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Recent studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of gastrointestinal cancer, and that cyclooxygenase-2 (COX-2) may be a target enzyme for the prevention or regression of cancer by the use of NSAIDs. Mucin histochemistry has made possible a clear distinction between the differentiated characteristics of gastrointestinal epithelial cells, and the possibility that phenotypic shifts from gastric- to intestinal-type in gastric carcinoma progression has been suggested. To evaluate the role of COX-2 in gastric cancer progression, we immunohistochemically investigated COX-2 expression, and examined its relationship to proliferative activity, mucin phenotype, and clinicopathological parameters in human advanced gastric carcinomas.Forty-five surgical specimens of advanced gastric carcinomas (invaded the muscularis propria or subserosa) were examined. Immunohistochemical staining was performed with monoclonal antibodies against COX-2, Ki-67, CD10 (brush border), MUC-2 (goblet-cell mucin), MUC-5AC (gastric foveolar mucin), and MUC-6 (pyloric mucin). COX-2 expression was scored by the percentage of COX-2-positive neoplastic cells, and proliferative activity was assessed by the Ki-67 labeling index at the deepest area of invasion. The mucin phenotype of the carcinomas was classified into three categories; gastric, intestinal, and unclassified.COX-2 staining was restricted to the cytoplasm, not only in cancer cells but also in intestinal metaplasia and some inflammatory cells and COX-2 expression in cancer cells varied greatly, but the staining in some samples was preferentially found at the invasive front. COX-2 positivity was found to correlate with Ki-67 labeling. The mean COX-2 scores were 2.29%, 2.71%, and 2.75%; and the Ki-67 labeling indices were 23.6%, 40.6%, and 56.5%, in gastric-, intestinal-, and unclassified- type carcinomas, respectively.A close relationship between COX-2 expression and proliferative activity was confirmed in the deepest areas of advanced gastric carcinoma, and the proliferative activity increased from gastric- to intestinal- and to unclassified- type gastric carcinoma, suggesting a role for COX-2 expression and differences in biological behavior according to mucin phenotype expression during gastric cancer progression.

In epithelial tissue, cell-matrix and cell-cell adhesive interactions have important roles in the normal organization and stabilization of the cell layer. The malignant conversion of epithelial cells involves alterations in the expression and function of these adhesion systems that enable a switch to a migratory phenotype in tumor invasion and metastasis. Fascin is an actin-crosslinking protein that is found in the core actin bundles of cell-surface spikes and projections that are implicated in cell motility. We demonstrate that fascin is not detectable in normal colonic epithelium, but is dramatically up-regulated in colorectal adenocarcinoma. To test the hypothesis that fascin could participate in tumor invasive behavior, we developed a cell culture model to examine the effect of fascin expression on the adhesive interactions, invasiveness, and differentiation of colonic epithelial cells. We report marked effects on the organization of cell-surface protrusions, actin cytoskeleton, and focal adhesions in the absence of alterations in the protein levels of the major components of these structures. These effects correlate with alterations in cell movements on two-dimensional matrix, and increased invasiveness in three-dimensional matrix. The cells also show increased proliferation and decreased capacity for normal glandular differentiation in collagen gels. We propose that up-regulation of fascin, by promoting the formation of protrusive, actin-based, cell-motility structures, could be a significant component in the acquisition of invasive phenotype in colonic carcinoma.

Recent studies have shown that cyclooxygenase-2 (COX-2) inhibitors may participate in the proliferation of cancer cells. Because the cadherin-catenin complex is not only a key component of the adherens junction but also has been suggested to regulate cell proliferation, modulation of these molecules may be a mechanism by which COX-2 activity affects cell proliferation. In this study, we evaluated the effect of a COX-2 inhibitor on the proliferation and expression of E-cadherin-complexes in gastrointestinal cancer cell lines. Methods: The gastrointestinal cancer cell lines Caco2, HT29, and MKN45 were grown for 24 h in the presence and absence of a selective COX-2 inhibitor, etodolac (10−5, 10−4, and 10−3 M). Cell proliferation was assessed by 3H-thymidine incorporation, and the expression of E-cadherin and catenins was assessed by Western blotting, Northern blotting, and immunofluorescence. Results: Etodolac induced a significant reduction in cell proliferation in Caco2 and MKN45 cells. E-cadherin expression was upregulated after stimulation with etodolac in Caco2 cells, whereas the expression of α-, β-, γ- and p120-catenins was not modified. The expression of E-cadherin mRNA was also upregulated in Caco2 cells, and was upregulated also in MKN45 cells, which did not express normal E-cadherin protein by the use of a mouse monoclonal antibody against human E-cadherin, HECD-1 antibody. Immunofluorescence revealed that the increased E-cadherin was localized at the cytoplasmic membrane. Conclusions: The inhibition of cell growth by etodolac in Caco-2 cells was associated with a dose-dependent upregulation and intense cytoplasmic localization of E-cadherin. No quantitative change in catenin expression was found in this phenomenon. These findings suggest that the COX-2 inhibitor affects the transcription of E-cadherin, or that there may be some homeostatic link between the cell cycle and E-cadherin transcription.

E-cadherin and its associated cytoplasmic proteins, alpha-, beta-, and gamma-catenins, play an essential role in the control of epithelial differentiation. We have previously shown that loss or down-regulation of E-cadherin/catenin correlates with poor survival in advanced gastric adenocarcinoma. The aim of this study was to assess the expression of E-cadherin and catenins in early gastric cancers (EGCs). Immunohistochemical staining for E-cadherin and alpha-, beta-, and gamma-catenins was performed on 41 paraffin-embedded gastrectomy specimens of EGC using an indirect immunoperoxidase technique. The pattern of expression and cellular localization of the E-cadherin/catenin complex in tumour cells were correlated with the macroscopic appearance of the tumour according to the Japanese Endoscopic Society classification. The tumours were classified as follows: three type I (protruding) and 38 type II (superficial), of which ten were type IIa (elevated), one was type IIb (flat), and 27 were type IIc (depressed). E-cadherin and alpha-, beta-, and gamma-catenins were expressed at the cell-cell junctions in normal mucosa. Forty out of 41 tumours showed abnormal expression (loss of membranous immunoreactivity and/or nuclear staining) of at least one component of the E-cadherin catenin complex. Loss of E-cadherin immunoreactivity was more frequently seen in type IIb (1/1, 100%) and type IIc (27/27, 100%) than in type I (1/3, 33%) and type IIa (1/10, 10%) (p

p120(ctn) is a substrate of the tyrosine kinase pp60 src. Tyrosine kinases such as src localize to the adherens junctions and phosphorylate junctional proteins in both normal and transformed cells.(1) p120(ctn) forms a complex with E-cadherin at the adherens junction and is phosphorylated by ligands such as epidermal growth factor receptor as well as pp60 src. Phosphorylation of p120(ctn) has been shown to correlate with cell transformation. The aim of this study was to investigate in vivo expression of p120(ctn) in gastric carcinoma and to examine any relationship to pathological characteristics and patient survival. Immunohistochemical staining for p120(ctn) was performed in 68 gastric carcinoma specimens (19 diffuse, 49 intestinal type), in 22 lymph node metastases, and in gastric mucosal biopsies from 16 patients with gastric dysplasia and ten healthy controls. Up-regulation of p120(ctn) cytoplasmic staining was seen in six (37 per cent) of the gastric dysplasia cases and in 45 (66 per cent) tumours (89 per cent of diffuse and 57 per cent of intestinal tumours). Loss of membranous distribution of staining for p120(ctn) was seen in 22 (32 per cent) tumours (52 per cent of diffuse and 24 per cent of intestinal tumours). The staining pattern in the primary tumour showed no correlation with tumour type, grade, or stage, or patient survival. Of 22 lymph node metastases examined, 13 (60 per cent) showed loss of membranous staining. In conclusion, staining for p120(ctn) in gastric carcinoma and dysplasia revealed marked up-regulation of cytoplasmic staining, sometimes associated with reduced membranous expression. Up-regulation of expression of p120(ctn) has not previously been described in human epithelial malignancy. The significance of these findings is uncertain, but they may reflect a change in tyrosine kinase signal transduction pathways, and a role for p120(ctn) in ligand-induced mitogenic signalling and cell transformation.

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo . In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin–catenin complex, a prime mediator of Ca 2+ dependent cell–cell adhesion, and the adenomatous polyposis coli (APC)–catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10 −9 M and 10 −8 M) for 20–24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, α-catenin and β-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, β-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.

Basic fibroblast growth factor-2 (FGF-2) has an important role in angiogenesis, and has been demonstrated to promote ulcer healing. However, the actual part played by FGF-2 in the process of ulcer healing is not well understood. In this study, we investigated expression of FGF-2 transcripts at each stage of gastric ulcer healing, using an acetic acid model in rats. We made ulcers in the rat stomach by direct application of acetic acid, and 3 days and 1, 2, 3, 4, 8 weeks after treatment, we examined expression of FGF-2 transcripts by in situ hybridization. On day 3, FGF-2 transcripts were detected in mononuclear cells infiltrating the submucosal layer around the ulcer. After 1 and 2 weeks, expression of FGF-2 transcripts was prominent in spindle-shaped mesenchymal cells and endothelial cells, which proliferated in the ulcerative region. Some of the spindle-shaped cells which expressed FGF-2 transcripts also showed immunoreactivity for alpha-smooth muscle actin. After 3 and 4 weeks, FGF-2 expression was seen mainly in endothelial cells of vessels. These results suggest that different cells produce FGF-2 during the process of gastric ulcer healing, and some of the spindle-shaped cells expressing FGF-2 transcripts in the early phase are myofibroblasts.

Dysfunction of the cadherin–catenin complex, a key component of adherens junctions, is thought to confer invasive potential to cells. The aim of this study is to examine the expression and function of the E-cadherin/catenin complex in gastric carcinoma cell lines. Expression of E-cadherin, α, β and γ-catenin and p120ctn, and of the adenomatous polyposis coli protein (APC), together with function of the cadherin–catenin complex was examined in a panel of gastric carcinoma cell lines, using immunocytochemistry, Western blotting and a cell–cell aggregation assay. Protein interactions were examined by sequential immunoprecipitation and immunoblotting with antibodies to E-cadherin, α, β and γ-catenin, p120ctn and APC. Abnormalities of E-cadherin, α- and β-catenin expression, were associated with disturbance of E-cadherin–catenin complex composition, loss of membranous localization and loss of calcium-dependent aggregation in six gastric carcinoma cell lines. APC protein expression and interaction with β-catenin was preserved in five cell lines. We demonstrate frequent abnormalities of expression and function of E-cadherin and catenins, and associated disturbance of E-cadherin-mediated intercellular adhesion in gastric carcinoma cell lines. These findings support the tumour suppressor role of the E-cadherin and its contribution to the development and progression of the neoplastic phenotype in gastric carcinoma. © 1999 Cancer Research Campaign

We examined the expression of FGF-2 mRNA in 16 early and 14 advanced gastric cancer by in situ hybridisation to elucidate its role in cancer progression. Anti-sense RNA probes were synthesized by transcribing the subcloned vector with T7 RNA polymerase in the presence of digoxigenin-labeled UTP. FGF-2 mRNA was located mainly in the cytoplasm around the nuclei of endothelial cells, fibroblasts and carcinoma cells. The expression was more frequently in the diffuse type carcinomas (4/7, 57%) than in the intestinal type tumours (5/23, 22%). The survival rates of advanced gastric cancers with FGF-2 mRNA expression were significantly lower than those without FGF-2 mRNA expression (p < 0.01). No significant correlation was seen with other clinicopathological factors. These results suggest that FGF-2 may play an important role for the growth of diffuse type gastric cancers, particularly at their advanced stage.