Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety

AbstractA mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the 3 GalCers, the mAb reacted with two GalCers which contained α-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid

Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I

<p>Abstract</p> <p>Background</p> <p>This study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H.</p> <p>Methods</p> <p>The expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR.</p> <p>Results</p> <p>Immunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (<it>P </it>< 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both <it>P </it>< 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by co-immunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (<it>P </it>< 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (<it>P </it>< 0.01). The mRNA level of CD44 in both cell lines was similar (<it>P </it>> 0.05).</p> <p>Conclusion</p> <p>Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.</p

Increase in Docetaxel-Resistance of Ovarian Carcinoma-Derived RMG-1 Cells with Enhanced Expression of Lewis Y Antigen

Epithelial carcinomas of the ovary exhibit the highest mortality rate among gynecologic malignancies. Studies found that the metabolism of glycolipids or carbohydrates is associated with acquirement of anticancer drug-resistance by cancer cells. This study was to characterize possible involvement of Lewis Y (LeY) antigen in the drug-resistance of cancer cells. We transfected the α1,2-fucosyltransferase gene into human ovarian carcinoma-derived RMG-1 cells and established RMG-1-hFUT cells with enhanced expression of LeY. We determined the effects of docetaxel on the survival of cells by MTT assaying and observed the apoptosis of cells in the presence of docetaxel by flow cytometric analysis and by transmission electron microscopy. Plasma membranes and intracellular granules in RMG-1-hFUT cells were stained with anti-LeY antibody, the intensity of the staining was higher than that in control cells. The RMG-1-hFUT cells exhibited higher resistance to docetaxel than the control cells with regard to the docetaxel concentration and time course. After treatment with 10 μg/mL docetaxel for 72 h, the control cells, but not RMG-1-hFUT, contained abundant positively stained cell debris due to disintegration of the cytoskeleton. On transmission electron microscopy, although the control cells treated with docetaxel as above showed the following morphology, i.e., absence of villi, cells shrunken in size, pyknosis, agglutinated chromatin and cell buds containing nuclei in the process of apoptosis, the RMG-1-hFUT cells showed only agglutinated chromatin and vacuoles in the cytoplasm. In summary, cells with enhanced expression of LeY were shown to acquire docetaxel-resistance, indicating the possible involvement of glycoconjugates in the drug-resistance

Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

<p>Abstract</p> <p>Background</p> <p>Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor's pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.</p> <p>Methods</p> <p>We constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.</p> <p>Results</p> <p>Our results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002.</p> <p>Conclusions</p> <p>Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.</p

The Stimulation of IGF-1R Expression by Lewis(y) Antigen Provides a Powerful Development Mechanism of Epithelial Ovarian Carcinoma

Objective: This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples. Methods: Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R. Results: In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT (P &lt; 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells (P &lt; 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells (P &gt; 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P &lt; 0.05), benign (33.00% and 53.33%, all P &lt; 0.01), and normal (0% and 40%, all P 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P &lt; 0.05). Conclusion: Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers

Study on the Expression and Clinical Significances of Lewis y Antigen and Integrin αv, β3 in Epithelial Ovarian Tumors

Objective: To detect the expression and clinical significances of Lewis y antigen and integrin αv, β3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, β3. Methods: Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, β3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, β3 in ovarian cancer. Results: In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (P &lt; 0.05) and benign (25%) (P &lt; 0.01) tumors, and normal ovarian tissues (0) (P &lt; 0.01). The expression rate of integrin αv, β3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline (45.94%, 40.54%) (both P &lt; 0.05), benign group (10.00%, 15.00%) (both P &lt; 0.01) and normal ovary group (5%, 15%) (both P &lt; 0.01). Conclusions: Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis

Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways

Abstract: Objective: To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. Methods: mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. Results: Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression. Conclusion: Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation. Int. J. Mol. Sci. 2012, 13 829 Keywords: Lewis(y) antigen; cell cycle; cyclin; cyclin-dependent kinases; cyclin-dependent kinase inhibitor

Lewis (y) Antigen Overexpression Increases the Expression of MMP-2 and MMP-9 and Invasion of Human Ovarian Cancer Cells

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion

Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues