alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Liver-Neoplasms

he colonization of the liver by colorectal cancer (CRC) cells is a complicated process which includes many stages, until macrometastases occur. The entrapment of malignant cells within the hepatic sinusoids and their interactions with resident non-parenchymal cells are considered very important for the whole metastatic sequence. In the sinusoids, cell connection and signalling is mediated by multiple cell adhesion molecules, such as the selectins. The three members of the selectin family, E-, P- and L-selectin, in conjunction with sialylated Lewis ligands and CD44 variants, regulate colorectal cell communication and adhesion with platelets, leucocytes, sinusoidal endothelial cells and stellate cells. Their role in CRC liver metastases has been investigated in animal models and human tissue, in vivo and in vitro, in static and shear flow conditions, and their key-function in several molecular pathways has been displayed. Therefore, trials have already commenced aiming to exploit selectins and their ligands in the treatment of benign and malignant diseases. Multiple pharmacological agents have been developed that are being tested for potential therapeutic applications.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Colorectal Neoplasms; Hexanes; Humans; Lewis X Antigen; Ligands; Liver Neoplasms; Mannose; Molecular Sequence Data; Molecular Structure; Protein Isoforms; Selectins; Sialyl Lewis X Antigen

Topics: Carcinoma; Cell Adhesion Molecules; Cell Movement; Colorectal Neoplasms; Glycosylation; Humans; Integrins; Kalinin; Liver Neoplasms; Neoplasm Invasiveness; Neoplasms; Oligosaccharides; Sialyl Lewis X Antigen

Expression of sialyl-Lewisx (sLex) antigen was studied immunohistochemically in 110 resected human gastric carcinomas using an anti-sLex monoclonal antibody. Lymph node, liver, and peritoneal metastases were clearly more prevalent in tumors expressing high levels of sLex than in those with no or low-level sLex expression. No correlation was found between sLex expression and histologic grade or histologic type of the Lauren classification. Among the tumors with lymph node metastasis, 44% expressed high levels of sLex in both the primary tumor and involved lymph nodes, and 14% of the metastatic lesions demonstrated increased sLex expression. The 5-year survival rate of the patients undergoing complete (R0) gastric resections was 60% in the sLex high-expression group, which was significantly lower than that of the sLex low-expression group (81%) and of the no-expression group (87%) (p < 0.05). These results suggest that high-level sLex expression is related to both an increased risk of metastasis and poor prognosis in gastric cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Gastrectomy; Humans; Immunohistochemistry; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Peritoneal Neoplasms; Prognosis; Risk; Sialyl Lewis X Antigen; Stomach Neoplasms; Survival Rate

The increased expression of sialyl-Lewisx (SLex) epitope on the surface of tumor cells has been known for decades. However, genetic manipulation of the expression of SLex and the role of SLex in cancer cell proliferation remains to be fully elucidated. The present study suggested that the monoclonal antibody of SLex (KM93) significantly inhibited the proliferation of human hepatocarcinoma (HCC) cells. The expression levels of three sialyl‑Lewis oligosaccharide antigens, SLex, SLea and dimeric SLex (SDLex), were determined on the cell surface of the MHCC97 human HCC cell line. The expression of SLex was markedly higher in MHCC97 cells than in normal liver cells. The expression of SDLex was also relatively high, however, no significant difference was observed between normal liver cells and HCC cells. The expression of SLea was only detected in trace quantities. Fucosyltransferase (FUT) is the key enzyme of the fucosylation step in the biosynthesis of sialyl‑Lewis oligosaccharide antigens. Therefore, the present study investigated the expression of FUTs. It was found that the mRNA and protein expression levels of FUT7 were high in the MHCC97 HCC cell line compared with levels in normal liver cells. FUT6 was also expressed at a high level, although the difference was not statistically significant between MHCC97 cells and normal liver cells. No expression of FUT3 was detected. The results were consistent with the change insialyl‑Lewis antigens. The effects of FUT7 small interfering (si)RNA transfection on the expression of FUT7, expression of SLex and MHCC97 cell proliferation were also examined. Following FUT7 siRNA transfection, the expression of FUT7 was markedly downregulated, as determined by western blot and reverse transcription‑quantitative polymerase chain reaction methods. The results from flow cytometry showed that the synthesis of SLex was also inhibited, which was consistent with the downregulated expression of FUT7. MHCC97 cell proliferation was also significantly inhibited following FUT7 siRNA transfection, which was correlated with suppression of the S‑phase in cell cycle progression. By using inhibitors of various signaling pathways, it was found that the knockdown of FUT7 inhibited the activation of phospholipase Cγ (PLCγ) by inhibiting the translocation and phosphorylation of PLCγ. In conclusion, the results suggested that FUT7 has animportant functional role in human HCC cell proliferation by controlling cell cycle progression via the PLCγ/

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Oligosaccharides; RNA Interference; RNA, Small Interfering; Sialyl Lewis X Antigen

Interactions between endothelial and tumor cells via E-selectin and sialyl Lewis x (sLex) have been suggested to play a significant role in the development of metastasis and tumor growth. In this work, we tested whether inhibition of E-selectin expression on the surface of endothelial cells might impair endothelial/tumor cells interactions and tumor growth of hepatocarcinoma cells in vitro and in vivo.. We used HepG2 cells that highly express sLex antigens and HuH7 cells that do not express sLex. Inhibition of E-selectin expression on the surface of endothelial cells was obtained by using cimetidine and amiloride treatment.. Cimetidine and amiloride inhibited, respectively, by 20 and 64 % E-selectin expression by activated endothelial cells and significantly subsequent adhesion of HepG2 cells to activated endothelial cells. Subcutaneous injection of cimetidine or amiloride resulted in a significant inhibition of HepG2 cells tumor growth in nu/nu mice but not of HuH7 cells. Thus, cimetidine and amiloride administration led to an inhibition of 57 and 75 % of HepG2 tumor growth in vivo, respectively. This effect was associated with an inhibition of vasculogenesis as demonstrated by anti-CD31 immunostaining.. Inhibition of E-selectin expression allows an anti-tumoral effect on sLex-expressing HCC tumors in vivo. This suggests that interactions between HCC cells and endothelial cells through sLex antigens and E-selectin might be a target for treatment of HCC. Further studies might evaluate the clinical impact of cimetidine and amiloride in the treatment of HCC patients alone or in combination with other anti-tumoral agents.

Topics: Amiloride; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cimetidine; E-Selectin; Endothelial Cells; Female; Hep G2 Cells; Humans; Lewis X Antigen; Liver Neoplasms; Mice; Neovascularization, Pathologic; Sialyl Lewis X Antigen

The metastasis of hematogenous cancer cells is associated with abnormal glycosylation such as sialyl lewis antigens. Although the hepatitis B virus X protein (HBx) plays important role in liver disease, the precise function of HBx on aberrant glycosylation for metastasis remains unclear.. The human hepatocellular carcinoma tissues, HBx transgenic mice and HBx-transfected cells were used to check the correlation of expressions between HBx and Sialyl lewis antigen for cancer metastasis. To investigate whether expression levels of glycosyltransferases induced in HBx-transfected cells are specifically associated with sialyl lewis A (SLA) synthesis, which enhances metastasis by interaction of liver cancer cells with endothelial cells, ShRNA and siRNAs targeting specific glycosyltransferases were used.. HBx expression in liver cancer region of HCC is associated with the specific synthesis of SLA. Furthermore, the SLA was specifically induced both in liver tissues from HBx-transgenic mice and in in vitro HBx-transfected cells. HBx increased transcription levels and activities of α2-3 sialyltransferases (ST3Gal III), α1-3/4 fucosyltransferases III and VII (FUT III and VII) genes, which were specific for SLA synthesis, allowing dramatic cell-cell adhesion for metastatic potential. Interestingly, HBx specifically induced expression of N-acetylglucosamine-β1-3 galactosyltransferase V (β1-3GalT 5) gene associated with the initial synthesis of sialyl lewis A, but not β1-4GalT I. The β1-3GalT 5 shRNA suppressed SLA expression by HBx, blocking the adhesion of HBx-transfected cells to the endothelial cells. Moreover, β1-3GalT 5 silencing suppressed lung metastasis of HBx-transfected cells in in vivo lung metastasis system.. HBx targets the specific glycosyltransferases for the SLA synthesis and this process regulates hematogenous cancer cell adhesion to endothelial cells for cancer metastasis.

Topics: Adult; Animals; CA-19-9 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Glycosyltransferases; Hepatitis B virus; Human Umbilical Vein Endothelial Cells; Humans; Lewis X Antigen; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Trans-Activators; Viral Regulatory and Accessory Proteins

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP-labeled Lewis lung carcinoma subline H-59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide-induced increase in H-59 cell adhesion. Although direct selectin-selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver.

Topics: Animals; Carcinoma, Lewis Lung; Cell Adhesion; Disease Progression; Endothelium, Vascular; Inflammation; Ligands; Lipopolysaccharides; Liver; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neutrophils; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; Tumor Cells, Cultured

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Topics: Bone Marrow; Bone Marrow Neoplasms; Carcinoembryonic Antigen; CD146 Antigen; Cell Adhesion; Cell Movement; E-Selectin; Fucosyltransferases; Humans; Hyaluronan Receptors; Liver; Liver Neoplasms; Male; Neoplasm Metastasis; Oligosaccharides; Prostatic Neoplasms; Sialoglycoproteins; Sialyl Lewis X Antigen

The role of alpha1,3fucosyltransferase-VII (alpha1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7,721 cells. After the cells were transfected with alpha1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that alpha1,3FucT-VII is a potential anti-apoptotic factor in H7,721 cells. After "alpha1,3FucT-VII" cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of alpha1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7,721 cells responsible to UV stress) when compared with the "Mock" cells. In contrast, "alpha1,3FucT-VII" cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X(L). The up regulation of alpha1,3FucT-VII mRNA and cell surface SLe(x) (alpha1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that alpha1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cells, Cultured; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Oligosaccharides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialyl Lewis X Antigen; Signal Transduction; Transfection; Tretinoin; Ultraviolet Rays

The glycoantigen sialyl-Lewis x (sLex) and its isomer sialy-Lewis a (sLea) are frequently associated with advanced states of cancer and metastasis. In a previous work, we have shown that hepatocarcinoma cells (HCC) HepG2 interact with the endothelial E-selectin exclusively through sLe(x) oligosaccharides, the synthesis of which could be completely prevented by the alpha(1,2)-fucosyltransferase-I (FUT1), thus resulting in a strong inhibition of adhesion and rolling on activated endothelial cells. The purpose of the present study was to evaluate the impact of inhibiting sLex synthesis and the subsequent E-selectin adhesion, on HCC tumor growth in nude mice. Four weeks after subcutaneous transplantation of cells, no FUT1-derived tumor could be detected, whereas 75% of control animals developed large size tumor nodules. Between the 4th and the 8th week postinoculation, 33% tumors arose from FUT1-transduced cells but showed a slow growth (nodule volumes less than 500 mm(3)), while more than 50% of control tumors reached volumes between 1,500 and 3,000 mm(3). Several parameters were examined, including cell division and proliferation, apoptosis, adhesion to extracellular matrix components and angiogenesis/vasculogenesis. We provide evidence that among all, vasculogenesis was the most clearly affected by FUT1 expression, suggesting that tumor angiomorphogenesis may, at least partly, depend on E-selectin-mediated interaction between HCC and endothelial cells, the inhibition of which remarkably retards tumor growth.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Division; Cell Proliferation; E-Selectin; Extracellular Matrix; Fucose; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Oligosaccharides; Sialyl Lewis X Antigen; Time Factors; Transduction, Genetic; Transplantation, Heterologous

Carbohydrate antigens with subterminal fucosylation have been implicated in the development and progression of several cancers, including hepatocellular carcinoma (HCC). Fluorescent sensors targeting fucosylated carbohydrate antigens could potentially be used for diagnostic and other applications. We have designed and synthesized a series of 26 diboronic acid compounds as potential fluorescent sensors for such carbohydrates. Among these compounds, 7q was able to fluorescently label cells expressing high levels of sLex (HEPG2) within a concentration range of 0.5 to 10 microM. This compound (7q) did not label cells expressing Lewis Y (HEP3B), nor cells without fucosylated antigens (COS7). This represents the first example of a fluorescent compound labeling cells based on cell surface carbohydrate structures.

Topics: Animals; Boronic Acids; Carcinoma, Hepatocellular; Cell Line; Fluorescence; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Molecular Structure; Oligosaccharides; Sialyl Lewis X Antigen

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.

Topics: Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cells, Cultured; Chromones; DNA, Complementary; Endothelium, Vascular; Fucosyltransferases; Humans; Insulin; Laminin; Liver Neoplasms; Morpholines; Neoplasm Metastasis; Oligosaccharides; Phenotype; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sialyl Lewis X Antigen; Signal Transduction; Transfection; Up-Regulation

The roles of terminal sialyl and fucosyl residues in cell surface glycans in the metastatic potential of H7721 cells, a human hepatocarcinoma cell line, were studied.. Neuraminidase and alpha-L-fucosidase were used to remove the sialyl and fucosyl residues, respectively. Cell adhesion to fibronectin (Fn), laminin (Ln), and human umbilical vein epithelial cell (HUVEC), as well as chemotactic cell migration and invasion, were selected as the parameters of metastatic potential ex vivo.. Sialyl residue is not essential for cell adhesion to Fn, but is important in cell adhesion to Ln and invasion, and is crucial in cell adhesion to HUVEC and migration. In contrast, fucosyl residue contributes more than sialyl residue to cell adhesion to Fn and Ln, but less to adhesion to HUVEC, and is not essential in chemotactic cell migration and invasion. Cell adhesion to HUVEC, migration, and invasion were inhibited by the monoclonal antibody of sialyl Lewis X, but not by the antibody of non-sialyl Lewis X.. Terminal sialyl residues on cell surface glycans are more important than fucosyl residues in mediating cell adhesion to HUVEC and cell migration/invasion, but the reverse is true in cell adhesion to Fn and Ln.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Chemotaxis; Epithelial Cells; Fibronectins; Fucose; Humans; Laminin; Lewis Blood Group Antigens; Liver Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; Oligosaccharides; Sialic Acids; Sialyl Lewis X Antigen; Tumor Cells, Cultured; Umbilical Veins

To study the significance of E-selectin and its ligand-sLeX in the metastasis of hepatocellular carcinoma (HCC).. Flow cytometry and immunohistochemistry were used to detect the expression of E-selectin and its ligand-sLeX in both HCC cell lines and human HCC tissues.. The positive rate of E-selectin in vascular endothelial cells adjacent to cancer was 67.9% (19/28). The expression of E-selectin in tumors accompanied with emboli or satellite foci was significantly higher than that in tumors without emboli or satellite foci (P<0.05), and it was not related to tumor size, tumor capsule, AFP content, and the degree of differentiation. The positive expression of sLeX in SMMU-7721, PLF/PRF/5 and HepGII cell lines was 7.03%, 63.35% and 97.29% respectively. The positive cells of sLeX mainly distributed in the margin of tumor tissues. The positive expression of sLeX in HCC cells in emboli or invasive tumor tissues was much higher than in Primary foci.. E-selectin and its ligand-sLeX are closely correlated with the metastasis of HCC.

Topics: Adult; Carcinoma, Hepatocellular; E-Selectin; Flow Cytometry; Humans; Immunohistochemistry; Ligands; Liver Neoplasms; Middle Aged; Oligosaccharides; Sialyl Lewis X Antigen

The relationship between the structures of glycans on the surface of H7721 cells, a human hepatocarcinoma cell line, and the cell behaviors was studied by using neuraminidase and alpha-L-fucosidase to remove the terminal sialyl or fucosyl residues of surface glycans respectively. The cell adhesion to fibronectin (Fn), laminin (Ln) and human umbilical vein epithelial cell (HUVEC), as well as cell chemotactic migration and invasion were selected as the parameters of the cell behaviors. It was found that sialyl residue was not essential for the cell adhesion to Fn, but was important in the cell adhesion to Ln and chemotactic cell invasion, and very crucial in the cell adhesion to HUVEC and chemotactic migration. In contrast, fucosyl residue was probably participate in cell adhesion to Fn, Ln and HUVEC, but not important in chemotactic migration and invasion. The cell adhesion to HUVEC, chemotactic migration and invasion were inhibited by the monoclonal antibody of sialyl Lewis X (SLex), but not by the antibody of non-sialyl Lewis X (Lex). This result supports the finding that sialyl residue is more important than fucosyl residue in the contribution to the above-mentioned three cell processes.

Topics: Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cells, Cultured; Chemotaxis; Fibronectins; Humans; Laminin; Lewis X Antigen; Liver Neoplasms; Polysaccharides; Sialyl Lewis X Antigen

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 E(2) 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. beta 3Gal-T5 and ST3Gal III and IV expression was inhibited by celecoxib and was enhanced by prostaglandin E(2) 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.

Topics: Animals; Caco-2 Cells; Cell Adhesion; Colonic Neoplasms; Cyclooxygenase 2; Dinoprostone; Endothelium, Vascular; Glycosyltransferases; HT29 Cells; Humans; Isoenzymes; Liver Neoplasms; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Oligosaccharides; Prostaglandin-Endoperoxide Synthases; Sialyl Lewis X Antigen

Sialyl Lewis(a) (CA19-9) and sialyl Lewis(x) antigens (SLX) may play a role in tumor metastasis by serving as functional ligands in the cell adhesion system. The authors examined preoperative serum levels of CA19-9 and SLX in 218 patients who underwent resection for gastric cancer to determine their prognostic value. The patients were divided into two groups, termed the low and high antigen groups, based on a value selected as a diagnostic cutoff. Correlation between the antigen serum levels, various established clinicopathologic factors, and prognosis were studied by univariate and multivariate analysis. The disease-specific interval for high CA19-9 and SLX groups was significantly shorter than that of their respective low groups (p = 0.0024 and p < 0.0001, respectively). Patients with stage III/IV tumors who had high serum SLX levels had shorter disease-specific intervals than those with low serum levels (p = 0.0017). A Cox's regression analysis revealed a high serum SLX level as an independent factor for worse outcome. In addition, logistic regression analysis revealed that a high serum SLX level was an independent predictor for liver metastasis. In conclusion, an elevated preoperative serum SLX level was a predictor for poor outcome after resection for gastric cancer, whereas CA19-9 was not.

Topics: Adult; Aged; CA-19-9 Antigen; Carcinoembryonic Antigen; Disease-Free Survival; Female; Humans; Liver Neoplasms; Logistic Models; Male; Middle Aged; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen; Stomach Neoplasms

Carbohydrate expression of cancer cells is closely related to the metastatic nature of colorectal cancer. In the present study we investigated the relevance of carbohydrate expression profiles of colorectal cancer cells in the primary lesion to metastatic distribution patterns as well as prognosis in 134 cases. Carbohydrate expression was estimated by histochemistry with 17 kinds of lectins and 3 kinds of Lewis-related monoclonal antibodies (MAbs), and correlations between the staining and clinicopathological parameters were examined. The results showed that lymphatic invasion, lymph node metastasis, and peritoneal metastasis correlated with staining with lectins that bind galactose/N-acetylgalactosamine residues (Gal/GalNAc) such as Maclura pomifera (MPA), Arachis hypogaea (PNA), Helix pomatia (HPA), and Vicia villosa (VVA). In contrast, hepatic metastasis correlated with staining with Anguilla anguilla lectin (AAA), anti-LewisX (LEX-2), anti-sialyl Lewisa (NS 19-9), and anti-sialyl-dimeric LewisX (FH-6) MAbs, all of which bind preferentially to fucosylated carbohydrate chains. The five-year survival rate of patients was related to the staining of cancers with MPA, HPA, FH-6 or NS19-9, and MPA- and FH-6 staining were independent prognostic factors. We conclude that carbohydrate expression profiles of cancer cells are relevant to the route of tumor cell dissemination, metastatic pattern as well as prognosis of colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates; Colorectal Neoplasms; Female; Humans; Lectins; Lewis X Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Oligosaccharides; Peritoneal Neoplasms; Prognosis; Prospective Studies; Sialyl Lewis X Antigen; Staining and Labeling

The expressions of Lewis antigens, alpha1,3 fucosyltransferase (alpha1,3 FucT)-VII, and the metastatic potential of the 7721 human hepatocarcinoma cell line after the transfection of the cDNA of nm23-H1, a known metastasis-suppressive gene, were studied using mock cells as the control, which were transfected with the pcDNA3 vectors.. Cell adhesion to human umbilical vein epithelial cells (HUVEC), chemotaxic cell migration through transwells, and invasion through matrigel were selected as the metastasis-related phenotypes to assess the metastatic potential at the cell level.. The results showed that the expression of SLe(x) was high, while the expression of Le(x), SDLe(x), and SLe(a) were very low on the surface of the mock cells. After transfection of nm23-H1, the expressions of SLe(x), alpha1,3 FucT-VII, and the cell adhesion to HUVEC, as well as cell migration and invasion were simultaneously decreased in all three clones of nm23-H1-transfected cells. Among different clones, the decreased expressions of SLe(x) and alpha1,3 FucT-VII were roughly correlated to each other, and also, in general, proportional to the ability of cell adhesion to HUVEC, cell migration, and invasion. The expressions of these metastasis-related phenotypes were lowest in clone 3 and highest in clone 4. Only the specific monoclonal antibody to SLe(x) (KM93) significantly abolished the cell adhesion, migration, and invasion, while other monoclonal antibodies against SDLe(x) or Le(x) and SLe(a) only slightly inhibited or entirely failed to inhibit the above-mentioned phenotypes. However, the rate of cell growth was not changed after the transfection of nm23-H1, and the ability of colony formation on the soft agar was only decreased in one clone.. These findings reveal that the down-regulation of alpha1,3 FucT-VII and its product, SLe(x), is one of the mechanisms to explain the metastasis-suppressive effect of the nm23-H1 gene.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Chemotaxis; DNA, Complementary; Down-Regulation; Epithelial Cells; Flow Cytometry; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Oligosaccharides; Phenotype; Sialyl Lewis X Antigen; Transcription Factors; Transfection; Tumor Cells, Cultured; Umbilical Cord

The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the 'neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and alpha1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe(x) expression on the surface of mock cells was high, whereas expression of SDLe(x), Lex and SLe(a) was absent or negligible. This is compatible with the abundant expression of alpha1,3-fucosyltransferase VII, very low expression of alphafucosyltransferase III/VI, and almost absent expression of alpha1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe(x) and alpha1,3-fucosyltransferase VII were simultaneously elevated, but that of alphafucosyltransferase III/VI was not altered. The expression of both SLe(x) and alpha1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells. These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe(x) and alpha1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe(x) (KM93) and SDLe(x) (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe(x) and SLe(a) did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of alpha1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of alpha1,3-fucosyltransferase VII and its specific product, SLe(x), and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c-erb

Topics: Antibodies; Carcinoma, Hepatocellular; Cell Adhesion; Cells, Cultured; DNA, Complementary; Endothelium, Vascular; Fibronectins; Fucosyltransferases; Genes, erbB-2; Humans; Liver Neoplasms; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Phenotype; RNA, Messenger; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured; Up-Regulation

The clinicopathological factors and expression of sialyl Lewis antigens which are the cell adhesion molecules to endothelial cells were compared in relation to the extent of the postoperative hepatic metastasis in 23 consecutive patients with pancreatic ductal adenocarcinoma whose clinical courses were carefully monitored and documented. The overall survival of cases with massive hepatic metastasis (MHM) was significantly poorer than that of those with local or no hepatic metastasis (p = 0.0453). Postoperative MHM was significantly correlated with the presence of duodenal invasion (p = 0.0418), the presence of portal vein invasion (p = 0.0435), the presence of extratumoral venous invasion (p = 0.0052) and high expression of sialyl Lewis x antigen (p = 0.0022). Multivariate analysis confined significant correlation between the high expression of sialyl Lewis x antigen and the development of MHM (p = 0.0402). Kaplan-Meier analysis revealed that the overall survival of patients with a high expression of sialyl Lewis x antigen was significantly poorer than that of patients with a low expression of the antigen (p = 0.0216). These results indicate that the overexpression of sialyl Lewis x antigen plays an important role in the development of MHM, and also predicts a poorer overall survival of these patients. Further studies with more cases are warranted to confirm these results.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Oligosaccharides; Pancreatic Neoplasms; Postoperative Period; Prognosis; Sialyl Lewis X Antigen; Survival Rate; Veins

In inflammation, hepatocytes secrete several proteins into serum, the so-called acute phase proteins. In addition to increased serum levels of alpha(1)-acid glycoprotein (AGP), there are also changes in the composition of its sugar chains. To investigate the cytokine-stimulated alteration of sugar chains on AGP, we cultured the human hepatoma cell line HuH-7 cells in serum-free medium (IS-RPMI) with and without IL-1beta and IL-6, and analyzed AGP secretion into the medium. AGP was increased during stimulation with both IL-1beta and IL-6, although the effect of IL-1beta was more pronounced. Lectin-binding assay for secreted AGP also indicated significant increases in the binding activities to Aleuria aurantia lectin (AAL), Sambucus sieboldiana agglutinin (SSA), and concanavalin-A (ConA). In particular, AAL binding activity increased with higher expression of the sialyl Lewis X (sLe(X)) antigen, NeuAc alpha2-3 Gal beta1-4 (Fuc alpha1-3) GlcNAc-R. Thus, the increase in AGP fucosylation may be correlated with the increase of sLe(X). The present results indicate that the serum-free culture of HuH-7 cells provides a useful model for investigating the secretion of proteins from hepatocytes, and the effects of cytokines on the changes in sugar chains of glycoproteins in vitro.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Hepatocellular; Culture Media, Serum-Free; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-1; Interleukin-6; Kinetics; Lectins; Liver Neoplasms; Molecular Sequence Data; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Although the beneficial effect of cimetidine on survival in cancer has been clinically demonstrated in colorectal cancer patients, the mode of action of cimetidine has not been elucidated. In this report, we have demonstrated for the first time that cimetidine can block the adhesion of a colorectal tumor cell line to the endothelial cell monolayer in cell culture and that it can suppress the metastasis of the tumor cell in a nude mouse model. We also demonstrated that these antimetastasis effects of cimetidine might occur through down-regulation of the cell surface expression of E-selectin on endothelial cells, a ligand for sialyl Lewis antigens on tumor cells. We found that the cimetidine-mediated down-regulation of E-selectin did not involve down-regulation of E-selectin mRNA or blocking of the nuclear translocation of nuclear factor kappaB, a transcriptional activator of E-selectin gene expression. Because two other histamine type 2 receptor antagonists, famotidine and ranitidine, did not show any similar effect, these actions of cimetidine probably do not occur via blocking of the histamine receptor. These observations support the idea that cancer metastasis can be blocked by cimetidine administration through blocking the adhesion of tumor cells to the endothelium when an interaction between E-selectin and sialyl-Lewis antigens plays a role.

Topics: Animals; Cell Adhesion; Cell Nucleus; Cimetidine; Colorectal Neoplasms; Dose-Response Relationship, Drug; Down-Regulation; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Famotidine; Histamine H2 Antagonists; Humans; Interleukin-1; Liver Neoplasms; Mice; Mice, Nude; Microscopy, Confocal; Neoplasm Transplantation; NF-kappa B; Oligosaccharides; Ranitidine; RNA, Messenger; Sialyl Lewis X Antigen; Transcriptional Activation; Tumor Cells, Cultured; Umbilical Veins

To improve the survival rate of patients with colon cancer, liver metastases must be eradicated in a clinically occult state. This study was designed to find a predictor for potential liver metastases or micrometastases in colon cancer.. Peripheral blood samples and tumor specimens were obtained from 36 patients with colon cancers. The blood samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the expression of sialylated carbohydrates was also investigated in the tumors immunohistochemically.. A carcinoembryonic antigen (CEA)-specific signal in the blood was detected in 9 of 12 (75%) patients with liver metastasis and in 8 of 24 (33%) patients without liver metastasis, respectively (P < 0.05). The positive rates of sialyl Lewis A (sLeA) and sialyl Lewis X (sLeX) were 36.3% and 40% in tumors without liver metastasis vs. 58.3% and 100% with liver metastasis, respectively. Within a year after surgery, liver metastases became clinically evident in three of the four patients without liver metastasis who showed a CEA-positive signal in their blood preoperatively and who had tumors with a strong expression of sLeX.. A combination of both markers may provide prognostic information for liver metastases in colon cancer.

Topics: Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lewis X Antigen; Liver Neoplasms; Oligosaccharides; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Sialyl Lewis X Antigen

Sialyl Lewis(x)antigen (SLX) is a carbohydrate antigen that serves as a ligand for selectin, an adhesion molecule expressed on vascular endothelial cells. The expression of SLX in 245 patients with advanced gastric carcinoma was examined immunohistochemically, and its clinicopathologic significance was analysed. We classified the patients with advanced gastric carcinoma into 91 with differentiated type and 154 with undifferentiated type. SLX expressed in 135 of 245 patients (55%), comprising 68 (75%) patients with differentiated carcinoma and 67 (44%) with undifferentiated carcinoma. The positive rate for SLX expression was significantly higher among patients with differentiated carcinoma than among those in undifferentiated carcinoma (P<0.0001). With differentiated carcinoma, the incidence of lymph node metastasis, advanced tumour stage (stage III and IV) and liver recurrence was significantly higher in SLX-positive patients than in SLX-negative ones (P<0.0001, P = 0.0065 and P = 0. 028, respectively). Moreover, the prognoses were better in patients with SLX-negative tumours than in those with SLX-positive tumours (P = 0.019). With undifferentiated carcinoma, there were no significant correlations between SLX expression and any clinicopathological features or prognoses. The clinicopathologic significance of SLX expression in gastric carcinoma patients depends on histologic type. SLX expression may be of great relevance in predicting liver metastases in patients with differentiated carcinoma.

Topics: Aged; Carcinoma; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Oligosaccharides; Peritoneal Neoplasms; Sialyl Lewis X Antigen; Stomach Neoplasms; Survival Analysis

To study the significance of E-selectin and its ligand-sLeX in the metastasis of hepatocellular carcinoma (HCC).. Flow cytometry and immunohistochemistry were used to detect the expression of E-selectin and its ligand-sLeX in HCC cell lines and in human HCC tissues.. The positive rate of E-selectin in vascular endothelial cells adjacent to cancer nest in tumors was 67.9% (19/28). In tumors accompanied with emboli or satellite foci, it was significantly higher than that without emboli or satellite foci (P < 0.05). The positive rate of E-selectin was not related to tumor size, tumor capsule, AFP, and the degree of differentiation. The positive expressions of sLeX in SMMU-7721, PLC/PRF/5 and HepGII cell lines were 7.03%, 63.35% and 97.29% respectively. The positive cells of sLeX were mainly distributed in the margin of tumors; the positive expression of sLeX in HCC cells in emboli or invasive tumor tissues was much higher than that in primary foci.. E-selectin and its ligand-sLeX are closely correlated with the metastasis of HCC.

Topics: Adult; Carcinoma, Hepatocellular; Cell Line, Tumor; E-Selectin; Female; Flow Cytometry; Humans; Immunohistochemistry; Ligands; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

The initial steps of leukocyte and tumor cell adhesion involve selectin receptor/ligand interactions. The selectin ligand components sialyl Lewis x and sialyl Lewis a are oncodevelopmental antigens involved in progression of adenocarcinoma. Interrupting biosynthesis of these surface glycans by inhibition of alpha(1,3)fucosyltransferase (FUT) gene expression is an attractive goal for functional and therapeutic studies. We report here the inhibition of E-selectin-mediated adenocarcinoma cell adhesion by stable transfection of antisense sequences directed at the human Lewis alpha(1,3/1,4)fucosyltransferase gene, FUT3. The metastatic parental cell line, HT-29LMM, expressed high levels of sialyl Lewis x, sialyl Lewis a, alpha(1,3/1,4)fucosyltransferase activity, and FUT3 transcript, but antisense transfectant cell lines did not. When injected into the spleens of nude mice, the stable antisense clones were unable to colonize the liver. These results provide target validation for inhibition of carcinoma metastasis with antisense FUT sequences and confirm the primacy of alpha(1,3)fucosyltransferases in the synthesis of selectin ligands.

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; CA-19-9 Antigen; Cell Adhesion; Cells, Cultured; Colonic Neoplasms; Disease Progression; DNA, Antisense; E-Selectin; Endothelium, Vascular; Female; Fucosyltransferases; Gangliosides; Genetic Vectors; Glycosylation; Humans; Injections; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Oligosaccharides; Protein Processing, Post-Translational; Sialyl Lewis X Antigen; Spleen; Transfection; Tumor Cells, Cultured

Curative resection does not guarantee long term survival for the patient with extrahepatic bile duct carcinoma because of the possibility of metastases to the liver (LM). Expression of mucin core protein-1 (MUC1), sialyl-Le(x), and sialosyl-Tn in bile duct carcinoma was determined and compared with LM and prognosis.. Immunohistochemical expression of MUC1, sialyl-Le(x), and sialosyl-Tn in 73 extrahepatic bile duct tumors was analyzed using the DF3, FH6, and TKH2 monoclonal antibodies, respectively. Scoring was based on the percentage of immunoreactive cells: negative, low expression (25%).. High expression of MUC1, sialyl-Le(x), and sialosyl-Tn was observed in 68.5%, 34.2%, and 54.8%, respectively. of 73 cases. Patients with tumors showing high expression of MUC1 had a higher rate of LM (48.9%) and a significantly shorter survival period (median survival time, 17.8 months) compared with patients with tumors showing low (incidence of LM, 9.1%; median survival time, >100 months) or negative (incidence of LM, 11.1%; median survival time, 52.9 months) expression of MUC1 (P < 0.01). However, the survival period of patients with tumors showing high, low, or negative expression of sialyl-Le(x) or of sialosyl-Tn did not differ significantly. High MUC1 expression correlated with LM by logistic regression analysis and emerged as an independent prognostic factor in stepwise multivariate analysis.. The results of the current study demonstrate that high expression of MUC1 correlates with LM and poor outcome in patients with extrahepatic bile duct carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Tumor-Associated, Carbohydrate; Bile Duct Neoplasms; Glycoproteins; Humans; Liver Neoplasms; Logistic Models; Middle Aged; Mucin-1; Neoplasm Proteins; Oligosaccharides; Peptide Fragments; Prognosis; Retrospective Studies; Sialyl Lewis X Antigen

Glycoconjugates bearing oligosaccharide Lex, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->3R, are found on the surface of several cell types. Although recent studies have indicated that Lexon both glycosphingolipids (GSL) and polylactosaminoglycans can mediate under certain experimental conditions Lex-Lexinteractions, cell-cell interactions based exclusively on LexGSLs have not been demonstrated. In this study we show that preincubation of nonaggregating rat basophilic leukemia (RBL) cells with purified LexGSLs resulted in incorporation of the GSLs into plasma membrane, as determined by immunostaining, and formation of aggregates in the presence of Ca2+; no aggregates were formed after preincubation of the cells with globoside or sphingomyelin. Lex-mediated aggregation was inhibited by removal of Ca2+or by addition of lactofucopentaose III but not by lactose or lacto-N-fucopentaose II. In a mixture of Lex-positive and Lex-negative RBL cells most of the aggregates were composed exclusively of Lex-positive cells. The combined data suggest that interactions between LexGSL on opposite cell surfaces are strong enough to allow formation of stable cell-cell contacts.

Topics: Animals; Carcinoma, Hepatocellular; Cell Adhesion; Cell Aggregation; Colonic Neoplasms; Glycosphingolipids; Humans; Leukemia, Basophilic, Acute; Lewis Blood Group Antigens; Liver Neoplasms; Oligosaccharides; Rats; Sialyl Lewis X Antigen; Tumor Cells, Cultured

We have previously reported that colon cancer cells metastasized to the liver expressed an increased amount of sialyl Lewis X (SLeX) antigen compared to their corresponding primary lesions. It is now well known that SLeX antigen and sialyl Lewis A (SLeA) antigen are ligands for the selectins expressed on the endothelial cells. Therefore, it is assumed that SLeX-rich colon cancer cells could be easily adhered to the endothelial cells that express selectins. In this report we have tried to induce selectin expression on the human liver sinusoidal endothelial cells and have examined the adhesion of SLeX-high or -low expressing colon cancer cells to the interleukin-1beta (IL-1beta)-treated liver specimens using Stamper-Woodruff assay. These human colon cancer cells are termed KM12HX or KM12LX cells, respectively. A significantly increased number of KM12HX cells adhered to the IL-1beta-treated liver specimens compared to KM12LX cells. The adhesion of KM12HX cells was inhibited by the pretreatment of tumor cells with anti-SLeX antibody or by the pretreatment of liver specimens with anti-selectin antibodies. Selectin expression on the liver sinusoidal endothelial cells and endothelial cells of blood vessels after IL-1beta treatment was confirmed by immunohistochemically using anti-selectin monoclonal antibodies (MAbs). These findings strongly suggest that SLeX-expressing cancer cells could adhere to the sinusoidal endothelial cells via an SLeX-selectin interaction system and this could be a first step for colon cancer cells that metastasize to the liver. The mechanism by which these selectins can be induced in vivo is the next problem to be considered.

Topics: Cells, Cultured; Colonic Neoplasms; Endothelium; Humans; Interleukin-1; Ligands; Liver; Liver Neoplasms; Neoplasm Metastasis; Oligosaccharides; Selectins; Sialyl Lewis X Antigen

To elucidate the early events of blood-borne metastasis under actual blood flow, real-time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117-P and liver-metastatic RAW117-H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time-dependently. The elimination rate of RAW117-H10 cells, however, was slower than that of RAW117-P cells, suggesting that RAW117-H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117-H10 cells metastasized in the liver to a greater extent than RAW117-P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117-H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117-H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117-H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117-H10 cells were found to express a much higher level of sialyl Lewis A than RAW117-P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117-H10 cells with the hepatic endothelium, leading to metastasis.

Topics: Animals; Antigens, Neoplasm; CA-19-9 Antigen; Cell Adhesion; Cell Movement; Female; Flow Cytometry; G(M1) Ganglioside; Gangliosides; Injections, Intravenous; Liver Neoplasms; Lung Neoplasms; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Neuraminidase; Portal Vein; Sialyl Lewis X Antigen; Tomography, Emission-Computed; Tumor Cells, Cultured

The expression of the sialyl Lewis x antigen (sLe(x)) on surgical specimens of primary gastric cancer correlates with the degree of differentiation and synchronous and metachronous liver metastasis. Multivariate analysis by means of Quantification theory II revealed that sLe(x) expression was an independent risk factor for liver metastasis from gastric cancer.

Topics: Aged; Animals; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; CA-19-9 Antigen; Female; Gangliosides; Gastric Mucosa; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Oligosaccharides; Prognosis; Risk Factors; Sialyl Lewis X Antigen; Stomach Neoplasms

Two factors potentially determining the consistent overexpression of sialyl-Le(x) antigen in colon carcinoma and metastases were investigated: (i) the expression of the mucins MUC1 and MUC2, known to carry sialyl-Le(x), by Northern blotting; (ii) the extent of sialic acid O-acetylation, by Western blotting and HPLC. RNA and sialyl-Le(x)-positive mucins were purified from normal colonic mucosa (N), primary carcinomas (T) and their liver metastases (M). Northern blots showed that mRNA expression both of MUC1 and of MUC2 decreases during the progression of the disease, and is lowest in metastatic tissue. The expression of mucin-bound sialyl-Le(x) increased strongly from N to T and, to a lesser extent, to M. After alkali treatment of the mucins these differences disappeared, indicating that the total amount of mucin-bound sialyl-Le(x) is the same in the 3 types of tissues. The O-acetylation of mucin-bound sialyl-Le(x) gradually decreased from N to M. HPLC analysis showed that in N about 70%, in T 45% and in M only 20% of mucin-bound sialic acids are O-acetylated. Thus, the increase of sialyl-Le(x) detectable during colon-carcinoma progression is due to diminished O-acetylation and not to increased expression of mucin protein cores. The decrease of O-acetylation is therefore the primary chemical alteration contributing to colon carcinoma-associated overexpression of sialyl-Le(x).

Topics: Acetylation; Carcinoma; Colorectal Neoplasms; Humans; Liver Neoplasms; Mucin-1; Mucin-2; Mucins; Oligosaccharides; RNA, Messenger; Sialyl Lewis X Antigen

A human colon cancer cell line, OCUC-LM1(LM), was established from a liver metastasis in our laboratory. Intrasplenic injection of LM into nude mice was repeated three and five times, and the daughter cell lines were designated as LM-H3 and LM-H5 respectively. The level of sialyl Lewis A (SLA) in the supernatant of LM-H3 and LM-H5 was 3 and 4.5 times higher than that of LM respectively. Flow cytometric analysis of SLA expression showed that the peak channel for LM was 113; for LM-H3, 126; and for LM-H5, 146. The mean fluorescence intensity of LM was 102.3 +/- 43.5; for LM-H3, 126.2 +/- 28.4; and for LM-H5, 144.8 +/- 23.4. In endothelial cell adhesion assays, the percentages of adherent LM-H3 and LM-H5 cells were significantly higher than for LM. The activity of alpha1-->4 fucosyltransferase was higher in LM-H3 and LM-H5 than in LM, but there was no difference in alpha2-->3 sialyltransferase activities for type 1 chain among the cell lines. Our results suggest that SLA expression is associated with acquisition of a high capacity for liver metastasis of colon cancer; increased SLA expression is due mainly to increased fucosyltransferase activity.

Topics: Animals; Antibodies; beta-Galactoside alpha-2,3-Sialyltransferase; Carbohydrate Conformation; Carbohydrate Sequence; Cell Adhesion; Cells, Cultured; Colonic Neoplasms; E-Selectin; Endothelium, Vascular; Flow Cytometry; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Liver Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Sialyltransferases; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Veins

Ten human colorectal cancer (CRC) cell lines were implanted orthotopically into the ceca and also into the livers, muscles and peritoneal cavities of SCID mice in order to analyze the characteristics regulating metastatic behaviors of CRCs. All the CRC cell lines formed tumors in the muscle and cecum, but they could be classified into two groups: (1) six cell lines with high tumorigenicity in the liver (HTLs) forming differentiated tumors, and (2) four with no tumorigenicity in the liver (NTLs) forming poorly differentiated tumors in SCID mice. After orthotopic implantation, NTLs never metastasized to the liver, whereas HTLs did. Therefore, intrahepatic tumorigenicity and differential status were closely associated with liver metastasis whereas differentiation was not associated with lung metastasis. The 6 HTLs demonstrated an inverse correlation between liver metastases and peritoneal dissemination, and immunohistochemistry indicated expression of sLeX, CA19-9 and carcinoembryonic antigen in tumors which correlated well with the liver metastatic rate. We found a strong correlation between liver metastasis and intrahepatic tumorigenicity and could reproduce the clinical correlations between the pattern of the metastatic spread and the differentiation phenotype of CRC in vivo. We consider further examination using this model will be useful for analyzing the complex mechanisms involved in clinically metastasizing CRCs.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; Carcinogenicity Tests; Colorectal Neoplasms; Disease Models, Animal; ErbB Receptors; Humans; Injections, Intraperitoneal; Liver Neoplasms; Male; Mice; Mice, SCID; Neoplasm Transplantation; Oligosaccharides; Peritoneal Neoplasms; Sialyl Lewis X Antigen; Tumor Cells, Cultured

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Topics: Animals; Base Sequence; CA-19-9 Antigen; E-Selectin; Fucosyltransferases; Gangliosides; Liver Neoplasms; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Transgenic; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Peptide Fragments; Recombinant Fusion Proteins; Sialyl Lewis X Antigen

Liver metastasis from colorectal carcinoma is an important problem in surgical treatment and profoundly affects the prognosis of patients. If it were possible to identify characteristic features in the primary lesion strongly related to liver metastasis, these could be used as prognostic markers for liver metastasis. To search for such features, the primary lesions of patients with colorectal carcinoma with liver metastasis were investigated.. Three groups of colorectal carcinoma were examined: Group A with synchronous liver metastases; Group B with only lymph node metastases without recurrence for 5 years; and Group C with recurrence of liver metastases. Groups A and B included 24 cases and Group C, 20. We focused on cancer cell morphology at the invasive front and expression of sialyl Lewis X (sialyl Lex) in the primary cancer.. At the invasive front in Group A it was frequently found that polygonal, not columnar, cancer cells with a single or solitary trabecular form with indistinct polarity, showed an infiltrative growth pattern. This type of morphology was termed "focal dedifferentiation" and graded four levels. Eleven of 24 cases (46%) had severe focal dedifferentiation in Group A, 1 of 24 (4%) in Group B, and 6 of 20 (30%) in Group C. Sialyl Lex staining was positive in 12 of 24 cases (50%) in Group A, in 3 of 24 cases (13%) in Group B, and in 7 of 20 cases (35%) in Group C in the primary carcinoma. In respect to the staining of (sialyl Lex) at focal dedifferentiation, it was positive in 17 of 24 cases (71%) in Group A, in 4 of 24 cases (17%) in Group B and in 11 of 20 cases (55%) in Group C. Focal dedifferentiation and sialyl Lex staining in the primary cancer showed a significant difference between Groups A and B. Sialyl Lex staining at focal dedifferentiation showed a significant difference between Groups A and B and Groups B and C. Other adhesion related molecules, sialyl LeA and CEA, showed no difference among Groups A, B, and C.. Both focal dedifferentiation and expression of sialyl Lex antigen in the primary lesion are considered good markers for assessing the metastatic proclivity of colorectal cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Oligosaccharides; Prognosis; Rectal Neoplasms; Sialyl Lewis X Antigen

E-selectin is an adhesion molecule of endothelial cells that binds to cancer cells mediated by sialyl Lewis A (sLea) or sialyl Lewis X (sLe(x)). It is suspected to be involved in hematogenous metastasis of tumors. Therefore, it is worth examining E-selectin expression in human colorectal cancer and its hepatic metastasis. In the present study, E-selectin was clearly revealed on the endothelial cells of small vessels adjacent to cancer nests both in primary and in metastatic nests in immunohistochemistry. In these tissues, E-selectin was observed on the endothelial cells lining the lumen of small vessels. Its expression adjacent to cancer nests appears to be induced through some stimuli by cancer cells, since its degree of expression is inversely correlated to the distance of the blood vessels from the cancer nests (p < 0.001). Endothelial cells adjacent to the metastatic lesion expressed E-selectin more extensively than those adjacent to the primary foci. This is also in line with the finding on serum E-selectin levels which were significantly elevated in the metastatic group as compared with the non-metastatic group. The serum E-selectin level may provide useful information in the diagnosis for hepatic metastasis of colorectal cancer, although the results are still tentative.

Topics: Aged; Aged, 80 and over; CA-19-9 Antigen; Cell Adhesion Molecules; Colon; Colorectal Neoplasms; E-Selectin; Endothelium, Vascular; Female; Gangliosides; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Male; Middle Aged; Oligosaccharides; Rectum; Sialyl Lewis X Antigen

Sialyl Lewis A (SLA) and sialyl Lewis X (SLX) have been shown to be specific ligands for endothelial leukocyte adhesion molecule-1 (ELAM-1), and may be involved in the process of adhesion between cancer cells and endothelium. We used immunohistochemical methods to study the expression of SLA, SLX and CEA in both primary tumors and matched metastatic liver lesions of colorectal carcinomas. Specimens from primary tumors and matched liver metastases from 24 patients with colorectal carcinomas were studied immunohistochemically. The degree of expression of CEA in liver metastases was similar to that in primary tumors, but SLA and SLX were expressed on a larger proportion of tumor cells in liver metastases than in primary tumors. Our findings suggest that colorectal carcinoma cells expressing SLA and/or SLX form metastatic liver tumors. They also suggest that expression of SLA and SLX in primary of colorectal carcinoma can be used as a prognostic indicator of metastasis.

Topics: Adult; Aged; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Gangliosides; Humans; Immunohistochemistry; Lewis Blood Group Antigens; Liver Neoplasms; Male; Middle Aged; Oligosaccharides; Sialyl Lewis X Antigen

We have investigated the inhibitory mechanism of the initial arrest of L5178Y-ML25 lymphoma cells in a target organ (liver) by using recombinant fibronectin fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Pretreatment of hepatic sinusoidal endothelial (HSE) cell monolayers with lymphoma cells or their conditioned medium for 4 to 6 h resulted in the enhancement of lymphoma cell adhesion to HSE cell monolayer. The increased tumor adhesiveness was completely abolished by preincubation of the conditioned medium with anti interleukin-1 beta monoclonal antibody (mAb). Synthetic sialyl Le(x) (SLe(x)) as a ligand for endothelial cell leukocyte adhesion molecule-1 (ELAM-1) adhesion receptor and anti ELAM-1 mAb blocked the conditioned medium-induced enhancement of tumor-endothelial cell interaction, while pretreatment of the activated HSE cell monolayer with anti vascular cell adhesion molecule-1 (VCAM-1) mAb did not affect the enhanced tumor cell adhesion. These results indicate that tumor cell interaction with the stimulated HSE cells is mediated by ELAM-1 molecules on HSE cells. However, the expression of SLe(x) and SLe(a) on the tumor surface was not observed by flow cytometric analysis. ELAM-1-mediated enhancement of tumor cell adhesion to HSE monolayer was also inhibited in a concentration-dependent manner by CH-271 fusion polypeptide or the sulfated chitin derivative sulfated carboxymethyl-chitin, which can bind to the heparin-binding domain of CH-271. In addition, CH-271 inhibited not only tumor-endothelium interaction but also tumor cell invasion into reconstituted basement membrane Matrigel in vitro.

Topics: Animals; Antibodies, Monoclonal; Basement Membrane; Carbohydrate Sequence; Cell Adhesion; Cell Adhesion Molecules; Culture Media, Conditioned; E-Selectin; Endothelium, Vascular; Fibronectins; Interleukin-1; Liver; Liver Neoplasms; Lymphoma; Mice; Molecular Sequence Data; Neoplasm Invasiveness; Oligosaccharides; Recombinant Fusion Proteins; Recombinant Proteins; Sialyl Lewis X Antigen; Tumor Cells, Cultured