To probe the potential contribution of ␤-galactoside-contributing epitopes and receptor proteins (gal-1 and gal-3) to colon malignancy, we first examined the expression of galectins and binding sites in clinical specimens by lectin and immunohistochemistry. Sixty-seven colonic surgical resections were studied, including 10 normal, 10 mild dysplasias, 10 severe dysplasias and 37 cancers. gal-1 and gal-3 were expressed in variable amounts in the epithelial cells and the connective tissue of normal colon. Their expression significantly increased with the degree of dysplasia, suggesting that gal-1 and gal-3 and their binding sites are related to malignant progression, while gal-8 has been associated with suppressor activity. To study the functional aspects, the influence of these galectins on the migration of 4 human colorectal cancer cell lines (HCT-15, LoVo, DLD-1, CoLo201) was studied. In agreement with histopathologic monitoring, these tumor cells were found to produce gal-3, while only Cell-surface epitopes have a conspicuous bearing on cellular features associated with malignancy. The concept that glycans of surface glycoconjugates can function in information storage and transfer by forming a sugar code 1 is supported by the rather ubiquitous presence of endogenous lectins. Since they are located at branch ends, ␤-galactoside-containing epitopes are spatially accessible for such an interaction. This point and the elaborate enzymatic machinery producing these epitopes imply functional relevance of these glycan sections. A complete family of endogenous lectins act as receptors for ␤-galactosides and derivatives; they are referred to as galectins and are involved in the various aspects of tumor biology, including invasion and metastasis. [2][3][4][5][6][7][8] The members of this family share a jelly roll-like folding and a common topology of the CRD with high-sequence homology scores in intermammal comparison, secretion via a nonclassical pathway and interaction with the extracellular glycans of laminins, fibronectins, integrins and other cell-surface components. 4 -9 With respect to colon tumor biology, gal-1 and gal-3 have been investigated histopathologically with different methods and reagents, yielding conflicting results on correlations with tumor progression. 4,10 -12 Two reports on transfected cells and tissue sections underscore the potential relevance of gal-3 for colon cancer metastasis. 10,13 However, the prototype (homodimeric) gal-1 is encountered less frequently in colon cancer. 11,14,15 This indication of functional divergence prompted us to extend the histopathologic monitoring and to use cell models to test in vitro if and to what extent these 2 galectins influence cell migration. Interestingly, the tandem-repeat-type gal-8 has previously been shown to display the features of a tumor suppressor based on cell-migration measurements and immunologic monitoring of expression. 16 Thus, our aims were to shed further light on the role of gal-1 and gal-3 in the biology of human colon cancers, to presen...

Background and aims: Galectins are β-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. Methods: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with α-and β-D-galactose, α-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. Results: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. Conclusions: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus o...

SUMMARY:In colon cancer, endothelial cell selectins can promote tumor cell attachment via interactions with sialylated Lewis antigens present at the surface of tumor cells, thereby facilitating tumor cell arrest and transmigration into the extravascular space. However, it is not known whether Lewis antigens interact with colon tumor cells and modify their migration. Our aim was to detect the presence of binding sites on human tumor cells for Lewis a/x antigens and their sialylated derivatives in vitro and in vivo and to analyze their influence on migration of colon cancer cells. The immunocytochemical and histochemical levels of expression of the four Lewis antigens were quantitatively determined in four human colon cancer cell lines and in in vivo nude mice xenografts. The levels of expression of specific binding sites for these sugar epitopes were determined by synthetic neoglycoconjugates. The influence of binding of these carbohydrate ligands on cancer cell migration was quantitatively evaluated by computer-assisted phase-contrast videomicroscopy performed on Matrigel culture supports either left uncoated or coated with neoglycoconjugate presenting synthetic Lewis a , sialyl Lewis a , Lewis x , or sialyl Lewis x antigens. The influence of the calcium concentration in the culture medium on the Lewis antigen-mediated effects was checked. Human colon cancer cells expressed significant amounts of specific binding sites detected by the synthetic probes in addition to the oligosaccharide epitopes. The expression levels differed considerably between the four cell lines and between in vitro and in vivo specimens. Cell migration analysis revealed that the four Lewis antigens markedly decreased the levels of migration of the HCT-15 and LoVo cancer cells. This effect depends on the calcium concentration in the culture medium. Binding sites for Lewis epitopes are present on colon cancer cells. The functional relevance of these sites is indicated by the negative influence on cell migration of a matrix containing the oligosaccharides as ligand parts. (Lab Invest 2003, 83:777-787).

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