g(m3)-ganglioside and Adenocarcinoma

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

To determine the effect of exogenous GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells.. A549 cells were treated with GM3 at different concentrations for 48 hours. MTT assay was used to detect the cell proliferation and flow cytometry was applied to analyze cell apoptosis. RT-PCR was used to detect the expression level of VEGF mRNA and confocal laser scanning microscopy was applied to observe the localization and fluorescence intensity of VEGF.. Comparing with the control, being treated with higher than 10 µmol/L GM3 significantly inhibited A549 cell proliferation (P < 0.05), and the suppressive effect could be enhanced following increasing doses. The IC(50) was 412 µmol/L. Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly promoted the apoptotic rate of A549 cells (P < 0.05). Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly decreased the VEGF mRNA level of A549 cells (P < 0.05), and the fluorescence intensity of VEGF distinctly weakened.. Exogenous ganglioside GM3 can inhibit the proliferation, promote apoptosis, and down-regulate the VEGF expression level in A549 cells. This may be considered as two mechanisms of GM3 for its anti-tumor effect by modulating cell apoptosis and angiogenesis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; G(M3) Ganglioside; Humans; Inhibitory Concentration 50; Lung Neoplasms; RNA, Messenger; Vascular Endothelial Growth Factor A

Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal, Humanized; Antigens, Neoplasm; Breast Neoplasms; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Female; G(M3) Ganglioside; Humans; Hybridomas; Immunotherapy; Mice; Mixed Function Oxygenases; Molecular Targeted Therapy; Mutagenesis, Site-Directed; Protein Engineering; RNA, Small Interfering

Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT-I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT-I mRNA as well as GM3 in 40 tumor tissues surgically removed from non-small cell lung cancer patients. Adenocarcinoma tissues expressed SAT-I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT-I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT-I mRNA and epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR-TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC(50) values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R(2) = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR-TK inhibitors, the SAT-I gene was introduced stably into a GM3-negative clone of murine 3LL lung cancer cells to produce GM3-reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3-reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT-I mRNA in lung biopsy samples from non-small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR-TK inhibitors.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; ErbB Receptors; G(M3) Ganglioside; Gefitinib; Humans; Immunoblotting; Immunoprecipitation; Lung Neoplasms; Mice; Middle Aged; Protein Kinase Inhibitors; Quinazolines; RNA, Messenger; Sialyltransferases; Tumor Cells, Cultured

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

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

Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

Topics: Adenocarcinoma; Benzylidene Compounds; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Membrane; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; G(M3) Ganglioside; Humans; Lung Neoplasms; Nitriles; Phosphorylation; Protein Tyrosine Phosphatases; Quinazolines; Radioligand Assay; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

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 patterns of gangliosides and phospholipids and their relation to TSH response were examined in twenty-six malignant thyroid tumors (4 follicular, 6 papillary, 5 medullary, 11 anaplastic carcinomas) and thirty-six hyperplastic goiters. Thirteen thyroid tissues adjacent to benign tumors with no evidence of macroscopic or microscopic abnormalities were used as normal tissue. In normal thyroids the major ganglioside was GD3 (44%) and GM3 was the second ganglioside (20%). In minor amounts GD1a (8.6%), GD1b (6.2%), GT1b (5.7%) and GM1 (5.6%) were present. In hyperplastic goiters and in follicular carcinomas the patterns of gangliosides were similar to that of normal tissue except for GM3 which, in the last tissue, was higher (34%). In papillary carcinomas low levels of GM3 (11%) and GT1b (0.8%) with a high level of GD1b (12.6%) were found. In anaplastic carcinomas GM3 was very high (47%) whereas GD3 was low (18%). In these tumors also a high percentage (14.0%) of GD1a was found. In medullary carcinomas the lowest levels of GM3 (4%) with the highest level of GD3 (64%) were found. Although large differences of the gangliosides distribution were clearly encountered in the various pathological human thyroid, no correlation between lack of TSH response and some individual ganglioside could be made. No differences in the individual phospholipid in the various tissues studied were found.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Papillary; G(M3) Ganglioside; Gangliosides; Humans; Phospholipids; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

Human lung carcinoma tissues with histological types of adenocarcinoma, squamous cell and small cell undifferentiated carcinomas were investigated for glycolipids. Carcinoma tissues, as well as normal adult and embryonic lungs contained ceramide mono-, di- and trihexosides, globoside and hematoside as major glycolipids. In addition to them, sulfatide which was identified as ceramide 3-sulfate-galactoside, was isolated in much lesser amount. The content of sulfatide was markedly increased in adenocarcinoma than that in other carcinomas and normal lung. Adenocarcinoma was also characterized by significantly lower level of total glycolipids which was largely due to the diminished contents of ceramide mono- and dihexosides, and hematoside, as compared to those in other two carcinomas. Squamous cell carcinoma had a characteristic pattern with an increment of hematoside. In small cell undifferentiated carcinoma, glycolipid contents were similar with those in squamous cell carcinoma but the relative composition of major glycolipids was markedly differed from that in other types. All the types of carcinoma examined showed marked increase of ceramide mono- and dihexosides (except for ceramide dihexoside in adenocarcinoma) compared to those in normal adult lung. Overall feature of glycolipids in embryonic lung appeared to be an intermediate between carcinomas and normal adult lung.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Female; G(M3) Ganglioside; Globosides; Glycosphingolipids; Humans; Lung; Lung Neoplasms; Pregnancy; Sulfoglycosphingolipids; Trihexosylceramides