Prostaglandin E2 Increases Growth and Motility of Colorectal Carcinoma Cells*

Hongmiao ShengDagger , Jinyi ShaoDagger , M. Kay Washington§, and Raymond N. DuBoisDagger ||**DaggerDagger

From the Departments of Dagger  Medicine, § Pathology, and || Cell Biology, the  Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center and the ** Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232-2279

Received for publication, October 23, 2000, and in revised form, March 12, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chronic use of nonsteroidal anti-inflammatory drugs results in a significant reduction of risk and mortality from colorectal cancer in humans. All of the mechanism(s) by which nonsteroidal anti-inflammatory drugs exert their protective effects are not completely understood, but they are known to inhibit cyclooxygenase activity. The cyclooxygenase enzymes catalyze a key reaction in the conversion of arachidonic acid to prostaglandins, such as prostaglandin E2 (PGE2). Here we demonstrate that PGE2 treatment of LS-174 human colorectal carcinoma cells leads to increased motility and changes in cell shape. The prostaglandin EP4 receptor signaling pathway appears to play a role in transducing signals which regulate these effects. PGE2 treatment results in an activation of phosphatidylinositol 3-kinase/protein kinase B pathway that is required for the PGE2-induced changes in carcinoma cell motility and colony morphology. Our results suggest that PGE2 might enhance the invasive potential of colorectal carcinoma cells via activation of major intracellular signal transduction pathways not previously reported to be regulated by prostaglandins.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 APCDelta 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, PGF2alpha , 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-3alpha /beta (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 [gamma -32P]ATP, and 10 µg of L-alpha -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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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).

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.

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).

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-3alpha /beta (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 [gamma -32P]ATP and 10 µg of L-alpha -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.

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.

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®.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PI3Kbeta 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.

    FOOTNOTES

* This work was supported in part by United States Public Health Services Grants RO1DK 47297, P01CA77839, and P30CA-68485 (all to R. N. D.); the T. J. Martell Foundation; and Katie Couric.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger Recipient of a Veterans Administration Research Merit Grant. To whom reprint requests should be addressed: Dept. of Medicine/GI, MCN C-2104, Vanderbilt University Medical Center, 1161 21st Ave. S., Nashville, TN 37232-2279. Tel.: 615-322-5200; Fax: 615-343-6229; E-mail: raymond.dubois@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M009689200

    ABBREVIATIONS

The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; EP, E-prostanoid; PG, prostaglandin; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription; PCR, polymerase chain reaction; COX, cyclooxygenase; PBS, phosphate-buffered saline; FAK, focal adhesion kinase; PKB, protein kinase B.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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