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INTRODUCTION |
There is a 40-50% reduction in the relative risk of colorectal
cancer and colorectal cancer-associated mortality in individuals taking
nonsteroidal anti-inflammatory drugs
(NSAIDs)1 (1-3). Inhibition
of cyclooxygenase-2 (COX-2) activity is thought to represent one of the
mechanisms by which NSAIDs exert their anti-neoplastic effects (Refs. 4
and 5; reviewed in Ref. 6). In support of this hypothesis, lack of the
COX-2 (prostaglandin endoperoxide synthase-2) gene results in a
reduction of the number of tumors which develop in mice heterozygous
for an APC
716 mutation by more than 7-fold
(7). Additionally, COX-2 expression in colorectal carcinoma cells
provides a growth and survival advantage (5, 8), and increases tumor
cell invasiveness (9). Treatment with selective COX-2 inhibitors
significantly reduces the adenoma burden in humans (10) and in animals
(11). There are two isoforms of prostaglandin endoperoxide synthase,
which are commonly referred to as COX-1 and COX-2. COX-1 is produced
constitutively in many different cell types and tissues (12), but its
expression can be regulated under some circumstances (13). COX-2 is
induced by cytokines, growth factors, and tumor promoters (reviewed in Ref. 14). In studies of human colorectal cancer, COX-2 levels are
increased in about 90% of cancers and ~50% of pre-malignant colorectal adenomas, but the enzyme is not usually detected in adult
intestinal tissues (15, 16). Cyclooxygenase catalyzes the conversion of
arachidonic acid to prostaglandin (PG) G2 and PGH2. PGH2 is subsequently converted to a
variety of prostaglandins, which include PGE2,
PGD2, PGF2
, PGI2, and
thromboxane A2 by each respective prostaglandin synthase.
Prostaglandins are synthesized by a wide variety of human tissues and
serve as autocrine or paracrine lipid mediators to signal changes
within their immediate environment. PGs are involved in diverse
biological processes, which include inflammation, blood clotting,
ovulation, implantation, initiation of labor, bone metabolism, nerve
growth, wound healing, kidney function, blood vessel tone, and immune
responses (reviewed in Ref. 17).
The precise contribution of increased biosynthesis of
prostaglandins by COX-2 to the progression of neoplasia is currently under evaluation. For example, PGE2 generated in
colorectal carcinomas may enhance cell survival and/or may affect other
aspects of epithelial cell behavior such as cell-cell or cell-substrate
adhesion (5). A link between the neoplastic effect of carcinogen
treatment and prostaglandin signaling was recently made by the
observation that genetic disruption of the E-prostanoid receptor
subtype 1 (EP1) results in a reduction in the number of
aberrant crypt foci that develop in mice following carcinogen treatment
(18). Based on these findings, we sought to determine the effects of
PGE2 on the biology of colorectal carcinoma cells. We found
that PGE2 stimulated an increase in the proliferation and
motility of colorectal carcinoma cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
LS-174 cells were purchased from ATCC
(Manassas, VA). The cells were maintained in McCoy's 5A medium
containing 10% fetal bovine serum. LY 294002 and wortmannin were
purchased from Calbiochem (La Jolla, CA). PGE2, butaprost,
sulprostone, and PGE1 alcohol were purchased from Cayman
Chemical (Ann Arbor, MI).
Immunoblot Analysis--
Immunoblot analysis was performed as
described previously (19). Cells were lysed for 30 min in
radioimmunoprecipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin, 1 mM sodium orthovanadate) and then
clarified cell lysates were denatured and fractionated by
SDS-polyacrylamide gel electrophoresis. After electrophoresis, the
proteins were transferred to nitrocellulose membranes and the filters
were incubated with the antibodies indicated and developed by the
enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
The anti-phosphorylated Akt antibody was purchased from New England
Biolabs (Beverly, MA), and the anti-active ERK1/2 antibody was from
Promega (Madison, WI).
Cell Growth in Matrigel®--
1 × 104 cells were suspended in 0.5 ml of 1:2 diluted
Matrigel® (Collaborative Biomedical Products, Bedford,
MA), and the mixture was plated into 24-well plates. PGE2
in fresh medium was added to the cell culture every 2 days. After the
plates were incubated for 10-15 days, they were photographed using a
camera attached to an inverted microscope. Relative colony size was
determined by measuring 10 random colonies in each slide (50 measurements/well). The mean for each treatment set was calculated
and compared with controls.
ERK Kinase Assay--
p42/p44 MAP kinase activity was measured
by determining the transfer of the phosphate group of adenosine
5'-triphosphate to a peptide that is a highly specific substrate for
p42/44 MAP kinase (Biotrak system, Amersham Pharmacia Biotech).
Akt Kinase Assay--
For determination of Akt kinase activity
we used the Akt kinase assay kit made by New England Biolabs (Beverly,
MA) according to the manufacturer's instructions. Serum-starved cells
were treated with PGE2 and then lysed at the indicated
times. Akt was immunoprecipitated using a monospecific Akt antibody.
The immunoprecipitate was then incubated with a GSK-3 fusion protein in
the presence of ATP. Phosphorylation of GSK-3 was measured by Western
blotting using an anti-phospho-GSK-3
/
(Ser21/9) antibody.
PI3K Assay--
PI3K assays were performed as described by Jiang
et al. (20). Cells were lysed and immunoprecipitated with
anti-Tyr(P) antibody (4G10, Upstate Biotechnology, Lake Placid, NY).
The activity of PI3K in immunoprecipitates was determined by incubating
the beads with reaction buffer (10 mM Hepes, pH 7.4, 10 mM MgCl2, 50 µM ATP, 20 µCi of
[
-32P]ATP, and 10 µg of
L-
-phosphatidylinositol-4-phosphate) for 20 min at
25 °C. Phosphorylated products were separated by thin layer
chromatography and visualized by autoradiography.
Immunofluorescence--
LS-174 cells were grown in 35-mm tissue
culture plates and fixed in methanol/acetone at
20 °C for 10 min.
Fixed cells were incubated with 10% normal donkey serum for 1 h
and then with anti-FAK or anti-paxillin antibody (Transduction
Laboratories, Lexington, KY) for 2 h at room temperature. After
washing the cells three times with PBS, they were incubated with
Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West
Grove, PA) for an additional 1 h. The cells were then washed with
PBS, mounted, and observed under fluorescent microscopy with
appropriate filters. For direct immunofluorescence, cells were fixed
with formaldehyde-Triton solution and then incubated with 10 nM fluorescent phalloidin for 30 min.
Cell Migration and Invasion Assays--
Cell migration and
invasion assays were carried out using Transwell chambers (8 µm,
Corning Costar Co., Cambridge, MA). 5 × 104 cells
suspended in 400 µl of serum-free McCoy's 5A medium were placed in
the uncoated (migration assay) or 1:10 diluted
Matrigel®-coated (invasion assay) upper chamber. The lower
chamber was filled with 1 ml of McCoy's 5A medium containing vehicle
or 0.1 µM PGE2. After an incubation period of
20 h at 37 °C, the cells on the upper surface of the filter
were removed with a cotton swab. The filters were fixed and stained
with 0.5% crystal violet solution. Cells adhering to the undersurface
of the filter were counted. Three independent experiments were carried
out, and the data are expressed as the mean ± S.E. of assays
performed in triplicate.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
RT-PCR was carried out using the RNA PCR kit from
PerkinElmer Life Sciences.
A set of specific PCR primers for EP receptor subtypes (GenBankTM
accession numbers NM000955, NM0000956, NM000957, and NM000958 for
EP1, EP2, EP3, and EP4,
respectively) have been designed as follows: EP1 fragment,
forward (5'-ACCGACCTGGCGGGCCACGTGA-3'; 321-342) and reverse
(5'-CGCTGAGCGTGTTGCACACCAG-3'; 750-729); EP2 fragment,
forward (5'-TCCAATGACTCCCAGTCTGAGG-3'; 169-190) and reverse
(5'-TGCATAGATGACAGGCAGCACG-3'; 642-621); EP3 fragment, forward (5'-GATCACCATGCTGCTCACTG-3'; 396-415) and reverse
(5'-AGTTATGCGAAGAGCTAGTCC-3'; 904-884); EP4 fragment,
forward (5'-GGGCTGGCTGTCACCGACCTG-3'; 565-585) and reverse
(5'-GGTGCGGCGCATGAACTGGCG-3'; 1050-1030).
One µg of total RNA was reverse-transcribed and amplified with
35 PCR cycles. The amplified products were visualized on 1.5% agarose gels.
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RESULTS |
Alterations in the Phenotype of LS-174 Cells following
PGE2 Treatment--
Constitutive expression of COX-2 has
been reported in 85-90% of colorectal carcinomas (15, 16). COX-2 is
expressed in both carcinoma and stromal cells (21). Therefore, it is
possible that carcinoma cells that do not express COX-2 could receive
paracrine signals from PGE2 produced by neighboring stromal
cells. In order to elucidate whether PGE2 might exert any
effect on the phenotype of colon cancer cells, LS-174 human colon
cancer cells were treated with PGE2. LS-174 cells do not
generate detectable prostaglandins, although COX-2 protein is detected
in this cell line (22). LS-174 cells are able to form "crypt-like"
aggregates when they are cultured in Matrigel®. We found
that exogenously added PGE2 exerted a growth-stimulatory effect on LS-174 cells (Fig.
1A). The size of LS-174
colonies in Matrigel® increased following PGE2
treatment in a dose-dependent manner (Fig. 1B).
Treatment with 10 nM PGE2 resulted in optimal
stimulation of LS-174 cell growth, causing a 2-fold increase in colony
diameter.

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Fig. 1.
Growth-stimulatory effect of PGE2
in LS-174 cells. A, the stimulatory effect of
PGE2 on the growth of LS-174 cells in
Matrigel®. 1 × 104 LS-174 cells were
suspended in 0.5 ml of 1:2 diluted Matrigel® (serum-free).
PGE2 (50 nM) in fresh medium was replaced every
2 days. DMSO, Me2SO. After the plates were
incubated for 15 days, colonies were photographed (magnification,
×40). B, concentration-dependent stimulation of
colony growth by PGE2. 1 × 104 LS-174
cells were suspended in 0.5 ml of 1:2 diluted Matrigel®
(serum-free) and treated with indicated concentration of
PGE2. After the plates were incubated for 15 days, relative
colony diameter was determined and compared with controls. The
mean ± S.E. of assays performed in triplicate are plotted. The
results were similar in three separate experiments.
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To our surprise, treatment with PGE2 caused a dramatic
change in the morphology of the LS-174 colonies. When grown in
extracellular matrix components (Matrigel®), LS-174 cells
formed well organized structures consisting of an outside layer of
cells with an acellular center (Fig.
2A, panel a). Positive Alcian Blue staining indicated that the LS-174
colonies were filled with colonic type mucin (data not shown). In
contrast, the LS-174 cells exposed to PGE2 formed irregular
solid clumps of cells with a poorly organized structure (Fig.
2A, panel b). When grown on plastic
culture dishes, LS-174 cells formed in "non-spreading" round clumps
(Fig. 2A, panel c). Addition of 10 nM PGE2 led to a rapid change in phenotype,
which included increased spreading of cells within 2-4 h (Fig.
2A, panel d). Fluorescent staining with rhodamine-phalloidin demonstrated that PGE2 treatment
for 24 h resulted in protruding actin filaments from the cell
periphery in the form of microspikes (Fig. 2B,
panel b, white arrows) and an increase in the number of stress fibers (Fig. 2B,
panel b, black arrows).
PGE2 treatment also increased focal adhesion complexes as
determined by immunostaining for focal adhesion kinase (FAK) and
paxillin. Normally, FAK and paxillin are localized to the cytoplasm in
LS-174 cells (Fig. 2B, panels c and
e), but following PGE2 treatment the proteins
accumulated into focal adhesions at the ends of actin stress fibers
(Fig. 2B, panels d and f,
arrows).

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Fig. 2.
PGE2 modulation of the morphology
of LS-174 cells. A, morphology of LS-174 cells. LS-174
cells were seeded in Matrigel® and treated with DMSO
(a) or 50 nM PGE2 (b) for
15 days. Colonies were photographed using an inverted microscope
(original magnification, ×100). LS-174 cells were seeded in 60-mm
plates and were grown in serum-deprived medium for 48 h prior to
PGE2 treatment. Pictures were taken after the cells were
treated with Me2SO (c) or 100 nM
PGE2 (d) for 24 h. The morphology of a cell
clump is depicted (original magnification, ×400). B, effect
of PGE2 on actin rearrangement and focal adhesion in LS-174
cells. LS-174 cells were serum-starved for 48 h and treated with
vehicle (panels a, c, and
e) or 0.1 µM PGE2
(panels b, d, and f) for
24 h. Immunofluorescence was carried out by using
rhodamine-phalloidin (panels a and b),
anti-FAK antibody (panels c and d), or
anti-paxillin antibody (panels e and
f). PGE2 treatment for 24 h results in
protruding actin filaments in the form of microspikes (panel
b, marked by white arrows), and an increase in
the number of stress fibers (panel b, marked by
black arrows). PGE2 treatment increased focal
adhesion complexes as determined by immunostaining for FAK
(panel d, marked by white arrows) and
paxillin (panel f, marked by white
arrows).
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To further examine the spreading behavior induced by PGE2,
we carried out experiments using a modified Boyden chamber. Treatment of cells with PGE2 resulted in a significant increase
(2-3-fold) in cell motility (Fig.
3A). Addition of 0.1 µM PGE2 also promoted the movement of LS-174
cells through a Matrigel®-coated polycarbonate membrane by
2-3-fold (Fig. 3B). Therefore, PGE2 altered the
behavior of LS-174 cells by stimulating an increase in their motility,
which could explain, in part, their change in cellular organization
when grown as multicellular colonies.

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Fig. 3.
Analysis of cell migration and invasion.
Modified Boyden chambers were uncoated (A) or coated with
1:10 diluted Matrigel® (B). 5 × 104 serum-starved LS-174 cells were seeded into the upper
chamber, and the assay was carried out for 20 h with vehicle or
0.1 µM PGE2 as attractant. Cells that
migrated to the bottom part of the upper chamber were counted per field
(×200) and averaged from three wells. The results shown are
representative of three separate experiments. CTR,
control.
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Evaluation of EP Receptor Subtypes--
We next determined
if LS-174 cells express EP receptors, which are known to bind
PGE2 with a high affinity (reviewed in Ref. 23). The
expression of EP receptors in LS-174 cells was determined by RT-PCR
using specific oligonucleotide primers. EP2,
EP3, and EP4 were clearly expressed in LS-174
cells (Fig. 4A), but mRNA for the EP1 receptor was barely detectable. To elucidate
the functional role of EP receptor subtypes in LS-174 cells, we treated
the cells with butaprost (1 µM, a selective
EP2 receptor agonist), sulprostone (5 µM, a
selective EP3 receptor agonist), and PGE1
alcohol (10 nM, a selective EP4 receptor
agonist). Treatment with butaprost or sulprostone did not cause
significant changes in cell morphology (data not shown). However,
treatment with the PGE1 alcohol (10 nM)
resulted in more rapid and significant cell spreading when compared
with the effect of PGE2 alone. Alterations in LS-174 cell
spreading were seen within 1 h following addition of the PGE1 alcohol (Fig. 4B). Thus, LS-174 cell
spreading and migration, stimulated by PGE2, may be
predominantly mediated through the EP4 signaling
pathway.

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Fig. 4.
Evaluation of the role of EP receptors.
A, expression of PGE2 receptors. One µg of
total RNA extracted from LS-174 was reverse-transcribed by using random
hexamers. The fragment was amplified by specific primers for
EP1, EP2, EP3, and EP4
for 35 PCR cycles. The amplified products were visualized on 1.5%
agarose gels. M, molecular weight marker.
B, the effect of EP receptor agonists. LS-174 cells were
serum-starved for 48 h prior to the treatment with vehicle
(Control) or PGE1 alcohol (0.1 µM). The pictures were taken 24 h after initiation
of treatment. The morphology of cell clumps is shown (original
magnification, ×200).
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Regulation of ERK and Akt Activity by PGE2--
A
number of signaling pathways is known to regulate cell growth and
motility. The MAP kinase/ERK kinase/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were evaluated following PGE2 treatment. Treatment
of LS-174 cells with PGE2 (100 nM) only had a
modest effect on the activity of ERK1/2. PGE2 treatment
slightly increased the levels of phosphorylated ERK1/2 as determined by
Western blotting analysis (Fig.
5A, upper
panel). The results of an ERK kinase assay confirmed this
finding (Fig. 5A, lower panel).

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Fig. 5.
Effect of PGE2 on ERK and Akt
activities in LS-174 cells. A, the activation of
ERK1/2. LS-174 cells were serum-starved for 48 h prior to
PGE2 (0.1 µM) treatment. Cellular protein was
collected at the indicated time points for determination of the levels
of phosphorylated ERK1/2 (upper panel). For ERK
kinase assays, LS-174 cells were serum-starved for 48 h and cell
lysates were prepared at the indicated time points. ERK kinase activity
was measured using the Biotrak system. B, the activation of
Akt. LS-174 cells were serum-deprived for 48 h prior to
PGE2 (0.1 µM) treatment. Cellular protein was
collected at the indicated time points for determining the levels of
phosphorylated Akt (upper panel). To determine
PGE2 regulation of Akt kinase activity, LS-174 cells were
serum-deprived for 48 h and treated with 0.1 µM
PGE2. Akt was immunoprecipitated using a monospecific Akt
antibody. The immunoprecipitate was then incubated with a GSK-3 fusion
protein in the presence of ATP. Phosphorylation of GSK-3 was measured
by Western blotting using an anti-phospho-GSK-3 / (Ser21/9)
antibody (lower panel). C, PI3K assay.
LS-174 cells were lysed and immunoprecipitated with anti-Tyr(P)
monoclonal antibody. PI3K activity in immunoprecipitates was determined
by incubating the beads with reaction buffer containing 20 µCi of
[ -32P]ATP and 10 µg of
L- -phosphatidylinositol-4-phosphate. Phosphorylated
products were separated by thin layer chromatography and visualized by
autoradiography. PIP2,
phosphatidylinositol 3,4-bisphosphate. D, inhibition
of PI3K. LS-174 cells were serum-deprived for 48 h. Wortmannin
(Wort., 0.1 µM) or LY 294002 (LY,
10 µM) were added 1 h prior to the PGE2
(0.1 µM) treatment. Cell lysates were collected after a
2-h incubation, and the levels of pAkt were analyzed by Western
blotting.
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On the other hand, treatment with PGE2 led to a
marked activation of the PI3K/Akt pathway. The levels of phosphorylated
(Ser-473) Akt/PKB were elevated following treatment with
PGE2 in LS-174 cells (Fig. 5B, upper
panel). Kinase assays, which measure the capacity to
phosphorylate GSK-3 kinase, demonstrated that Akt kinase activity
was greatly increased following PGE2 treatment of LS-174
cells (Fig. 5B, lower panel). It is
known that Akt/PKB can be activated by G protein-coupled signaling in
both a PI3K-dependent and -independent manner. Kinase
assays failed to detect any PI3K activity in serum-starved LS-174
cells, and treatment with PGE2 resulted in rapid induction
of PI3K activity, as determined by the conversion of
phosphatidylinositol 4-phosphate to phosphatidylinositol 3,4-bisphosphate (Fig. 5C). To confirm the involvement of
PI3K in PGE2 activation of Akt/PKB, we evaluated two
inhibitors of this pathway (wortmannin (0.1 µM) and LY
294002 (10 µM)) and found that they both completely
blocked PGE2-induced phosphorylation of Akt/PKB (Fig.
5D).
The Role of PI3K/Akt in PGE2-induced Pro-neoplastic
Effects--
To determine whether the activation of Akt/PKB by
PGE2 altered the phenotype of LS-174 cells, the cells were
treated with PGE2 in the presence of specific PI3K
inhibitors, LY 294002 (5 µM) and wortmannin (0.1 µM). Both LY-294002 and wortmannin, at low
concentrations, have been demonstrated to specifically target PI3K
activity (24). LS-174 cells were grown on plastic dishes and subjected
to serum deprivation for 48 h. The cells were then treated with
PGE2 (0.1 µM) in the presence or absence of
LY 294002 or wortmannin for 24 h. DNA synthesis was evaluated by
[3H]thymidine incorporation assays. PGE2
treatment resulted in a 70% increase in [3H]thymidine
incorporation in LS-174 cells, and addition of 5 µM LY
294002 completely blocked the PGE2-induced increase in DNA synthesis. The presence of 0.1 µM wortmannin also
abolished the PGE2 effect on DNA synthesis (Fig.
6A). Although inhibition of PI3K/Akt activity blocked PGE2-induced growth effects, LY
294002 and wortmannin (0.1 µM) also prevented the
PGE2-induced cell spreading in LS-174 cells (Fig.
6B and data not shown). Next, we evaluated the role of
PI3K/Akt activity on cell motility. Modified Boyden chamber assays
demonstrated that PGE2 treatment resulted in a 2-2.5-fold
increase in cell motility, and 5 µM LY 294002 or 0.1 µM wortmannin completely abolished this effect (Fig. 6,
C and D).

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Fig. 6.
Roles of PI3K/Akt in cell proliferation, cell
morphology, and cell migration. A, cell proliferation
and inhibition of PI3K/Akt. LS-174 cells (2.5 × 104)
were placed in 24-well plates and grown in serum-deprived medium for
48 h. The indicated treatments along with 1 µCi of
[3H]thymidine were added into the cultural medium 24 h prior to harvesting cells for determination of
[3H]thymidine incorporation. PGE2,
0.1 µM PGE2; LY, 5 µM LY 294002; W, 0.1 µM
wortmannin. The experiment was carried out in quadruplicate and
repeated three times. The results were normalized to the amount of
radioactivity in control cells and plotted as mean ± S.E.
B, cell morphology and inhibition of PI3K/Akt. LS-174 cells
were grown in 60-mm plates and treated with vehicle (panel
a), 5 µM LY 294002 (panel
b), 0.1 µM PGE2 (panel
c), or 0.1 µM PGE2 plus 5 µM LY 294002 (panel d) for 48 h in serum-free medium (original magnification, ×100). C
and D, cell migration and inhibition of PI3K/Akt. 5 × 104 LS-174 cells were seeded into the upper chamber of
modified Boyden chambers, and the assay was carried out for 20 h
with indicated treatment as attractant in the lower chamber.
PGE2, 0.1 µM PGE2;
LY, 5 µM LY 294002; wort., 0.1 µM wortmannin. Migrating cells were counted per
microscope field (times]200) and averaged from three wells. The
results shown are representative of three separate experiments.
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Since PGE2 treatment dramatically altered the growth and
morphology of LS-174 colonies in Matrigel®, it was of
interest to determine the effects of PI3K/Akt activity on LS-174 cells
grown in Matrigel®. As demonstrated in Fig.
7A, LY 294002 impaired the
ability of LS-174 cells to grow in Matrigel® whereas
PGE2 significantly increased the size and altered the morphology of LS-174 colonies. Interestingly, addition of LY 294002 completely blocked the PGE2 effects on cells grown in
Matrigel® by inhibiting colony growth and the invasive
morphology. Wortmannin exerted similar effects but to a lesser degree
on LS-174 cells grown in Matrigel® (Fig. 7B and
data not shown).

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Fig. 7.
Inhibition of PI3K/Akt and cell growth in
Matrigel®. A, LY 294002 effect on LS-174 cell
growth in Matrigel®. 1 × 104 LS-174
cells were suspended in 0.5 ml of 1:2 diluted Matrigel®.
Cells were treated with vehicle (CTR), LY 294002 (LY, 5 µM), PGE2 (0.1 µM), or LY 294002 plus PGE2
(PGE2 + LY). After the plates were incubated for
15 days, colonies were photographed (×40). Colony size in diameter was
determined and compared with controls, and the mean ± S.E. of
assays performed in triplicate are plotted. The results shown are
representative of two separate experiments. B, wortmannin
(wort., 0.1 µM) effect on LS-174 cell growth
in Matrigel®.
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DISCUSSION |
It is now clear that COX-2 plays a role in the promotion of
colorectal cancer (6). However, the effects of prostaglandins generated
by COX-2 have largely been unexplored. Here we provide evidence that
prostaglandin-mediated signaling affects cell proliferation, motility,
and morphogenesis and that activation of the PI3K/Akt pathway is
essential for the PGE2-induced changes in neoplastic potential.
To evaluate the effect of prostaglandins on the behavior of colorectal
carcinoma cells, we employed several approaches. Treatment with
PGE2 stimulated DNA synthesis and cell spreading in LS-174 cells grown on plastic cultures. LS-174 cells form well differentiated multicellular colonies in Matrigel®, mimicking tumor
growth in animals. Treatment of LS-174 colonies with PGE2
led to a significant disruption of their cellular organization with
increased motility. The stimulation of cell migration by PGE2 has been observed previously in mesangial,
endothelial, and T cells (25-27). Forced expression of COX-2 in colon
carcinoma cells results in increased invasiveness compared with the
parental cells (9). These findings suggest that a prostaglandin
product, such as PGE2, might stimulate cell motility and
invasiveness under certain circumstances. In the present study, we show
that addition of PGE2 to serum-deprived LS-174 cells
results in increased cell spreading accompanied by polymerization of
actin and assembly of stress fibers, indicating that PGE2
induced cytoskeletal reorganization. A role for the actin cytoskeleton
has been implicated in many cellular functions, including motility,
chemotaxis, cell division, endocytosis, and secretion (28-30). Our
data also demonstrate that PGE2 treatment caused
aggregation of FAK and paxillin, promoting the formation of focal
adhesion complexes, which are known to be essential for cell
migration (31, 32).
PGE2 acts via specific transmembrane G protein-coupled
receptors (EP receptors) (23). Four EP receptor subtypes have been identified and are designated EP1, EP2,
EP3, and EP4. EP1 signals via
increased Ca2+, which leads to vasoconstriction.
EP3 can also serve to stimulate vasoconstriction and
inhibits the generation of cAMP, whereas EP2 and
EP4 are known to mediate vasorelaxation by stimulating an
increase in cAMP levels. Our results show that both sulprostone (EP3 agonist) and butaprost (EP2 agonist) (33,
34) did not mimic the effect of PGE2 to increase cell
spreading. However, both the PGE1 alcohol and misoprostol
(relatively selective EP4 agonist, data not shown) (33-35)
induced significant cell spreading. These findings suggest that
signaling via the EP4 receptor is, at least in part,
responsible for the PGE2-induced changes in LS-174 cell behavior.
Evidence suggests that the PI3K/Akt pathway promotes growth
factor-mediated cell survival and inhibits apoptosis (36). PI3K/Akt also plays a key role in the regulation of cell adhesion and actin rearrangement (37, 38). These observations suggest that the PI3K/Akt
pathway is oncogenic and involved in the neoplastic transformation of
mammalian cells. PI3K can be activated by growth factors, oncogenes, and is involved in the transmission of signals from certain G protein-coupled receptors (39-41). Akt is stimulated by a variety of
agonists acting on G protein-coupled receptors (42-44). Murga et
al. (41) recently reported that PI3K
is necessary and
sufficient to transmit signals from G proteins to Akt/PKB. Akt/PKB may
also be activated by cyclic AMP-dependent protein kinase in
a wortmannin-insensitive manner (42, 45). Here, we found that treatment
with PGE2 rapidly increased the kinase activity of Akt/PKB
and that wortmannin and LY 294002 blocked PGE2-induced
phosphorylation of Akt/PKB, suggesting the involvement of PI3K. Thus
far, the mechanism by which prostaglandin activates Akt/PKB is not
clear, and, to our knowledge, this represents the first report of
Akt/PKB modulation by PGE2. However, we have not
established a direct link between the EP receptor and PI3K activation
in the present study.
Our data further demonstrate the involvement of Akt/PKB activity in the
PGE2-induced increase in cell proliferation and motility. LY 294002, at low concentrations (5-20 µM), specifically
targets PI3K activity (24). The observation that both LY-294002 and wortmannin (structurally unrelated PI3K inhibitors) exerted similar effects on LS-174 cells indicates that PI3K is the likely target of
these compounds (reviewed in Refs. 46 and 47). We found that the growth
of LS-174 cells (either on plasic or in Matrigel®) was
significantly impaired by LY294002 (5 µM), suggesting
that basal levels of PI3K/Akt activity are required for continuous growth of LS-174 cells. Specific inhibitors of PI3K that blocked the
activation of Akt/PKB did inhibit PGE2-induced changes in cell behavior, suggesting that both PGE2-induced growth
stimulation and cytoskeletal reorganization involve the activation of
the PI3K/Akt pathway. Several studies demonstrated that Akt/PKB pathway plays an extremely important role in cell cycle progression via modifying the expression of cell cycle proteins, such as cyclin D1 and
p27kip (48-51). Activation of the PI3K/Akt
pathway is thought to be essential for cytoskeletal reorganization
under certain circumstances (37). These previous studies strongly
support our findings that the PI3K/Akt activity is required for
PGE2-induced increases in the growth and invasiveness of
LS-174 cells.
Although COX enzyme activity is proposed to play a pro-neoplastic role
in colorectal carcinogenesis, the downstream signaling that mediates
these effects is poorly understood. Our results demonstrate that
PGE2 can induce significant phenotypic alterations in
colorectal carcinoma cells. These changes include increased motility,
changes in cell shape, and stimulation of cell growth. We found the
PI3K/Akt signaling pathway to be involved in the regulation of
morphogenic and proliferative changes. This work establishes a role for
PGE2 in the stimulation of tumor cell motility and reveals
an additional cellular target, the EP4 receptor, which appears to be involved in this process.