g(m2)-ganglioside and Colonic-Neoplasms

Previous studies have shown that (GM3), a ganglioside, suppresses hepatoma cell motility and migration by inhibiting phosphorylation of EGFR and the activity of the PI3K/AKT signaling pathway. Therefore, the aim of the present study was to investigate whether the combined treatment of CD82 with gangliosides can exert a synergistic inhibitory effect on cell motility and migration. Epidermal growth factor receptor (EGFR) signaling was studied for its role in the mechanism through which CD82 and gangliosides synergistically inhibit the motility and migration of SW620 human colon adenocarcinoma cells. GM3 and/or GM2 treatment, and/or overexpression of CD82 was performed in SW620 cells. High-performance thin layer chromatography, reverse transcription-quantitative PCR, western blotting and flow cytometry assays were used to confirm the content changes of GM2, GM3 and CD82. In addition, the phosphorylation of EGFR, MAPK and Akt were evaluated by western blot analysis. SW620 cell motility was investigated using wound healing analysis and chemotaxis migration assay. The combination of GM3 and GM2 with CD82 was found to markedly suppress EGF-stimulated SW620 cell motility compared with the individual factors or combination of GM2 or GM3 with CD82 by inhibiting the phosphorylation of EGFR. The results suggested that CD82 in combination with either GM2 or GM3 can exert a synergistic inhibitory effect on cell motility and migration; however, the synergistic mechanisms elicited by GM2 or GM3 with CD82 differ.

Topics: Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Down-Regulation; ErbB Receptors; G(M2) Ganglioside; G(M3) Ganglioside; Humans; Kangai-1 Protein; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Signal Transduction; Transfection; Tyrosine

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Topics: Caco-2 Cells; Cell Hypoxia; Cell Line, Tumor; Colon; Colonic Neoplasms; Culture Media; Epithelial Cells; G(M2) Ganglioside; Humans; Immunohistochemistry; Mixed Function Oxygenases; N-Acetylneuraminic Acid; Organic Anion Transporters; Symporters; Transfection

The content of the GM2 ganglioside and the activity of UDP-GalNAc: GM3 beta-1,4N-acetylgalactosaminyltransferase (beta-1,4GalNAcT), which synthesizes GM2, increased in gastric cancer tissues and gastric cancer cell lines as compared with that in normal gastric mucosa.. Expression of beta-1,4GalNAcT mRNA and a concentration of GM2 in the human gastrointestinal tissues were examined. Beta-1,4GalNAcT mRNA in human surgical specimens, which was not detectable with Northern blotting because of the paucity of absolute amounts expressed, was detected with competitive reverse transcription-polymerase chain reaction (PCR) method using an internal standard cRNA that could be amplified by the same primers as target mRNA in PCR. The quantification of GM2 was examined using immunostaining of thin-layer chromatography.. In 10 of 10 gastric carcinomas and 6 of 13 colonic carcinomas, mRNA expression was more enhanced than that in the normal mucosa of each patient. The alteration of GM2 content in carcinoma from normal tissue generally was correlated to the change in the expression of beta-1,4GalNAcT mRNA with a few exceptions. One gastric cancer sample had a higher level of mRNA with a lower GM2 content than the corresponding normal tissue, and two colonic carcinoma tissue specimens had a lower level of mRNA with a higher GM2 content.. These results suggest that expression of the beta-1,4GalNAcT gene is a key step in the molecular mechanisms underlying the regulation of cancer-associated GM2 expression in the stomach and the colon.

Topics: Adenocarcinoma; Base Sequence; Blotting, Northern; Colonic Neoplasms; G(M2) Ganglioside; Gastric Mucosa; Gene Expression; Humans; Intestinal Mucosa; Molecular Sequence Data; N-Acetylgalactosaminyltransferases; Polymerase Chain Reaction; Polypeptide N-acetylgalactosaminyltransferase; RNA-Directed DNA Polymerase; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms

A glycolipid detected in human gastric mucosa with anti-GM2 monoclonal antibody was characterized to be GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc beta 1-3Gal 1-4Glc-Cer (NGM-1), which was lost in gastric cancer tissue with complementary increase of GM2 sharing the same terminal carbohydrate structure as NGM-1 (Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Oshima, M. (1990) J. Biol. Chem. 265, 7880-7885). The study on differential expression of NGM-1 in gastric fundic mucosa, pyloric mucosa, gastric cancer, and various other tissues indicated that NGM-1 existed specifically in fundic mucosa. The content of GM3 and sialylparagloboside (SPG), which are the substrates for the synthesis of GM2 and NGM-1, respectively, were not significantly different in these tissues. Therefore, the presence of two kinds of beta 1,4GalNAc transferases having different substrate specificity was considered to be critical for the expression of NGM-1 and GM2. The activity of beta 1,4GalNAc transferase which synthesizes GM2 or NGM-1 was determined by detecting the products with specific monoclonal antibodies. The activity of beta 1,4GalNAc transfer to SPG was high in fundic mucosa, while it was absent in pyloric mucosa or cancer. On the other hand, the increased activity of beta 1,4GalNAc transfer to GM3 was observed in cancer tissues and cancer cell lines which were rich in GM2. Our conclusion is that the limited expression of NGM-1 in fundic mucosa and the increase of GM2 in cancer are attributed to two types of beta 1,4GalNAc transferases localized in each region with different substrate specificity; the one in fundic mucosa transfers GalNAc to SPG but not to GM3, and the other one enhanced in cancer transfers GalNAc to GM3 but not to SPG.

Topics: Antibodies, Monoclonal; Cell Line; Chromatography, Thin Layer; Colonic Neoplasms; G(M2) Ganglioside; Galactosyltransferases; Gangliosides; Gastric Fundus; Gastric Mucosa; Humans; Kinetics; N-Acetylgalactosaminyltransferases; Organ Specificity; Polypeptide N-acetylgalactosaminyltransferase; Pyloric Antrum; Stomach Neoplasms

This paper reports the presence of GM2 ganglioside containing N-glycolylneuraminic acid (NeuGc) in human colon cancer tissues. GM2(NeuGc) was detected by two-dimensional thin layer chromatography (2d-TLC)/enzyme-immunostaining using affinity-purified chicken antibody against GM3(NeuGc) and horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody. Like usual GM2 ganglioside containing N-acetylneuraminic acid (NeuAc) isolated from Tay-Sachs brain, GM2(NeuGc) in colon cancer could be converted into GM3(NeuGc) by human kidney beta-N-acetylhexosaminidase A in the presence of a GM2-specific activator protein isolated from guinea pig kidney. Three of 7 specimens of Hanganutziu-Deicher (HD) antigen-positive human colon cancer tissues so far examined expressed this unique ganglioside. In order to detect and determine specifically GM2(NeuGc) on human colon cancers, specific antibody against GM2 (NeuGc) has been prepared by immunizing chickens. By a sensitive TLC/immunostaining method using the antibody, the amounts of the antigen were determined to be 0.3-3% of total lipid-bound sialic acid. NeuGc-containing gangliosides were also detected in meconium and fetal intestinal tissues. Three species of antigenic gangliosides in pooled meconium were tentatively identified as GM3(NeuGc), sialylparagloboside and sialylhexaosylceramide on the basis of their migration positions on 2d-TLC and the results of endo-beta-galactosidase treatment. GM3(NeuGc) was the sole HD-active ganglioside in fetal intestinal tissue from one of 3 individuals tested; the other two showed no HD-active ganglioside at all. GM2(NeuGc), however, could not be detected in either meconium or fetal tissues so far examined, suggesting that this unique ganglioside is a tumor-specific antigen, at least for human intestinal tissues.

Topics: Antibodies; Antigens, Heterophile; Antigens, Neoplasm; Colonic Neoplasms; G(M2) Ganglioside; Gangliosides; Humans; Intestines; Meconium; Neuraminic Acids

Topics: Antibodies; Chromatography, Affinity; Chromatography, Thin Layer; Colonic Neoplasms; G(M2) Ganglioside; Glycosphingolipids; Humans; Immunoenzyme Techniques; Staining and Labeling

Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.

Topics: Antigens, Heterophile; Colonic Neoplasms; Densitometry; G(M2) Ganglioside; Gangliosides; Glycolipids; Humans