7Peroxisome Proliferator-Activated Receptor γ Ligand Inhibits
Cell Growth and Invasion of Human Pancreatic Cancer Cells
Koji Hashimoto, Richard T. Ethridge, and B. Mark Evers*
Department of Surgery, The University of Texas Medical Branch, Galveston, TX
Summary
Background. Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in certain human can-
cers; ligand-induced PPARγ activation can result in growth inhibition and differentiation in these cells.
However, the precise mechanism for the antiproliferative effect of PPARγ ligands is not entirely known.
Aim of Study. The purpose of this study was to examine the effect of PPARγ ligands on pancreatic cancer
cell growth and invasiveness.
Methods. The effect of two PPARγ ligands, 15 deoxy-∆12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone,
on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was
assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-
PGJ2 on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined.
Results. Both 15d-PGJ2 and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a
dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ2 resulted in a time-dependent
decrease in cyclin D1 expression associated with a concomitant induction of p21waf1 and p27kip1. In addi-
tion, 15d-PGJ2 treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancre-
atic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ2,
which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity.
Conclusions. These results demonstrate that PPARγ ligands have the dual advantage of inhibiting pan-
creatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for
these agents in the adjuvant treatment of pancreatic cancer.
Key Words: Pancreatic cancer; peroxisome proliferator-activated receptor γ; cell cycle; apoptosis; tumor
invasion.
Introduction
Pancreatic cancer is the fifth leading cause of
cancer death in the United States (1). In 2001, it is
estimated that approx 29,000 new cases of pancre-
atic cancer will be diagnosed with almost as many
deaths from this disease. The ability to intervene sur-
gically in pancreatic cancer is predicated on its early
detection. However, the lack of specific signs and
symptoms of the disease and its relative infrequency
make early detection by mass screening impossible.
Moreover, the high mortality associated with pan-
creatic cancer is related to its propensity for exten-
sive local invasion and early metastasis. Pancreatic
cancer cells are usually resistant to chemotherapy,
radiotherapy, and immunotherapy (2,3). Although
new chemotherapeutic agents for pancreatic cancer
International Journal of Gastrointestinal Cancer, vol. 32, no. 1, 7–22, 2002
© Copyright 2002 by Humana Press Inc.
All rights of any nature whatsoever reserved.
0169-4197/02/32:7–22/$20.00
*Author to whom all correspondence and reprint requests
should be addressed: Department of Surgery, The University
of Texas Medical Branch, 301 University Boulevard, Galve-
ston, TX 77555-0536. E-mail: mevers@utmb.edu
Review Article
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8 Hashimoto, Ethridge, and Evers
International Journal of Gastrointestinal Cancer Volume 32, 2002
are being evaluated in clinical trials (4), more effec-
tive adjuvant therapies are needed in the treatment
of this lethal disease.
Peroxisome proliferator-activated receptor γ
(PPARγ) is a member of the superfamily of ligand-
dependent nuclear receptors, which includes recep-
tors for steroid, retinoid, and thyroid hormones (5).
PPARγ has a critical role in adipogenesis and glu-
cose metabolism (6,7) and exerts anti-inflammatory
effects on monocytes and macrophages (8,9). Sev-
eral ligands have been identified, including the syn-
thetic anti-diabetic thiazolidinedione drugs, certain
nonsteroidal anti-inflammatory drugs (NSAIDs),
and naturally occurring ligands such as 15d-PGJ2
(10–12). The thiazolidinedione drugs, such as rosigli-
tazone and pioglitazone, are now widely used in the
treatment of insulin-resistant diabetes mellitus.
In addition to the critical role of PPARγ on insulin
sensitization, fatty acid metabolism, and inflamma-
tion, recent studies indicate that activation of PPARγ
can inhibit the growth and/or trigger the differenti-
ation process of several cancer cell lines. For exam-
ple, ligands for PPARγ have been shown to induce
cellular changes consistent with differentiation and
reversal of the neoplastic phenotype in liposarcoma,
colon, breast, and lung cancer cells (13–16).
Although these results suggest PPARγ ligands as
potential therapeutic agents in these malignancies,
the molecular mechanisms for their antiproliferative
activity have not been well-defined. Increased
cyclooxygenase-2 (COX-2) expression has been
shown to correlate with cancer development and pro-
gression (17–19). PPARγ ligands can indirectly sup-
press COX-2 expression by interfering with the
NF-κB signaling pathway (20) or, conversely,
directly activate COX-2 transcription (21). However,
the relationship between COX-2 regulation and the
biological effects of PPARγ on tumor cells has not
been previously investigated.
In this study, we determined the effect of treat-
ment with PPARγ ligands on pancreatic cancer cell
growth and COX-2 protein expression. In addition,
we assessed the potential role of the cell-cycle and
apoptotic-related proteins in pancreatic cancer inhi-
bition. Finally, the effect of these agents on pancre-
atic cancer cell invasiveness was determined. We
show that the PPARγ ligands, ciglitazone, and 15d-
PGJ2, inhibited pancreatic cancer cell growth through
a COX-2-independent mechanism. Treatment with
15d-PGJ2 decreased cyclin D1 expression, induced
expression of p21waf1 and p27kip1, and activated
caspase-8, -9, and -3 suggesting that the effect of
15d-PGJ2 was mediated through G1 cell-cycle reg-
ulation and caspase-dependent apoptosis. Moreover,
15d-PGJ2 decreased pancreatic cancer cell inva-
siveness and reduced matrix metalloproteinase-2
(MMP-2) and MMP-9 levels. Taken together, these
results suggest a potential role for PPARγ ligands in
the adjuvant treatment of pancreatic cancers.
Materials and Methods
Materials
The PPAR ligands, ciglitazone and 15d-PGJ2,
were purchased from Biomol (Plymouth Meeting,
PA) and Calbiochem (San Diego, CA), respectively,
and dissolved in dimethyl sulfoxide (DMSO). Tissue-
culture media and reagents were obtained from
Gibco-BRL (Grand Island, NY). Immobilon P mem-
branes for Western blots were from Millipore Corp.
(Bedford, MA). The enhanced chemiluminescence
(ECL) system for Western immunoblot analysis was
purchased from Amersham Corp. (Arlington
Heights, IL). Concentrated protein assay dye reagent
was purchased from Bio-Rad (Hercules, CA). Rabbit
polyclonal anti-PPARγ (H-100), goat polyclonal
anti-PPARα (N-19), rabbit polyclonal anti-cyclin
D1 (H-295), rabbit polyclonal anti-p27kip1 (C-19),
rabbit polyclonal anti-caspase-9 (H-170), rabbit
polyclonal anti-α-catenin (H-297), and rabbit poly-
clonal anti-β-catenin (H-102) antibodies were pur-
chased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Rabbit polyclonal anti-caspase-3 and
mouse monoclonal anti-PARP (clone 4C10-5) anti-
bodies were purchased from PharMingen (San
Diego, CA). Mouse monoclonal anti-caspase-8
(clone 5F7) antibody was purchased from Upstate
Biotechnology (Lake Placid, NY). Mouse mono-
clonal anti-MMP-2 (clone 147-6D11), mouse mon-
oclonal anti-MMP-9 (clone 56-2A4), and rabbit
polyclonal anti-p21waf1 antibodies were purchased
from Oncogene Research Products/Calbiochem
(Cambridge, MA). Mouse monoclonal anti-E-
cadherin antibody (clone 36) was purchased from
Transduction Laboratories (Lexington, KY). Mouse
monoclonal anti-COX-2 antibody was purchased
from Cayman Chemical (Ann Arbor, MI). Rabbit
anti-actin antibody was purchased from Sigma (St.
Louis, MO). CellTiter 96® AQueous One Solution
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