g(m3)-ganglioside and Colonic-Neoplasms

Topics: Adenocarcinoma; Apoptosis; Colonic Neoplasms; G(M3) Ganglioside; Glycosylation; Humans; Neuraminidase; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

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

Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2alpha-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between -1175 and -1077 from the translational initiation site, which contains the AP-2alpha binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2alpha is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2alpha diminished the enhancement of AP-2alpha and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2alpha also results in the induction of PTEN transcription in AP-2alpha-negative colon cancer cells. Additionally, GM3 induced AP-2alpha-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.

Topics: Base Sequence; Cell Line, Tumor; Colonic Neoplasms; G(M3) Ganglioside; Genes, erbB-1; Humans; Molecular Sequence Data; Promoter Regions, Genetic; PTEN Phosphohydrolase; RNA, Small Interfering; Transcription Factor AP-2; Transcriptional Activation

Sialidase expression levels are inversely correlated with the metastatic potential of mouse colon adenocarcinoma 26 sublines, as assessed by activity assays and RT-PCR, irrespective of total and cell surface sialic acid contents. Compared with low metastatic NL4 and NL44 cell lines, the highly metastatic NL17 and NL22 cells exhibit low expression of sialidases, accompanied with higher levels of sialylLe(x) and GM3. To investigate whether these properties of NL17 cells can be altered by sialidase overexpression, we transfected a cytosolic sialidase gene into NL17 cells. The result was markedly inhibited lung metastasis, invasion and cell motility with a concomitant decrease in sialylLe(x) and GM3 levels, in line with the case of spontaneously low metastatic sublines having relatively high endogenous sialidase levels, implying that sialidase level is a determining factor affecting metastatic ability. Treatment of the cells with antibodies against sialylLe(x) and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis.

Topics: Adenocarcinoma; Animals; Cell Line; Chromatography, Thin Layer; Colonic Neoplasms; DNA Primers; Female; Flow Cytometry; G(M3) Ganglioside; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Oligosaccharides; Reverse Transcriptase Polymerase Chain Reaction; Sialyl Lewis X Antigen; Sialyltransferases; Transfection; Tumor Cells, Cultured

We have found that an increase in the ganglioside GM3 is a prerequisite for the induction of terminal differentiation, cuhninating in death by apoptosis, of human colonic carcinoma cells in vitro. To evaluate the therapeutic effect of increasing GM3 in human colonic carcinoma cells, we examined whether treated cells lose their tumorigenic activity and whether this approach is effective against cancer cells growing in vivo. Cells of the human colonic carcinoma cell line HCT 116 not only differentiated but also lost their tumorigenic activity by an artificial increase in GM3. When HCT 116 tumors growing in nude mice were treated with a drug that increases GM3, an appreciable increase in GM3 and induction of apoptosis were clearly observed. The growth of treated tumors was greatly suppressed. These results suggest that the modulation of ganglioside expression to introduce gangliosides with biological activity into cancer cells could be a novel effective approach for cancer therapy.

Topics: Adenocarcinoma; Animals; Apoptosis; Brefeldin A; Cell Differentiation; Cell Division; Colonic Neoplasms; G(M3) Ganglioside; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Tumor Cells, Cultured

A cooperative inhibitory effect of GM3, together with CD9, on haptotactic cell motility was demonstrated by a few lines of study as described below. (i) Haptotactic motility of colorectal carcinoma cell lines SW480, SW620, and HRT18, which express CD9 at a high level, is inhibited by exogenous GM3, but not by GM1. (ii) Motility of gastric cancer cell line MKN74, which expresses CD9 at a low level, was not affected by exogenous GM3. Its motility became susceptible to and inhibited by exogenous GM3, but not GM1, when the CD9 level of MKN74 cells was converted to a high level by transfection with CD9 cDNA. Findings i and ii suggest that haptotactic tumor cell motility is cooperatively inhibited by coexpression of CD9 and GM3. (iii) This possibility was further demonstrated using cell line ldlD 14, and its derivative expressing CD9 through transfection of its gene (termed ldlD/CD9). Both of these cell lines are defective in UDP-Gal 4-epimerase and cannot synthesize GM3 unless cultured in the presence of galactose (Gal(+)), whereas GM3 synthesis does not occur when cells are cultured in the absence of Gal (Gal(-)). Haptotactic motility of parental ldlD cells is low, and shows no difference in the presence and absence of Gal. In contrast, the motility of ldlD/CD9 cells is very high in Gal(-) whereby endogenous GM3 synthesis does not occur, and is very reduced in Gal(+) whereby endogenous GM3 synthesis occurs. (iv) Photoactivatable (3)H-labeled GM3 added to HRT18 cells, followed by UV irradiation, causes cross-linking of GM3 to CD9, as evidenced by (3)H labeling of CD9, which is immunoprecipitated with anti-CD9 antibody. These findings suggest that CD9 is a target molecule interacting with GM3, and that CD9 and GM3 cooperatively downregulate tumor cell motility.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Cell Migration Inhibition; Cell Transformation, Neoplastic; Chemotaxis; CHO Cells; Clone Cells; Colonic Neoplasms; Cricetinae; Cross-Linking Reagents; Culture Media, Conditioned; G(M3) Ganglioside; Galactose; Humans; Membrane Glycoproteins; Stomach Neoplasms; Tetraspanin 29; Transfection; Tritium; Tumor Cells, Cultured; Ultraviolet Rays

We studied ganglioside expression in 12 human metastatic brain tumors metastasized from colon (4), renal (3), lung (2), esophagus (1), pancreas (1), and mammary (1) carcinomas. GM3 was the major common ganglioside expressed in brain metastatic tumor tissues, and GT1b was also present in all the metastatic brain tumor tissues. The latter was identified by TLC-immunostaining and characterized structurally by secondary ion mass spectrometry combined with 'Far-Eastern blot'. The immunohistochemical analysis of frozen tissue sections confirmed localization of GT1b in the tumor cell membrane or cytosol. GT1b was shown to be expressed both in the primary colon carcinoma and the metastasis of a single patient by immunohistochemical procedure. In systemic carcinomas without brain metastasis, GM3 was a common major component, but no GT1b was detected. These findings indicate that GT1b is a brain metastasis-associated ganglioside. We speculate that the presence of GT1b would be a useful marker for estimating metastatic potentials to the brain.

Topics: Adenocarcinoma; Biomarkers, Tumor; Brain Neoplasms; Chromatography, Thin Layer; Colonic Neoplasms; Frozen Sections; G(M3) Ganglioside; Gangliosides; Humans; Immunohistochemistry; Kidney Neoplasms; Mass Spectrometry; Neoplasm Metastasis

In this study we report the characterization of a human monoclonal antibody (HuMab), L612, that reacts with ganglioside GM3 and has therapeutic application for the treatment of human neoplasms, particularly melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed B-cell line L612 was established. L612 HuMab bound specifically to neoplastic cell lines in culture and in tissue biopsy specimens such as melanoma, colon, breast, and lung cancer. The antibody did not bind to normal cells or biopsy tissue. HuMab L612 showed the highest reactivity to melanoma cells, particularly to those with high concentrations of GM3. Immunostaining on high-performance thin-layer chromatography plates demonstrated that L612 HuMab bound to GM3 purified from melanoma cells. Removal of the sialic acid from GM3 abolished antibody binding. HuMab L612 also reacted to GM4 purified from egg yolk, indicating that it recognizes an NeuAc alpha 2-3 galactose antigen determinant. HuMab L612 heavy and light chains were sequenced and determined to belong to the mu heavy chain variable subgroup III and kappa chain variable subgroup IV families, respectively. The studies indicate that the L612 HuMab has significant therapeutic potential for a wide variety of human cancers.

Topics: Adenocarcinoma; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; B-Lymphocytes; Base Sequence; Breast Neoplasms; Carbohydrate Sequence; Cell Line, Transformed; Cloning, Molecular; Colonic Neoplasms; DNA Primers; Female; G(M3) Ganglioside; Gangliosides; Herpesvirus 4, Human; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Lung Neoplasms; Melanoma; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; Recombinant Proteins; Skin Neoplasms

The appearance of Hanganutziu and Deicher (HD) antibody in the sera of patients suffering from various diseases, including malignancies of some organs and liver disorders, was investigated by enzyme-linked immunosorbent assay using N-glycolylneuraminyl-lactosylceramide (HD3) and 4-O-acetyl-HD3 as the antigenic molecules. More than 25% of sera from patients suffering from malignancies, cholelithiasis and liver cirrhosis had HD antibody, whereas none of 41 sera from healthy persons had HD antibody. The percentage of HD antibody-positive patients was similar in stages I, II and III of gastric cancer and recurrence cases. Antibody titers of the positive patients in each stage were also not different from those in each other stage. These results indicated that HD antigenic expression on cancerous tissue is not dependent on the cancerous malignancy. The HD antibody level was elevated after surgical removal of cancerous tissues in 5 of 6 patients examined, indicating that tumor growth absorbed the serum antibody. Serum antibody against 4-O-acetyl-HD3 was detected independently of HD3 antibody in some cases; however, in most cases, correlation between the two antibody titers was observed.

Topics: Aged; Antibodies, Heterophile; Antigens, Neoplasm; Breast Neoplasms; Cholelithiasis; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Esophageal Neoplasms; Female; G(M3) Ganglioside; Humans; Liver Cirrhosis; Lymphoma; Male; Middle Aged; Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms

Antibody to 4-O-acetyl-N-glycolylneuraminyl lactosylceramide [GM3(Neu4AcGc)] was prepared by immunizing chicken with the glycosphingolipid antigen. The specific antibody was purified by affinity chromatography columns of Octyl-Sepharose linked to the homologous immunogen and its deacetylated analogue [N-glycolylneuraminyl lactosylceramide, GM3(NeuGc)], respectively. The specificity of the purified antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) and inhibition of equine erythrocyte hemagglutination using authentic glycosphingolipids as antigens. The results indicated that the antibody recognized both 4-O-acetyl and N-glycolyl groups of terminal sialic acid residue as the immunodeterminants. The purified specific antibody was applied in the confirmation of the presence of GM3(Neu4AcGc) in ganglioside fractions of human colon cancer tissues, which were suspected to have this antigen by studies of alkaline, periodate or neuraminidase treatment [Higashi et al. (1985) Cancer Res. 45, 3796-3802.], by thin-layer chromatography (TLC)-immunostaining technique.

Topics: Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Colonic Neoplasms; G(M3) Ganglioside; Gangliosides; Humans; Immune Sera; Immunodiffusion; Immunoenzyme Techniques

The gangliosides in six colorectal and two pancreatic carcinomas were examined. Their concentration in the primary tumour and the metastases was 5-10 fold higher than in normal colon mucosa. This increase involved the simple gangliosides, GM3 and GD3, as well as complex mono- and disialogangliosides. Some complex monosialogangliosides were detected in all the colorectal and pancreatic carcinomas but neither in normal colon mucosa and pancreas nor in kidney and lung carcinomas.

Topics: Aged; Colon; Colonic Neoplasms; Female; G(M3) Ganglioside; Gangliosides; Humans; Intestinal Mucosa; Kidney Neoplasms; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Pancreatic Neoplasms; Rectal Neoplasms