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

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

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

We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.

Topics: Acute-Phase Proteins; Amino Sugars; beta-Galactoside alpha-2,3-Sialyltransferase; Carcinoma, Hepatocellular; Cell Line, Tumor; Fucosyltransferases; Gene Expression Regulation; Humans; Interleukin-1beta; Oligosaccharides; RNA, Messenger; Sialyl Lewis X Antigen; Sialyltransferases; Up-Regulation

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

Sialyl Lewis X (SLEX) antigen, Neu5Acalpha2-3Galbeta1-4 (Fucalpha1-3) GlcNAc-R, plays important roles in cell-to-cell interaction: for example, the E- and P-selectin-mediated influx of SLEX expressing leukocytes into inflamed areas. A human hepatocellular carcinoma cell line, HepG2 cells, was highly expressed SLEX on secreted glycoproteins and cell surface, in contrast with HuH-7 cells. We identified SLEX expressing glycoproteins in HepG2 cultured medium by two-dimensional polyacrylamide gel electrophoresis, followed by in gel digestion and peptide mass fingerprint using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS), including transferrin, alpha1-antitrypsin, alpha2-HS glycoprotein and beta-glycoprotein. We analyzed N-glycans of these glycoproteins by MALDI-TOFMS in combination with exoglycosidase digestion; our results indicate increases in poly-fucosylated and high-branched N-glycans. High alpha1,3-fucosylation in glycoproteins would be caused by increased expression of alpha1,3-fucosyltransferase activities in HepG2 cells.

Topics: Carcinoma, Hepatocellular; Glycoproteins; Humans; Lewis X Antigen; Oligosaccharides; Proteomics; Sialyl Lewis X Antigen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Cells, Cultured

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

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLex expression induced by FSK, since only the monoclonal antibody of SLex showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLex via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (a-PKC) might also participate in the up-regulation of SLex expression by FSK, and cAMP/PKA pathway is a negative regulator of SLex expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.

Topics: Antibodies, Monoclonal; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Line; Cell Line, Tumor; Cell Movement; Cells, Cultured; Chemotaxis; Colforsin; Cyclic AMP; DNA, Complementary; Endothelium, Vascular; Enzyme Inhibitors; Flow Cytometry; Humans; Laminin; Neoplasm Metastasis; Oligosaccharides; Phenotype; Phosphatidylinositol 3-Kinases; Protein Kinase C; 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

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

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

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

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