Medicine, National Taiwan University, Taipei, Taiwan. E-mail ad-
dress: cryang@ntu.edu.tw                                                                 progression.
3
  Abbreviations used in this paper: DcR3, decoy receptor 3; Ct, cycle threshold; DN,        Human pancreatic carcinoma is a highly malignant cancer.
dominant negative; FasL, Fas ligand; IGF, insulin-like growth factor; IKK, I␬B ki-       This disease is usually diagnosed at a late, incurable stage, and
nase; mFasL, membrane-bound FasL; PDTC, pyrrolidine dithiocarbamate; sFasL, sol-         the 5-year survival rate is less than 5% (22). Pancreatic cancer
uble FasL; siRNA, small interfering RNA; SRB, sulforhodamine B.
                                                                                         is relatively resistant to cytotoxic therapy (22) and radiation
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00   treatment (23). In addition, there is increasing evidence that

www.jimmunol.org
8442                                                         DcR3 EXPRESSION VIA PI3K-, Akt-, AND NF-␬B-DEPENDENT PATHWAY

many cancers, including pancreatic cancer, develop different
methods of evading destruction by the immune system, such as
resistance to FasL-Fas interaction-mediated apoptotic signals,
despite expressing Fas (24). Furthermore, a recent study dem-
onstrated that human pancreatic adenocarcinomas show high
expression of DcR3, which blocks the growth inhibition signals
mediated by FasL (4). However, the underlying mechanisms
involved in modulating DcR3 expression are poorly understood.
Kim et al. (25) suggested that LPS treatment of human intesti-
nal epithelial cells induces DcR3 release via activation of
ERK1/2/JNK and the transcription factor NF-␬B. However, the
signaling pathway involved in DcR3 expression in tumor cells
is still unclear. In this study, we identified the signal transduc-
tion pathway of DcR3 expression in human pancreatic adeno-
carcinoma cells. Moreover, using small interfering RNA
(siRNA) to knockdown DcR3 levels, we evaluated whether re-
duced DcR3 expression increases the cytotoxic activity of FasL.
A clearer understanding of the mechanisms involved in DcR3                    FIGURE 1. Immunoprecipitation and ELISA of DcR3 protein expression
expression will help in developing therapeutic strategies for hu-             in the human pancreatic cancer cell lines AsPC-1 and PANC-1. A, Equal
man malignancies.                                                             amounts of concentrated medium from cells were immunoprecipitated with 1
                                                                              ␮g of anti-DcR3 Ab, followed by immunoblot analysis using the same Ab.
                                                                              HT-29 or SW480 cells were used as the negative or positive control for DcR3
Materials and Methods                                                         expression, respectively. The blots shown are representative of those obtained
Materials                                                                     in three separate experiments. B, Cells (1 ⫻ 105) were cultured in 24-well
                                                                              plates, the supernatants were collected at the indicated times, and DcR3 levels
Rabbit polyclonal Abs against phospho-Akt (Ser473), Akt, phospho-I␬B
                                                                              were measured by ELISA. The data are the mean ⫾ SEM for five separate
kinase (IKK)␣ (Ser180)/␤ (Ser181), or phospho-p65 (Ser536) and mouse anti-
phospho-I␬B␣ (Ser32/36) mAb were purchased from Cell Signaling Tech-          experiments. ⴱ, p ⬍ 0.05 compared with the control group; ⴱⴱ, p ⬍ 0.01
nology. Rabbit polyclonal Abs against insulin-like growth factor (IGF)-       compared with the control group.
1R␤; mouse mAbs against DcR3, I␬B␣, GAPDH, or FasL; and protein
A/G-PLUS agarose were purchased from Santa Cruz Biotechnology.                Immunoblot analysis
Mouse mAb against phosphotyrosine (clone 4G10) was obtained from Up-
state Biotechnology. Rabbit polyclonal Abs against IKK␣ and mouse mAb         Cells were incubated for 10 min at 4°C in lysis buffer (20 mM HEPES (pH
against p65 were purchased from BioVision. HRP-conjugated goat anti-          7.4), 2 mM EGTA, 50 mM ␤-glycerophosphate, 0.1% Triton X-100, 10%
mouse IgG, FITC-conjugated goat anti-mouse IgG, and HRP-conjugated
goat anti-rabbit IgG Abs were obtained from Jackson ImmunoResearch
Laboratories. The human DcR3 ELISA kit was purchased from R&D Sys-
tems. Recombinant human soluble FasL (sFasL) was purchased from
PeproTech Asia. I␬B␣M, a dominant-negative (DN) mutant of I␬B␣, was
provided by B.-C. Chen (Taipei Medical University, Taipei, Taiwan). Myr-
Akt (constitutively activated Akt), DN-Akt (a DN mutant of Akt), pGL2-
ELAM-␬B-luc, the empty expression vector pUSEamp⫹, and the
pEGFP-N1 plasmid were provided by C.-M. Teng (National Taiwan Uni-
versity, Taipei, Taiwan). Stealth siRNA for DcR3 (AF104419), nonsilence
control RNA, and transfection reagents were purchased from Invitrogen.
The dual-luciferase reporter assay kit and pGL4.74[hRluc/TK] vector were
obtained from Promega. LY294002 and pyrrolidine dithiocarbamate
(PDTC) were purchased from Sigma-Aldrich. Herbimycin A, PD98059,
wortmannin, SP600125, AG1478, rapamycin, AG1024, and AG1295 were
purchased from Calbiochem. GM6001 was obtained from Millipore. The
17-AAG was obtained from Tocris Cookson. All other chemicals were
from Sigma-Aldrich.

Cell culture
The AsPC-1 human pancreatic adenocarcinoma cells, PANC-1 human pan-
creatic epithelioid carcinoma cells, HT-29 human colon adenocarcinoma
cells, and human T cell leukemia Jurkat clone E6-1 cells were obtained
from American Type Culture Collection, and cultured in the medium rec-
ommended by the vendor (RPMI 1640 medium for AsPC-1 and Jurkat
cells, DMEM for PANC-1 cells, and MEM Eagle for HT-29 cells) sup-
plemented with 10% (v/v) FBS (Invitrogen Life Technologies), 100 U/ml
penicillin, and 100 ␮g/ml streptomycin (Biological Industries) at 37°C in
a humidified atmosphere of 5% CO2 in air.

Cell viability assay
                                                                              FIGURE 2. Effects of various inhibitors on DcR3 expression in AsPC-1
Cell viability was measured by the colorimetric MTT assay. Cells (1 ⫻         cells. Cells (1 ⫻ 105) were cultured in 24-well plates for 24 h and treated
104) in 100 ␮l of medium in 96-well plates were incubated with vehicle or
                                                                              with different inhibitors at the indicated concentration for another 24 h;
test compound for 48 h. After various treatments, 1 mg/ml MTT was add-
ed; the plates were incubated at 37°C for an additional 2 h; the cells were   then the culture medium was collected and DcR3 levels were measured by
pelleted and lysed in 100 ␮l of DMSO; and the absorbance at 550 nm was        ELISA. Cell viability compared with the control group was estimated using
measured on a microplate reader. Each experiment was performed in du-         the MTT assay. The data are the mean ⫾ SEM for four separate experi-
plicate and repeated five times.                                              ments. ⴱ, p ⬍ 0.05 compared with the control group.
The Journal of Immunology                                                                                                                          8443

FIGURE 3. IGF-1-mediated acti-
vation of the PI3K/Akt/NF-␬B path-
way is involved in the modulation of
DcR3 levels in AsPC-1 cells. A,
Equal amounts of total cell lysates
from AsPC-1 or PANC-1 cells were
immunoprecipitated with 1 ␮g of anti-
IGF-1R Ab, followed by immunoblot
analysis using anti-phosphotyrosine
Ab. B, Cells (1 ⫻ 106) were incu-
bated for 24 h with different inhibi-
tors at the indicated concentration
(left panel) or transfected with 0.8 ␮g
of DN-Akt, I␬B␣ (I␬B␣M), or empty
vector (EV) for 24 h (right panel);
then the cells were harvested and
whole cell extracts were prepared for
Western blot analysis for the indi-
cated proteins. In both A and B, the
blots shown are representative of
those obtained in three separate ex-
periments. C, Cells (5 ⫻ 105) were
transiently transfected with 0.8 ␮g of
DN-Akt, I␬B␣ (I␬B␣M), or EV in
the presence of 0.8 ␮g of pGL2-
ELAM-Luc for 24 h; then luciferase
activity was measured, as described
in Materials and Methods. Cells
treated with 10 ng/ml TNF-␣ for 24 h
were used as positive control. D,
AsPC-1 cells (1 ⫻ 106) were trans-
fected with 0.8 ␮g of DN-Akt, I␬B␣
(I␬B␣M), or EV for 36 h; then the
culture medium was collected and
DcR3 levels were measured by
ELISA. The data are the mean ⫾
SEM for four separate experiments.
ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared
with the control group, respectively.

glycerol, 1 mM DTT, 1 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, 1 mM PMSF,         Transient transfection assays and reporter gene assay
and 1 mM sodium orthovanadate), then were scraped off, incubated on ice
for a further 10 min, and centrifuged at 100 ⫻ g for 30 min at 4°C. The      A total of 1 ⫻ 106 cells was seeded in 6-well plates in 1 ml of medium
whole cell extract (120 ␮g of proteins) was mixed with an equal volume of    without serum 1 day before transfection. Following the manufacturer’s
SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 1% glycerol,           protocol, 5 ␮l of Lipofectamine 2000 (Invitrogen) in 50 ␮l of Opti-MEMI
300 mM 2-ME, and 0.00125% bromphenol blue), the mixture was heated           reduced serum medium was incubated for 5 min, then 2 ␮g of plasmid
at 95°C for 5 min and electrophoresed on 10% SDS gels, and the proteins      DNA, pEGFP-N1 plasmid, and pGL4.74[hRluc/TK] vector in 50 ␮l of
were transferred onto polyvinylidene fluoride membranes. Immunoblotting      Opti-MEMI reduced serum medium was added and the mixture was incu-
was performed using the relevant rabbit or mouse Ab and the correspond-      bated for 20 min at room temperature and added to the cells, which were
ing HRP-conjugated second Ab, followed by detection using ECL reagents       then incubated for 36 h. Transfection efficiency, determined by fluores-
(Amersham Biosciences) and exposure to photographic film.                    cence microscopy, was ⬎60% in all experiments. For the reporter gene
                                                                             assay, 50 ␮l of reporter lysis buffer (Promega) was added to each well, and
Immunoprecipitation assay                                                    the cells were scraped off the dishes, the samples were centrifuged at
                                                                             16,200 ⫻ g for 30 s at 4°C, and the supernatants were collected. Aliquots
Cell culture supernatants were collected and concentrated 30-fold (v/v) on   of cell lysates (5 ␮l) containing equal amounts of protein (10 –20 ␮g) were
an Amicon Ultra centrifugal filter device (Millipore), and then 5 mg of      placed in the wells of an opaque black 96-well microtitreplate, and 5 ␮l of
concentrated supernatant was immunoprecipitated overnight at 4°C with 1      luciferase substrate (Promega) was added and the luminescence immedi-
␮g of mouse anti-DcR3 mAb and A/G-agarose beads. The precipitated            ately measured in a microplate luminometer (Packard Instrument). To take
beads were washed three times with 1 ml of ice-cold cell lysis buffer, and   into account possible differences in transfection efficiency, the luciferase
the immune complex was resolved by 10% SDS-PAGE gel electrophoresis,         activity value was normalized using the luminescence from the cotrans-
followed by immunoblotting assay using anti-DcR3 Ab.                         fected Renilla pGL4.74[hRluc/TK] vector (Promega).

ELISA
                                                                             siRNA suppression assay
Cell culture supernatants were collected at various time points, and DcR3
levels were measured using commercial ELISA kits (R&D Systems), ac-          Cells (1 ⫻ 106) were plated in 6-cm dishes in 2 ml of medium without
cording to the vendor’s instructions.                                        serum 1 day before transfection. The cells were transfected with 160 nM
8444                                                       DcR3 EXPRESSION VIA PI3K-, Akt-, AND NF-␬B-DEPENDENT PATHWAY

FIGURE 4. DcR3 knockdown increases sFasL-
induced apoptosis in AsPC-1 cells. A, AsPC-1
cells (1 ⫻ 105) were transiently transfected with
160 nM DcR3 siRNA or nonsilence control
siRNA; then supernatants were collected at the
indicated time and DcR3 levels were measured
by ELISA. The data are the mean ⫾ SEM for
five separate experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01 compared with the control group, respec-
tively. #, p ⬍ 0.05; ⫹, p ⬍ 0.01 for the indicated
groups, respectively. B, AsPC-1 cells (1 ⫻ 104)
were transfected as indicated for different time
periods, and viable cell numbers were measured
using the MTT assay. The data are the mean ⫾
SEM for five separate experiments. C, AsPC-1
cells (1 ⫻ 106) were transfected with 160 nM
DcR3 siRNA (SiR) or nonsilence control siRNA
(NC) for 48 h; then sFasL was added for another
24 h (left columns) or 48 h (right columns), and
the cells were fixed and stained with propidium
iodide to analyze the DNA content by FACScan
flow cytometry. The cell cycle phase (sub-G1,
G0/G1, S, G2/M) is indicated. The sub-G1 phase
is indicative of apoptosis. The experiment was
performed three times with similar results.

DcR3 siRNA duplexes or with DcR3 nonsilence control using Lipo-             three times with FACS washing buffer; then the fluorescence of the cells
fectamine 2000. The siRNA-transfected cells were incubated for 48 h after   was analyzed using a FACScan flow cytometer (BD Biosciences). To de-
transfection before analysis. The 21-mer siRNAs were synthesized by In-     tect cell cycle progression, the cells were incubated with or without the
vitrogen. The DcR3 siRNA sequences were as follows: sense sequence,         indicated agent for 24 h, washed twice with ice-cold PBS, collected by
5⬘-GCC AGG CUC UUC CUC CCA UdTdT-3⬘; antisense sequence, 5⬘-                centrifugation, and fixed in 70% (v/v) ethanol for at least 2 h at ⫺20°C.
AUG GGA GGA AGA GCC UGG CdTdT-3⬘. The nonsilence control                    The cells were then incubated with 0.2 ml of DNA extraction buffer (0.2 M
siRNA sequences were as follows: sense sequence, 5⬘-GCC CGC UUU             Na2HPO4 and 0.1 M citric acid buffer (pH 7.8)) for 30 min at room tem-
CCC UCA GCA UdTdT-3⬘; antisense sequence, 5⬘-AUG CUG AGG GAA                perature, centrifuged at 3500 ⫻ g for 1 min at 25°C, resuspended in 1 ml
AGC GGG C-3⬘.                                                               of propidium iodide staining buffer (0.1% Triton X-100, 100 ␮g/ml RNase
                                                                            A, and 80 ␮g/ml propidium iodide in PBS), incubated at 37°C for 30 min
Flow cytometry
                                                                            in the dark, sorted by flow cytometry (FACScan; BD Biosciences), and
Cells were harvested, washed twice with FACS washing buffer (1% FBS         analyzed using CellQuest software (BD Biosciences). The cell cycle
and 0.1% NaN3 in PBS), incubated with Abs at 4°C for 30 min, and washed     distribution is shown as the percentage of cells containing G0/G1, S, G2,
The Journal of Immunology                                                                                                                       8445

FIGURE 5. Transfection of AsPC-1
cells with DcR3 siRNA increases
mFasL-mediated apoptosis. A, Jurkat
T cells were treated in the absence
(solid black area) or presence (gray
line) of 10 ␮g/ml PHA for 16 h, then
were incubated with anti-FasL Ab to
detect surface expression of FasL by
FACScan flow cytometry. Jurkat T
cells marked with FITC-coupled anti-
mouse IgG Ab served as the negative
control (dashed gray line in right
panel). B, AsPC-1 cells (1 ⫻ 106)
were transfected with 160 nM DcR3
siRNA (SiR) or nonsilence control
siRNA (NC); then, after 48 h, the me-
dium was replaced with medium con-
taining 6 ⫻ 105 paraformaldehyde-
fixed activated Jurkat T cells for 24 or
48 h, and analysis of DNA content
was performed by FACScan flow cy-
tometry. The sub-G1 phase is indica-
tive of apoptosis. The results shown
are representative of those obtained in
three separate experiments.

and M DNA, as judged by propidium iodide staining. The apoptotic            to analyze the results. The Ct value, which is inversely proportional to
population was determined as the percentage of cells with a sub-G1          the initial template copy number, is the calculated cycle number in
(⬍G1) DNA content.                                                          which the fluorescence signal emitted is significantly above background
                                                                            levels. The mRNA expression level of target genes was normalized to
RT-PCR analysis                                                             GAPDH using the 2–⌬⌬Ct method, in which ⌬Ct ⫽ target gene Ct –
                                                                            GAPDH Ct, and ⌬⌬Ct ⫽ ⌬Ct treatment – ⌬Ct control.
Total RNA was isolated from cells using TRIzol reagent (Invitrogen). Sin-
gle-strand cDNA for a PCR template was synthesized from 10 ␮g of total      Preparation of nuclear extracts and EMSA
RNA using random primers and Moloney murine leukemia virus reverse
transcriptase (Promega). The oligonucleotide primers used for the ampli-    As previously described (9), nuclear extracts were prepared and were sub-
fication are as follows: human DcR3 (GenBank Accession AF104419)            jected to the EMSA Gel Shift kit (Panomics), according to the manufac-
sense (⬎284 –306) 5⬘-TGC CGC CGA GAC AGC CCC ACG AC-3⬘ and                  turer’s specifications. Briefly, biotin-labeled NF-␬B-specific probes were
antisense (723–745) 5⬘-GAC GGC ACG CTC ACA CTC CTC AG-3⬘,                   incubated with 10 ␮g of nuclear extract at 15°C for 30 min to allow the
which produced a product of 461 bp; human IGF-1R (GenBank Accession         formation of protein (transcription factor)/DNA complexes. The complexes
NM000875) sense (⬍688 –708) 5⬘-AAA TGT GCC CAA GCA CGT                      were run by 6% nondenaturing PAGE in 0.5⫻ TBE at 4°C at 120 V, and
GTG-3⬘ and antisense (1105–1125) 5⬘-TGC CCT TGA AGA TGG TGC                 then transferred onto Biodyne B nylon membrane. Detection of signals was
ATC-3⬘, which produced a product of 437 bp. GAPDH was used as an            obtained using an ECL imaging system.
internal control. The GAPDH (GenBank Accession NM_002046) primers
used were sense (949 –972) 5⬘-TCC TCT GAC TTC AAC AGC GAC
                                                                            Sulforhodamine B (SRB) assay
ACC-3⬘ and antisense (1134 –1156) 5⬘-TCT CTC TTC CTC TTG TGC                Cells (1 ⫻ 105) were inoculated into 24-well plates. After an overnight
TCT TG-3⬘, which produced a product of 207 bp. Equal amounts of each        culture, cells were transfected with 0.8 ␮g of DN-Akt, I␬B␣M, empty
reverse-transcription product (1 ␮g) were PCR amplified using Taq poly-     vector, or 160 nM nonsilence control siRNA, DcR3 siRNA for 48 h. Then,
merase in 35 cycles consisting of 1 min at 95°C, 1 min at 58°C, and 1 min   three wells of cells were fixed with 10% TCA to terminate reaction (time
at 72°C. The amplified cDNA was run on 1% agarose gels and visualized       zero); other cells were incubated with or without sFasL (50 ng/ml) or
under UV light following SYBR Safe DNA gel stain (Invitrogen). The          PHA-activated Jurkat cells (membrane-bound FasL (mFasL)) for another
band intensity was quantified using densitometer. The intensities of the    24 or 48 h. After incubation, 0.4% SRB (Sigma-Aldrich) in 1% acetic acid
cDNA bands were normalized to GAPDH band intensities.                       was added to each well for 15 min, the plates were washed, and dyes were
                                                                            dissolved by 10 mM Tris buffer. Then, the absorbance was read at a wave-
Real-time RT-PCR with SYBR Green detection                                  length of 515 nm. Using the following absorbance measurements, such as
The isolated RNA subjected to RT-PCR was treated with DNase to avoid        time zero (T0), control growth (C), and cell growth in the presence of
amplification of DNA contaminants. The forward and reverse primers were     various treatments (Tx), the percentage of cell growth was calculated as
as follows: human DcR3 (GenBank Accession AF104419), CTT CTT                ((Tx ⫺ T0)/(C ⫺ T0)) ⫻ 100 for Tx ⱖ T0.
CGC GCA CGC TG and ATC ACG CCG GCA CCA G; human IGF-1R                      Data analysis
(GenBank Accession NM000875), TGG AGT GCT GTA TGC CTC TG
and CAC CTC CCA CTC ATC AGG A; and GAPDH (GenBank Acces-                    The data are expressed as the mean ⫾ SEM, and were analyzed statistically
sion NM_002046), ATT CCA CCC ATG GCA AAT TC and TGG GAT                     using one-way ANOVA. When ANOVA showed significant differences
TTC CAT TGA TGA CAA G. The cycle threshold (Ct) method was used             between groups, Tukey post hoc test was used to determine the specific
8446                                                                     DcR3 EXPRESSION VIA PI3K-, Akt-, AND NF-␬B-DEPENDENT PATHWAY

FIGURE 6. Akt is involved in modulation of DcR3 expression in PANC-1 cells. PANC-1 cells were seeded at different densities (A, 1 ⫻ 106 on 6-well
plates; B, 5 ⫻ 105 on 24-well plates) and transfected with 0.8 ␮g of constitutively active Akt (Myr-Akt) or empty vector (EV) for 24 h. A, Cells were
harvested and whole cell extracts were prepared for Western blot analysis with the indicated Abs. The blots shown are representative of those obtained in
three separate experiments. B, The culture medium was collected, and DcR3 levels were measured by ELISA. The data are the mean ⫾ SEM for five
separate experiments. ⴱ, p ⬍ 0.05 compared with the control group. C, PANC-1 cells were seeded onto 6-well plates at a density of 1 ⫻ 106/well, and then,
after 24 h, the medium was replaced for 48 h with conditioned medium from AsPC-1 cells that had undergone the indicated transfections, after which DNA
content was analyzed by FACScan flow cytometry. The sub-G1 phase is indicative of apoptosis. The results shown are representative of those obtained in
three separate experiments.

pairs of groups showing statistically significant differences. A p value of          PI3K/Akt or MAPK signaling pathways, which play critical roles
less than 0.05 was considered statistically significant.                             in tumor growth and development. We next asked whether growth
                                                                                     factor-induced tyrosine kinase activation was involved in modu-
Results                                                                              lation of DcR3 expression. To address this question, we treated
DcR3 expression in human pancreatic carcinoma cells                                  AsPC-1 cells with herbimycin A (a tyrosine kinase inhibitor),
Because DcR3 lacks a transmembrane sequence and is a soluble                         PD98059 or SP600125 (MAPK inhibitors), LY294002 or wort-
protein, we used an immunoprecipitation assay to determine the                       mannin (PI3K inhibitors), rapamycin (a mammalian target of rapa-
distribution of DcR3 in different human pancreatic cancer cells. As                  mycin inhibitor), AG1478 (an epidermal growth factor receptor
shown as Fig. 1A, high levels of endogenous DcR3 protein ex-                         inhibitor), AG1024 (an IGF-1 inhibitor), AG1295 (a platelet-de-
pression were seen in the human pancreatic adenocarcinoma cell                       rived growth factor inhibitor), or PDTC (an NF-␬B inhibitor). Af-
line AsPC-1, but not in the human pancreatic epithelioid carci-                      ter treatment for 48 h, only herbimycin A (1 ␮M), LY294002 (20
noma cell line PANC-1. The human colon cancer cell line HT-29                        ␮M), wortmannin (20 ␮M), AG1024 (10 ␮M), or PDTC (50 ␮M)
was used as the negative control and line SW480 as the positive                      significantly inhibited DcR3 expression (Fig. 2). If DcR3 level was
control for DcR3 expression. Using ELISA, expression in AsPC-1                       normalized with viable cell number, similar inhibitory effects were
cells peaked after 48-h incubation (6.46 ⫾ 0.32 ng/ml) and re-                       observed after above-mentioned inhibitor treatment (Supplemental
mained at this level till at least 60 h (6.24 ⫾ 0.27 ng/ml). Similar                 Fig. 2).4 None of the treatments had any significant effect on cell
result was observed if DcR3 level was normalized with viable cell                    viability, assessed using the MTT assay (Fig. 2). These data sug-
number (Supplemental Fig. 1).4 In addition, no DcR3 was detect-                      gest that growth factor (IGF-1)-mediated tyrosine kinase activation
able after 60-h incubation in PANC-1 cells (Fig. 1B).                                and PI3K/Akt and NF-␬B play a role in DcR3 expression in
                                                                                     AsPC-1 cells. To make further study, we detected IGF-1R mRNA
PI3K/Akt and NF-␬B are involved in DcR3 expression
                                                                                     or protein distribution levels in human pancreatic cancer cells
Extensive studies (26, 27) have demonstrated that growth factors,                    (Supplemental Fig. 3).4 High levels of IGF-1R were detected in
such as IGF-1, cause tyrosine kinase activation and trigger the                      AsPC-1 cells, but lower in PANC-1 cells. Using an immunopre-
                                                                                     cipitation assay, we observed different levels of tyrosine-phospho-
4
    The online version of this article contains supplemental material.               rylated IGF-1R in different human pancreatic cancer cells (Fig.
The Journal of Immunology                                                                                                                8447

3A), levels being high in AsPC-1 cells and much lower in PANC-1
cells. To examine whether there was a connection between the
IGF-1-induced signals (PI3K/Akt and NF-␬B activation) and
DcR3 expression, we treated AsPC-1 cells with LY294002 (20
␮M), wortmannin (20 ␮M), PDTC (50 ␮M), or AG1024 (10 ␮M),
and examined levels of phosphorylated and nonphosphorylated
Akt, IKK␤, IKK␣, I␬B␣, and p65 using Western blotting. As
shown in Fig. 3B (left panel), constitutive Akt phosphorylation
was seen in AsPC-1 cells. LY294002, wortmannin, or AG1024
treatment not only significantly reduced Akt phosphorylation at
Ser473 residue, but also suppressed phosphorylation of IKK␤ at
Ser181, IKK␣ at Ser180, I␬B␣ at Ser32/36, and p65 at Ser536. How-
ever, PDTC treatment suppressed phosphorylation of I␬B␣ and
p65, but not of Akt and IKK␣/␤ (Fig. 3B). As shown in Fig. 3B
(right panel), transfection of AsPC-1 cells with 0.8 ␮g of DN-Akt
significantly decreased phosphorylation of Akt, IKK␣/␤, I␬B␣,
and p65, whereas transfection with a DN mutant of I␬B␣
(I␬B␣M), which prevents I␬B␣ phosphorylation, only inhibited
I␬B␣ and p65 phosphorylation. To directly examine NF-␬B acti-
vation after blocking Akt and I␬B␣ phosphorylation, AsPC-1 cells
were transiently transfected with pGL2-ELAM-␬B-luciferase and
the turning on of the luciferase gene by NF-␬B used as an indicator
of NF-␬B activation. As shown as Fig. 3C, transfection with DN-
Akt and I␬B␣M for 24 h markedly reduced ␬B-luciferase activity.
Similar result was observed in EMSA (Supplemental Fig. 4).4 Us-
ing RT-PCR (Supplemental Fig. 5A)4 and real-time PCR assay
(Supplemental Fig. 5B),4 DcR3 mRNA levels significantly were
down-regulated by transfection with DN-Akt or I␬B␣ plasmids.
Furthermore, as shown in Fig. 3D, transfection of AsPC-1 cells
with DN-Akt or I␬B␣M for 36 h significantly reduced DcR3 ex-
pression (left panel), with no effect on cell viability (right panel)
or cell growth (Supplemental Fig. 6).4 These results clearly dem-
onstrated that endogenous IGF-1 activation of the PI3K/Akt/
NF-␬B signal pathway is involved in DcR3 expression.

siRNA knockdown of DcR3 expression significantly enhances
the cytotoxic effect of FasL                                            FIGURE 7. Schematic summary of the signal transduction pathway in-
                                                                        volved in inducing DcR3 expression in AsPC-1 cells. IGF-1 activates the
Resistance to apoptosis is believed to be one of the reasons for the
                                                                        PI3K/Akt, which, in turn, induces IKK␣/␤ phosphorylation, p65 phosphor-
failure of cancer treatments. Previous studies have demonstrated        ylation, and NF-␬B activation, which leads to DcR3 expression in human
that DcR3, acting as a decoy receptor, neutralizes the FasL-medi-       pancreatic adenocarcinoma cells.
ated apoptotic signal (1, 4). We therefore examined whether FasL-
induced apoptosis of pancreatic cancer cells benefited from knock-
down of DcR3 expression in AsPC-1 cells. Fig. 4A shows that             treatment groups (4.80 and 4.57%) (Fig. 5B). Using MTT, SRB
transfection of AsPC-1 cells with DcR3 siRNA significantly re-          assay, or direct cell counting, significant decreasing cell viability
duced DcR3 levels, this effect being first seen at 36 h and main-       or cell growth was observed in DcR3 siRNA transfection-com-
tained for at least 72 h. Fig. 4B shows transfection had no effect on   bined sFasL/mFasL groups when compared with each treatment
cell viability when compared with control group. In addition,           alone group (Supplemental Fig. 7).4 These results suggest that
FACScan analysis of cell cycle distribution (Fig. 4C) showed that       siRNA knockdown of DcR3 expression increases the cytotoxic
DcR3 siRNA transfection (SiR panels) or recombinant human               effect of FasL in AsPC-1 cells.
sFasL (50 ng/ml) (sFasL panels) alone had no effect on the number
of AsPC-1 cells in sub-G1 phase, whereas, when cells were trans-        Akt signals are involved in DcR3 expression in PANC-1 cells
fected with DcR3 siRNA for 24 or 48 h, then were treated with           We next examined whether DcR3 was expressed in PANC-1
sFasL for 24 h (SiR ⫹ sFasL panels), an increase in the number of       cells, which did not normally express DcR3, if the cells con-
cells in sub-G1 phase was seen (35.74 and 44.31%, respectively, at      stitutively expressed active Akt. Transfection of PANC-1 cells
24 and 48 h compared with controls). Moreover, mFasL is the             with constitutively active Akt (Myr-Akt) resulted in marked
primary mediator of apoptosis in the immune system (28). A pre-         phosphorylation of Akt and I␬B␣ in 24 h (Fig. 6A), with no
vious study reported that PHA (10 ␮g/ml) stimulates expression of       effect on cell viability (data not shown). After transfection with
mFasL in null Jurkat cells (29), and we therefore used this estab-      constitutively active Akt, DcR3 expression increased from non-
lished system to evaluate the effect of DcR3 siRNA on mFasL. Fig.       detectable levels at 24 h to 0.076 ng/ml at 36 h and 0.445 ng/ml
5A shows significant FasL staining was seen following PHA treat-        at 48 h (Fig. 6B). Again, these data suggest that the PI3K/Akt/
ment of Jurkat cells. Cell cycle distribution analysis indicated that   NF-␬B pathway is involved in the modulation of DcR3 expres-
combined DcR3 siRNA/mFasL treatment resulted in the accumu-             sion. We next examined whether incubation of PANC-1 cells in
lation of a significant number of cells in sub-G1 phase (15.81 and      AsPC-1 cell-conditioned medium (DcR3 rich) could change
19.16% at 24 and 48 h, respectively) compared with the single           FasL-induced apoptosis. Fig. 6C shows that treatment of
8448                                                     DcR3 EXPRESSION VIA PI3K-, Akt-, AND NF-␬B-DEPENDENT PATHWAY

PANC-1 cells with sFasL (100 ng/ml) resulted in a significant            Besides inducing the expression of these antiapoptotic proteins,
number of cells in sub-G1 phase (increase from 3.93 to                   NF-␬B seems to have other antiapoptosis effects, because Kajino
12.51%), and that this effect was markedly inhibited by replac-          et al. (43) found that IL-1␤, a potent NF-␬B inducer, blocked
ing the growth medium with AsPC-1 cell-conditioned medium,               TNF-␣-induced apoptosis and this effect was not abolished by pre-
but not using conditioned medium from AsPC-1 cells trans-                treatment of the cells with the protein synthesis inhibitor cyclo-
fected with DN-Akt, I␬B␣M, or DcR3 siRNA (8.67, 11.45, or                heximide or by blocking NF-␬B transcription using NF-␬B decoy
11.33%, respectively). Results from detection of cell viability,         oligonucleotides, showing that the antiapoptotic effect of NF-␬B
cell growth, and cell number also support these FACS data                does not need de novo protein synthesis and suggesting that
(Supplemental Fig. 8).4                                                  NF-␬B has a complex antiapoptic effect. In this study, PI3K in-
  Taken together, our results demonstrated that IGF-1 activation         hibitors or DN-Akt treatment significantly attenuated NF-␬B acti-
of the PI3K/Akt/NF-␬B signaling pathway is involved in endog-            vation (Fig. 3, B and C). In addition, treatment with DN-Akt or
enous DcR3 expression in AsPC-1 cells (Fig. 7), and that different       I␬B␣ (I␬B␣M) also significantly reduced DcR3 mRNA and pro-
DcR3 levels alter FasL-mediated apoptosis in human pancreatic            tein levels in AsPC-1 cells (Supplemental Fig. 5;4 Fig. 3D). These
adenocarcinoma cells.                                                    results suggest that the PI3K/Akt/NF-␬B pathway plays an impor-
                                                                         tant role in modulating DcR3 levels. However, we have not com-
Discussion                                                               pletely ruled out the possibility that other pathways may also be
In this study, we investigated the mechanisms of endogenous DcR3         involved.
expression and the potential therapeutic application of reducing DcR3       Resistance to apoptosis is believed to be one of the hallmarks of
levels combined with FasL treatment in human pancreatic adenocar-        cancer cells (44). Recent studies have shown that several cancer
cinoma. Our data demonstrate, for the first time, that PI3K/Akt-de-      cells, including pancreatic adenocarcinomas, have developed
pendent IKK␣/␤ phosphorylation, p65 phosphorylation, and NF-␬B           mechanisms making them resistant to FasL/Fas-mediated apopto-
activation are involved in DcR3 expression in AsPC-1 cells, and that     tic signals despite expressing Fas (24, 45). DcR3 is a decoy re-
reducing DcR3 expression by siRNA transfection significantly en-         ceptor of FasL, and several studies (46, 47) have demonstrated that
hances FasL-induced AsPC-1 cell apoptosis.                               there is a significant correlation between DcR3 overexpression and
   Recent studies have revealed that the PI3K/Akt pathway plays          resistance to Fas ligand-mediated apoptosis in cancer cells. Thus,
an important role in tumor progression (30, 31). This pathway is         a better understanding of the molecular mechanism of DcR3 ex-
stimulated by the aberrant activation of upstream signals, such as       pression would help in developing potential therapeutic strategies
growth factor receptor tyrosine kinase (32). IGF-1 is one growth         to increase the apoptosis of FasL-resistant cancer cells by blocking
factor that activates the PI3K/Akt or MAPK pathway to drive cell         DcR3 expression by cancer cells. Following up this idea, we have
survival and growth of different tumor cells (27). In 1994, Sell         developed several natural compounds that reduce DcR3 levels and
et al. (33) showed that fibroblasts derived from IGF-1R null mice        enhance the apoptotic effect of FasL on FasL-resistant pancreatic
cannot be transformed by several oncogenes. Our results showed           cancer cells (data not shown).
that levels of tyrosine-phosphorylated IGF-1R were different in             In summary, our study demonstrates that PI3K/Akt-mediated
two different pancreatic cancer cells, being high in AsPC-1 cells        IKK␣/␤ phosphorylation, p65 phosphorylation, and NF-␬B acti-
and much lower in PANC-1 cells. This implies that different sig-         vation are involved in the modulation of endogenous DcR3 levels
naling pathways might exist in these cells. In addition, treatment of    in AsPC-1 human pancreatic adenocarcinoma cells, and that re-
AsPC-1 cells with AG1024 (a specific IGF-1 inhibitor), herbimy-          ducing DcR3 levels significantly enhances the FasL-mediated ap-
cin-A (a tyrosine kinase inhibitor), LY294002 or wortmannin              optotic effect. These results indicate that DcR3 could be a potential
(PI3K inhibitors), or PDTC (an NF-␬B inhibitor) significantly re-        therapeutic target in human pancreatic cancer.
duced DcR3 expression, suggesting that IGF-1 activation of the
PI3K/Akt/NF-␬B pathway is involved in modulating DcR3 ex-                Disclosures
pression in these cells. Indeed, several studies (34, 35) have sug-      The authors have no financial conflict of interest.
gested that constitutive activation of PI3K/Akt and NF-␬B is seen
in many malignancies, including pancreatic cancer. Moreover, a           References
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