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[Cancer Research 62, 1567-1572, March 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Cyclooxygenase-2 Activity Altered the Cell-Surface Carbohydrate Antigens on Colon Cancer Cells and Enhanced Liver Metastasis

Yoshimi Kakiuchi1, Shingo Tsuji, Masahiko Tsujii, Hiroaki Murata, Naoki Kawai, Masakazu Yasumaru, Arata Kimura, Masato Komori, Takanobu Irie, Eiji Miyoshi, Yutaka Sasaki, Norio Hayashi, Sunao Kawano and Masatsugu Hori

Department of Internal Medicine and Therapeutics [Y. K., S. T., M. T., H. M., N. K., M. Y., A. K., M. K., T. I., M. H.], Departments of Biochemistry [E. M.], and Molecular Therapeutics [Y. S., N. H.], Osaka University Graduate School of Medicine, and Department of Clinical Laboratory Sciences, School of Allied Health Science, Faculty of Medicine, Osaka University [S. K.], Suita, Osaka 565-0871, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase-2 (COX-2) was recently reported (M. Tsujii and R. N. DuBois, Cell, 83: 493–501, 1995) to affect the metastatic potential of cells. Previous studies (M. Fukuda, Cancer Res., 56: 2237–2244, 1996) indicated that sialyl Lewis antigen expression is correlated with hematogenous metastasis of colon cancer. In the present study, we investigated the interaction between COX-2 activity, expression of sialyl Lewis antigens, in vitro cancer cell adhesion to endothelial cells, and in vivo metastatic potential. Effects of COX-2 activity and prostaglandin E2 on cell adhesion, expression of sialyl Lewis antigens, and glycosyltransferase genes were determined in Caco-2-m (COX-2 low level), Caco-2-COX-2 (programmed to overexpress COX-2), and HT-29 (COX-2 high level) cells. Metastatic spread of these cells to the liver was also investigated. Caco-2-COX-2 cells had increased SPan-1 levels and increased adherence to endothelial cells via SPan-1 compared with Caco-2-m cells. HT-29 cells expressed sialyl Lewis a and adhered to endothelial cells via sialyl Lewis a. Treatment with a COX-2 inhibitor, celecoxib, decreased SPan-1 and sialyl Lewis a expression and adherence to endothelial cells. ß3Gal-T5 and ST3Gal III and IV expression was inhibited by celecoxib and was enhanced by prostaglandin E2 treatment. Caco-2-COX-2 and HT-29 cells metastasized to the liver, whereas Caco-2-m cells did not. Pretreatment with celecoxib reduced the metastatic potential as well as anti-sialyl Lewis antibodies. Our results indicate a direct link between COX-2 and enhanced adhesion of carcinoma cells to endothelial cells, and enhanced liver metastatic potential via accelerated production of sialyl Lewis antigens. COX-2 inhibitors may suppress metastasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NSAIDs2 are currently being evaluated for their effectiveness as chemopreventive and chemotherapeutic agents (1 , 2) . COX is a major target of NSAIDs and the inducible cyclooxygenase, COX-2, is up-regulated in gastrointestinal cancers (3 , 4) . Therefore, it is likely that COX-2 has an important role in gastrointestinal carcinogenesis. We previously reported (5) that overexpression of COX-2 leads to phenotypic changes involving increased adhesion to the extracellular matrix and inhibition of apoptosis in rat intestinal epithelial cells, which could enhance their tumorigenic potential. Constitutive expression of COX-2 can also lead to alterations in the invasive potential of colorectal cancer cells (6) , and COX-2 may be involved in tumor angiogenesis (7) . Several reports recently suggested that COX-2 expression has an important role in hematogenous metastasis of colon carcinomas to the liver (8) . The precise mechanisms underling the role of COX-2 in this process are unknown.

The liver is a target organ of metastasis from gastrointestinal malignancies. Among the various steps in hematogenous metastasis to the liver, the initial adhesion of tumor cells to endothelial cells has a crucial role (9) . To resist the substantial wall shear stress exerted by blood flow, metastasizing colon carcinoma cells have to form adhesive contacts with endothelial cells, which prevents detachment and the colon carcinoma cells ability to reenter the circulation (10) . Adhesion of circulating cells to endothelial cells is mediated by a variety of cell adhesion molecules. Membrane-anchored selectins and carbohydrates initiate tethering and rolling of flowing tumor cells on endothelial cells during metastasis. Cell surface glycoproteins have essential roles in maintaining the function as well as the structure of cells (11) . Among carbohydrates of these glycoproteins, sLea and sLex are human tumor-associated antigens. Tumor makers sLea and sLex are also ligands for E-selectin (12 , 13) . Highly metastatic colonic carcinoma cells bind more strongly to E-selectin expressed on activated human endothelial cells than do low-metastatic counterparts (14) . Poorly metastatic tumor cells expressing low levels of sLex adhere more strongly to E-selectin after being genetically engineered to increase the amount of sLex (15) . These studies strongly suggest that the abundance of sLea and sLex is a key factor in metastatic spread.

The aim of the present study was to investigate the effect of constitutive COX-2 expression in human colon carcinoma cells on adherence to endothelial cells and the metastatic potential of these cancer cells. In the present study, using colon cancer cell lines that are programmed to express low and high levels of COX-2 and sialyl Lewis antigens (Caco-2 and HT-29 cell lines, respectively), we investigated the influence of COX-2 expression, COX-2 specific inhibition, and a COX-2 product, PGE2, on in vitro adhesion to endothelial cells, expression of sialyl Lewis antigens and glycosyltransferases, and in vivo metastatic potential. COX-2 expression or PGE2 treatment induced the expression of some glycosyltransferases and type-I sialyl Lewis antigens, leading to enhanced tumor-cell adhesion to endothelial cells and to liver metastasis. Celecoxib, a COX-2-specific inhibitor, suppressed these phenotypic changes induced by COX-2 activity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
PGE2 was purchased from Cayman Chemical (Ann Arbor, MI). Celecoxib, a COX-2 specific inhibitor, was a kind gift from Searle (St. Louis, MO). Antimouse MAbs against sLea (MAB2095) sLex (MAB2096) were purchased from Chemicon International, Inc. (Temecula, CA). Anti-SPan-1 MAb was kindly provided by Dr. Hirakawa (Osaka City University, Osaka, Japan) and Dainabot Co. Ltd. (Tokyo, Japan).

Cell Culture.
Caco-2 and HT-29 cells derived from human colon cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FBS (JRH Biosciences, Lenexa, KS), 1% antibiotics and antimycotics (Life Technologies, Inc., Grand Island, NY), in an atmosphere of 95% air and 5% CO2 at 37°C. HUVECs were obtained from Kurabou Co. (Osaka, Japan) and were maintained in Daigo’s T medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 2 ng/ml of recombinant basic fibroblast growth factor (Takeda Pharmaceutical Co., Osaka, Japan) and 10% FBS. In the studies of the effects of celecoxib, the cells were cultured for 48 h in DMEM supplemented with 10% FBS in the presence of 10 µM celecoxib. For investigation of the effect of PGE2, after 48-h starvation, the cells were treated by various concentration of PGE2 for 24 h.

Stable Transfection.
A 2.1-kb fragment containing the open reading frame for the rat COX-2 gene was isolated and cloned into the eukaryotic expression vector pCB6. This vector was constructed so that transcription of the cDNA was controlled by the cytomegalovirus promoter. This vector also contains a neomycin-resistant gene that allows for selection of transfected cells by the addition of G418 to the cell culture medium. Caco-2 cells obtained from the American Type Culture Collection were transfected using LipofectAMINE (Life Technologies, Inc.), as described previously (16) , and cultured in DMEM containing 1.5 mg/ml G418 (Sigma Chemical Co.), supplemented with 10% FBS. Five independently derived controls and COX-2 transfected cell lines were characterized and found to have similar phenotypes.

PGE2 Measurements.
PGE2 was measured by ELISA (Cayman Chemical). PGE2 is the major metabolite of arachidonic acid metabolism in Caco-2, Caco-2-COX-2, and HT-29 cells. These measurements were made in triplicate and repeated in three experiments.

Adhesion of Cancer Cells to Endothelial Cells.
HUVECs were maintained in a collagen-coated dish with E300 medium in a humidified atmosphere of 5% CO2 at 37°C. HUVECs at passages 2 through 5 were plated at a density of 1 x 105 cells/well 24 h before the assay and were grown to a confluent monolayer in six-well plates. The HUVECs were stimulated with 1 ng/ml of IL-1ß (Otsuka Pharmaceutical Co., Tokushima, Japan) for 4 h before the assay. The medium was replaced with DMEM dissolved in 1% BSA and incubated for 30 min. After preincubation, 100 µl of [3H]thymidine-labeled Caco-2-m, Caco-2-COX-2, or HT-29 cell suspension (1 x 106 cells/ml, 0.5 µCi/ml) were added and incubated with rotation (90 rpm) for 30 min at 37°C. To the HUVECs in six-well plates, the tumor cells (1 x 105 cells/well) were plated and incubated. After gently washing three times with PBS [137 mM NaCl, 8.10 mM Na2HPO4·12H2O, 2.68 mM KCl, 1.47 mM KH2PO4 (pH7.4)] to remove unattached cells, the attached cells were solubilized in 0.1 N NaOH, and the radioactivity was measured. The attaching potential was estimated by the ratio of attaching cell counts:totalcounts applied to the culture. For the celecoxib-treated group, cancer cells were treated with 10 µM celecoxib for 48 h before labeling. For inhibition assays using antibodies, tumor cells were preincubated with anti-sLea (50 µg/ml), sLex (100 µg/ml), or SPan-1 (100 µg/ml) antibody for 30 min at 37°C before the adhesion assay.

Flow Cytometry Analysis and ELISA for Sialyl Lewis Antigen Expression.
Flow cytometric analysis was performed using FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The indirect immunofluorescence method was applied to stain the tumor cells with anti-sLea, anti-sLex, or anti-SPan-1 MAb at 10 µg/ml as the primary antibody (incubated for 30 min at room temperature), followed by the addition of FITC-labeled antimouse immunoglobulin (Silenus Lab., Hawthorn, Australia) as the secondary antibody. For ELISA, cells were washed in PBS, harvested with a rubber scraper, washed once again in PBS, and pelleted by centrifugation. Cell pellets were solubilized by brief sonication in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100. After determination of the protein concentration with a Micro BCA protein assay reagent kit (Pierce, Rockford, IL), solubilized protein (10 µg) from each sample was subjected to ELISA using the same antibodies as were used in the FACScan. Samples were measured in triplicate, and this experiment was repeated five times.

Expression of a Series of Glycosyltransferase mRNAs.
Caco-2-m, Caco-2-COX-2, and HT-29 cells cultured in DMEM containing 10% FBS were treated with celecoxib (10 µM) for 6 h. For investigation of the effect of PGE2, the cells were starved for 48 h and treated with PGE2 (0.2 µM) for 6 h. Total cellular RNA was isolated using ISOGEN (Nippon Gene Co., Toyama, Japan), and cDNAs were synthesized with an oligo(dT) primer from 3 µg of DNase I-treated total RNA using a Superscript preamplification system for first-strand cDNA synthesis (Invitrogen Japan K. K., Tokyo, Japan). The sequences of primers used for PCR are given in Table 1Citation . PCR was performed in a GeneAmp PCR system 2400 (PE Applied Biosystem, Roissy, France) on 1 µl of the sample cDNA in a final volume of 25 µl containing 25 pmol of sense and antisense primers, and 2 units Taq DNA polymerase (AmpliTaq Gold; Perkin-Elmer, Wellesley, MA) under the following conditions: 1 cycle at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min. A 10-µl aliquot of the RT-PCR mixture was subjected to electrophoresis in a 2% agarose gel and the bands were visualized by ethidium bromide staining.


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  		Table 1 Oligonucleotide primers used for RT-PCR analysis

 
Liver Metastasis.
Six-week-old male BALB/c nu/nu nude mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). All of the mice were maintained under specific pathogen-free conditions at the Center for Animal Experimentation, Osaka University Graduate School of Medicine. Regular laboratory food and tap water for drinking were freely available. Mice were anesthetized with ethyl ether. The abdominal wall was incised and the spleen exposed. Viable Caco-2-m, Caco-2-COX-2, or HT-29 cells, 1 x 107 in 100 µl PBS were injected into the spleen. In the celecoxib-treated group, these cells were preincubated with 10 µM celecoxib for 48 h and then injected into the spleen. In the antibody-treated groups, the cells were preincubated with MAb against SPan-1 (100 µg/ml) or sLea (50 µg/ml) for 60 min before inoculation. The mice were killed 12 weeks after inoculation of the cancer cells to the spleen. The liver was removed and checked for the presence of hepatic metastasis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PGE2 Production.
The concentration of PGE2 in culture medium of each cell line treated with or without celecoxib is shown in Fig. 1Citation . The highest level of COX-2 metabolite in all of the cell lines was PGE2. The level of PGE2 in Caco-2-COX-2 cells was elevated by ~7.5-fold compared with Caco-2-m cells. HT-29 cells produced the same amount of PGE2 as Caco-2-COX-2 cells did. Celecoxib at 5 µM inhibited PGE2 production by 25% of control Caco-2-COX-2 and HT-29 cells, and with 10 µM or more, PGE2 production was significantly inhibited to the levels produced by COX-2-m cells.



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  		Fig. 1. COX-2 activity of Caco-2-m, Caco-2-COX-2, and HT-29 cells. ELISA for PGE2 using supernatants from Caco-2-m, Caco-2-COX-2, and HT-29 cells, pretreated with celecoxib at various concentrations for 48 h. *, P < 0.01, significant suppression of PGE2 production by celecoxib pretreatment compared with untreated control by Student’s t test. Results are from one experiment with five replicate samples and are representative of three experiments.

 
Effect of COX-2 Activity and PGE2 in Colon Cancer Cells on Adhesion to Endothelial Cells.
To quantify the adherence of colon cancer cells to endothelial cells, Caco-2, Caco-2-COX-2, and HT-29 cells were assayed for adhesion to HUVECs (Fig. 2)Citation . Each cell line was tested for adhesion to 1 ng/ml IL-1ß-stimulated HUVECs. None of the cell lines adhered to untreated HUVECs, and adherence of tumor cells to IL-1ß-stimulated HUVECs was significantly blocked by anti-E-selectin antibody (data not shown), which suggested that E-selectin was involved in these cell-cell interactions. As shown in Fig. 2ACitation , the adhesion of Caco-2-COX-2 cells to IL-1ß-stimulated HUVECs was significantly increased, compared with Caco-2-m cells. Anti-SPan-1 MAb significantly inhibited the adhesion, although anti-sLea or anti-sLex MAb did not. HT-29 adhered to stimulated HUVECs, and the adhesion of HT-29 was inhibited by anti-sLea MAb, but not by anti-sLex or anti-SPan-1 MAb. Celecoxib inhibited cancer cell adhesion to stimulated HUVECs in a dose-dependent manner. Adhesion of the cells that were grown in serum-free conditions for 48 h was reduced (Fig. 2B)Citation . When the serum-starved cells were treated with PGE2 for 24 h, however, the adhesion of Caco-2-m and HT-29 cells was increased by 4- and 2.5-fold, respectively, whereas there was no significant change in the adhesion of Caco-2-COX-2 cells. Our results suggest that SPan-1 and sLea are involved in the adhesion of Caco-2-m and HT-29 cells enhanced by PGE2 treatment, because these antibodies suppressed the adhesion. These results indicate PGE2, produced by COX-2, up-regulates SPan-1 and sLea expression in Caco-2 cells and HT-29 cells, respectively.



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  		Fig. 2. Adhesion assay of cancer cells to a monolayer of HUVEC. In A, Caco-2-m, Caco-2-COX-2, or HT-29 cells were treated with celecoxib for 2 days and then labeled with [3H]thymidine. In B, Caco-2-m, Caco-2-COX-2, or HT-29 cells were starved for 48 h, treated with PGE2 for 24 h, and then labeled with [3H]thymidine. Cancer cells (1 x 105 cells/well) were incubated with IL-1ß-stimulated HUVECs for 30 min at 37°C with rotation (90 rpm). After the nonadhering cells were washed out, the cpm of the cells on the plates were counted. For inhibition of cell adhesion, MAb against sLea, SPan-1, or sLex was preincubated with cancer cells for 30 min before application of the monolayer of endothelial cells. *, P < 0.01, significant suppression or enhancement of adhesion compared with controls. #, P < 0.01, significant inhibition in adhesion compared with 0.2-µM PGE2-treated groups by Student’s t test.

 
Effect of COX-2 Activity in Colon Cancer Cells on Sialyl Lewis Antigen Expression.
The results of flow cytometric analysis and ELISA of sialyl Lewis antigens of Caco-2 and HT-29 cells cultured in DMEM/10% FBS with or without 10 µM celecoxib are shown in Figs. 3ACitation and 4ACitation , respectively. Neither Caco-2-m nor Caco-2-COX-2 cells expressed sLea. Both Caco-2-m and Caco-2-COX-2 cells expressed sLex, and there was no significant difference between the two cell lines. Celecoxib did not have a significant effect on sLex expression in Caco-2-m or Caco-2-COX-2 cells. Caco-2-COX-2 cells express more SPan-1 than do Caco-2-m cells, and 10 µM celecoxib significantly inhibited SPan-1 expression in Caco-2-COX-2 cells. In HT-29 cells, sLea expression was detected using FACScan and ELISA, and 10 µM celecoxib significantly inhibited its expression. Regarding SPan-1 expression in HT-29 cells, there was no significant difference between control and celecoxib-treated group, although the data from FACScan suggested that SPan-1 expression was slightly suppressed by 10 µM celecoxib treatment. sLex expression was detected, but celecoxib did not have a significant effect on the levels.



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  		Fig. 3. Flow cytometric analysis of cell surface carbohydrates on colon cancer cells. In A, Caco-2-m, Caco-2-COX-2, and HT-29 cells, cultured in DMEM containing 10% FBS, were treated with celecoxib (10 µM) for 48 h. In B, the cells, starved for 48 h, were treated with PGE2 for 24 h. Cells were stained with primary (MAbs) and secondary antibodies, and analyzed by flow cytometry. Shaded peaks in A and B, the results of staining with mouse IgG for the first antibody as a negative control. Black lines, control conditions without celecoxib or PGE2. Dotted lines, the pattern with 10 µM celecoxib or 0.2 µM PGE2. Samples were measured in triplicate, and this experiment was repeated three times.

 


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  		Fig. 4. Production of sLea, Span-1, and sLex in Caco-2-m, Caco-2-COX-2, and HT-29 cells. In A and B, the cells were prepared the same as in Fig. 3, A and BCitation . ELISA was performed for each carbohydrate, using cell lysates of the cells and probing with specific antibodies. *, P < 0.01, significant increase or decrease in carbohydrate production by celecoxib or PGE2 treatment compared with controls by Student’s t test.

 
Effect of PGE2 in Colon Cancer Cells on Sialyl Lewis Antigen Expression.
The results from FACScan and ELISA of the cells treated with or without PGE2 after 2-day starvation in serum-free media are shown in Figs. 3BCitation and 4BCitation , respectively. PGE2 (0.2 µM) significantly induced SPan-1 expression in Caco-2-m cells, although it did not have a significant effect on sLex expression. In Caco-2-COX-2 cells, there was no significant change in SPan-1 or sLex expression before or after PGE2 treatment. The PGE2 concentration in the culture medium of Caco-2-COX-2 cells was ~0.35 µM, and the effect of PGE2 was maximal in Caco-2-COX-2 cells. Neither Caco-2-m nor Caco-2-COX-2 cells expressed sLea even after PGE2 treatment. In HT-29 cells, PGE2 induced sLea expression, although it did not have a significant effect on sLex or SPan-1 levels.

Effect of COX-2 Activity in Colon Cancer Cells on Glycosyltransferases to Form sLea.
The synthesis of sLex and sLea epitopes at the reducing terminal ends of the carbohydrate chains require a set of several glycosyltransferases (ß1,3GnT, ß1,4GalT, ST3Gal, and {alpha}1,3Fuc-T) for sLex synthesis and (ß1,3GnT, ß1,3GalT, ST3Gal, and {alpha}1,4Fuc-T) for sLea synthesis. The gene encoding a ß1,3GnT, which is required for the synthesis of type I and type II chains, has not yet been cloned. The gene encoding a ß1,4GalT has been cloned (17) . ST3Gal I-IV are suggested to be involved in carbohydrate synthesis and carcinogenesis in many tissues (18) . A ß1,3Gal-T, ß3Gal-T5, and Fuc-T III are the most probable candidates responsible for the synthesis of the type I Lewis antigens (19) . The sLex and sLea antigens in colorectal carcinoma tissue are carried mainly by mucins with O-linked glycosylated chains (20) . core2 GnT catalyzes the transfer of GlcNAc residue of Galß1,3-GalNAc-Ser/Thr with a ß1,6 linkage to synthesize one of the branched structures, the core 2 structure, at the root of the mucin O-glycans. To identify the key enzymes responsible for the apparent SPan-1 or sLea antigen expression that was enhanced by COX-2 expression and inhibited by celecoxib, the quantities of the eight glycosyltransferase transcripts determined using RT-PCR were compared. As shown in Fig. 5ACitation , there was no difference in the expression of core2 GnT or ß1,4GalT between control and celecoxib-treated groups, although their RT-PCR products were present in Caco-2 and HT-29 cells. As shown in Fig. 5BCitation , core2 GnT or ß1,4GalT levels were not different between control and PGE2-treated groups. ß3Gal-T5, ST3Gal III, and ST3Gal IV expression, which was detected in all cell lines, was inhibited by celecoxib and induced by PGE2 treatment or COX-2-programmed expression. ST3Gal I was expressed in all of the cell lines examined. COX-2-programmed expression or PGE2 treatment reduced ST3Gal I expression, whereas celecoxib up-regulated its expression. ST3Gal II expression was not detected in any cell line. Fuc-T III expression was not detected in Caco-2-m or Caco-2-COX-2 cells. Although Fuc-T III expression was present in HT-29 cells, celecoxib or PGE2 had no effect.



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  		Fig. 5. RT-PCR analysis of glycosyltransferases. In A, Caco-2-m, Caco-2-COX-2 (Caco-2-c), and HT-29 cells, cultured in DMEM containing 10% FBS, were treated with celecoxib (10 µM) for 6 h. In B, the cells were starved for 48 h and then treated with PGE2 for 6 h. After treatment, 3 µg of total RNA was prepared, and cDNA was obtained. After the PCR reaction, aliquots of the products were electrophoresed on 2% agarose gel in the presence of ethidium bromide. Cont, the group under the control condition. Cel, the celecoxib-treated group.

 
Effect of COX-2 Activity on Liver Metastasis.
The number of metastatic foci after the intrasplenic injection of tumor cells is shown in Fig. 6Citation . Caco-2-m cells did not produce gross tumors in the livers of nude mice. Caco-2-COX-2 cells were highly metastatic to the liver after inoculation to the spleen. Pretreatment with MAb against SPan-1 inhibited the number of metastatic foci. HT-29 cells also demonstrated the capacity to metastasize to the liver after inoculation of cancer cells into the spleen. Pretreatment with anti-sLea MAb markedly inhibited the metastasis of HT-29 cells to the liver. Celecoxib pretreatment significantly inhibited liver metastases after injection of Caco-2-COX-2 and HT-29 cells.



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  		Fig. 6. Liver metastasis by Caco-2-COX-2 and HT-29 cells. The colon cancer cells were treated with celecoxib at 10 µM for 48 h or with MAb against sLea or SPan-1 for 60 min. Then, 1 x 107 cells were inoculated into the spleen. The mice were killed and examined for the presence of hepatic metastasis 12 weeks after the intrasplenic inoculation. §, a ratio of mice showing tumor growth:mice given injections of tumor cells. #, P < 0.05, significant decrease in tumor incidence compared with control by {chi}2 test. *, P < 0.05, significant decrease in number of tumor nodules compared with controls by Mann-Whitney’s U test analysis.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hematogenous metastasis of tumor cells to the liver is one of the major causes of death in patients with colorectal cancer. Recent studies indicate an involvement of COX-2 expression in colorectal tumors and in their metastatic spread to distant organs. Several steps are thought to be involved in metastasis. We previously demonstrated that COX-2 expression in colon cancer cells reduces E-cadherin levels and activates matrix metalloproteinase-2, leading to an acceleration of cellular invasion (6) . We also reported that COX-2 expression, partially via PGE2, accelerates the production of angiogenic factors and enhances tumor angiogenesis (7) . Each one of these effects is an important step for the development of metastatic foci in the liver. Adhesion of tumor cells to endothelial cells represents another important step in metastasis. Carbohydrates on tumor cells have an important role in the first stage of the cell adhesion cascade (tethering, rolling) by interacting with selectins (9 , 10) . In an effort to better understand the role of COX-2 in hematogenous metastasis, we prepared colon cancer cells programmed to express COX-2 and investigated the effect of COX-2 expression and PGE2 treatment on the adhesion of tumor cells to endothelial cells, carbohydrate expression, and metastatic spread to the liver. The present study was designed to evaluate the role of COX-2 in the early stages of liver metastasis, i.e., attachment to endothelial cells. Therefore, celecoxib treatment was initiated just before intrasplenic injection. In a preliminary study, PGE2 synthesis recovered soon after the discontinuation of celecoxib treatment, and resistance to apoptosis, angiogenic factor production, and matrix metalloproteinase-2 activation was reduced by celecoxib and restored within 3 to 6 h after removal of the drug. Therefore, it appears that the cells pretreated with celecoxib recovered to levels equivalent to those of nontreated cells in mice within 6 h. The present study indicates that COX-2 activity may regulate expression of sialyl Lewis antigens, resulting in enhancement of liver metastasis via accelerated adhesion to endothelial cells.

The present study demonstrates that sLea and SPan-1 might be involved in tumor cell adhesion to endothelial cells and liver metastasis, whereas sLex might not. Cumulative data indicate that sialylation of the terminal structure of the carbohydrate antigens (sialyl Lewis antigens) on the tumor cell surface confer an adhesion and metastasis-prone phenotype, based on clinicopathological and experimental studies (21) . sLea and SPan-1 on the surface of colorectal cancer constitute a major ligand for E-selectin on activated endothelial cells, and the sLea (SPan-1)-E-selectin system contributes to the establishment of hematogenous metastasis (22 , 23) . These findings are consistent with our results. On the other hand, sLex is also recognized by the endothelial selectins that mediate metastasis of tumor cells (12) . Both cell lines (Caco-2 and HT-29) were strongly positive for sLex. Our results, however, indicated that anti-sLex antibodies did not have an inhibitory effect on the adhesion to endothelial cells. Carbohydrate ligands involved in the adhesion of cancer cells to endothelial cells differ, corresponding to the type of cancer. sLea has a functional role that mediates the adhesion of cancer cells to endothelial cells in colon carcinoma, and the adhesion of cancer cells to endothelial cells that is mediated by sLex is weaker than that mediated by sLea (22 , 24) , although coexpression of sLea and sLex is common.

In the present study, we demonstrated for the first time that cell surface carbohydrates are altered by COX-2 activity or PGE2 treatment. Celecoxib suppressed type I Lewis antigen expression and liver metastasis. To clarify the mechanism responsible for this effect, we investigated the relationship between COX-2 expression, celecoxib, or PGE2 treatment and glycosyltransferase expressions. Sialyl Lewis epitopes produced in cancer cells are mainly carried on mucin O-glycans (20) . core2 GnT is essential for the synthesis of the sialyl Lewis antigens in mucin-type oligosaccharides (25) . ß1,4GalT is an enzyme responsible for the type II Lewis antigens, sLex (18) . COX-2 expression, celecoxib, or PGE2 had no influence on the expression of these two enzymes.

The ß3Gal-T5 gene is the most probable candidate responsible for the synthesis of type I Lewis antigens in gastrointestinal tumor cells, and it is a key enzyme that determines the amount of type I Lewis antigens (19) . Its mRNA expression was suppressed by celecoxib and induced by COX-2 expression and PGE2 in Caco-2 and HT-29 cells. Because its transcriptional regulation is not yet clarified, the precise mechanism by which COX-2 activity and PGE2 are involved in its regulation is still obscure.

Sialic acid is transferred to galactose in Galß1–3GlcNAc/Galß1,4GluNAc/-R by ST3Gals, the expression of which is significantly increased in carcinoma tissue compared with that in normal mucosa (18) . In the present study, COX-2 expression or PGE2 treatment enhanced, and celecoxib inhibited, ST3Gal III and IV expression, whereas COX-2 expression or PGE2 reduced, and celecoxib increased, ST3Gal I expression. AP-2 has a critical role in the epithelial-cell-specific transcriptional regulation of the ST3Gal IV gene (26) . Because PGE2 induces specific binding activity to CRE and AP-2 DNA elements (27 , 28) , COX-2 and PGE2 are thought to be involved in ST3Gal IV expression via transcriptional regulation of AP-2. Among these ST3Gals, ST3Gal III (29) and ST3Gal IV (30) are responsible for the biosynthesis of sLea and sLex antigens, because ST3Gal III and ST3Gal IV transfer sialic acid to the precursor structures of sLex and sLea with good efficiency, whereas ST3Gal I and ST3Gal II are inefficient (31) , and transfection of ST3Gal I or II to colon cancer cells do not alter the level of sialyl Lewis antigens (32) .

Fuc-T III is required for sLea synthesis (33) , whereas Fuc-T III, V, VI, and VII are responsible for sLex synthesis (33, 34, 35, 36) . Therefore, the absence of expression of sLea synthesis in Caco-2 cells is attributable to a lack of Fuc-T III. In HT-29, Fuc-T III expression was detected; and COX-2 expression, celecoxib, or PGE2 had no effect on its expression.

Celecoxib or PGE2 treatment influenced expression, not of sLex, but of type I sialyl Lewis antigens in Caco-2 or HT-29 cells. ß1,3Gal-T5 is a key responsive enzyme for type I Lewis antigen synthesis (19) and ST3Gal III uses the type-I structure more efficiently than type II (29) , although ST3Gal IV exhibits high activity with type II compared with the type I structure (30) . Therefore, these results suggest that the key enzymes responsible for the augmented expression of sLea and SPan-1 by COX-2 activity or PGE2 are ß3Gal-T5 and ST3Gal III.

In conclusion, we observed a direct link between COX-2 expression and alterations of cell surface carbohydrate levels via changes of glycosyltransferases, leading to an increased metastatic potential. The inhibition of COX-2 may suppress the metastatic spread of colorectal carcinoma cells to the liver, which might have important therapeutic value in humans with metastatic disease.


   ACKNOWLEDGMENTS
 
We thank Dr. K. Hirakawa (Osaka City University, Osaka, Japan) and Dainabot Co. Ltd. (Tokyo, Japan) for the gift of monoclonal SPan-1 antibody and Drs. S. Nishihara (Soka University, Tokyo, Japan), M. Watanabe (Keio University, Tokyo, Japan), and R. Kannnagi (Aichi Cancer Research Institute, Nagoya, Japan) for their valuable suggestions.


   FOOTNOTES
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3636; Fax: 81-6-6879-3639; E-mail: kakiuchi{at}medone.med.osaka-u.ac.jp. Back

2 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; sLea, sialyl Lewis a; sLex, sialyl Lewis x; PG, prostaglandin; MAb, monoclonal antibody; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; IL, interleukin; RT, reverse transcription; ß1,3GnT, ß1,3-N-acetylglucosaminyltranferase; GalT, galactosyltransferase; ST3Gal, {alpha}2,3-sialyltransferase; Fuc-T, fucosyltransferase; core2 GnT,ß1,6-N-acetylglucosaminyltransferase. Back

Received 11/14/01. Accepted 12/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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