g(m3)-ganglioside and Lung-Neoplasms

Racotumomab (originally known as 1E10 mAb) is an anti-idiotype murine IgG1 directed to membrane glycoconjugates expressed in aggressive solid tumors. It was developed as a mirror image of the idiotype of another antibody against N-glycolyl-containing molecules, such as the NeuGcGM3 ganglioside. After a successful phase II/III study, racotumomab formulated in alum was conditionally approved in Latin American countries as maintenance therapy for advanced non-small cell lung cancer.. This review analyzes the biology of the target antigen, summarizes preclinical studies and discusses clinical trials in adults and the pediatric experience with racotumomab.. Proper patient selection and combination with chemotherapy, radiotherapy or checkpoint inhibitors appear to be critical issues to maximize the effects of racotumomab vaccination in lung cancer. In a recent phase I clinical trial in children with relapsed or resistant neuroectodermal malignancies, racotumomab was well tolerated and immunogenic, and its evaluation as immunotherapy for high-risk neuroblastoma is warranted.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Child; G(M3) Ganglioside; Humans; Lung Neoplasms

Racotumomab is a murine gamma-type anti-idiotype monoclonal antibody that specifically induces an antibody response against Neu-glycolyl GM3 ganglioside (NeuGcGM3), which is overexpressed in several solid tumors. It is adjuvanted with aluminum hydroxide for intradermal administration as a cancer vaccine (racotumomab-Alum, known commercially as Vaxira®). Racotumomab is currently being evaluated for a number of cancer indications, including melanoma, breast and lung cancer. In early clinical trials, racotumomab demonstrated high immunogenicity and low toxicity and it advanced to further clinical testing as a treatment for patients with non-small cell lung cancer (NSCLC). On the basis of promising results in a phase II/III study, racotumomab was launched in 2013 in Cuba and Argentina as an intradermal injection for the treatment of patients with advanced stage NSCLC.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; G(M3) Ganglioside; Humans; Lung Neoplasms; Neoplasms

Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. This clinical trial was conducted to provide a preliminary estimate of efficacy and safety of racotumomab as switch maintenance for patients with advanced non-small cell lung cancer (NSCLC).. Patients with stage IIIb/IV NSCLC who have at least stable disease after first-line chemotherapy were randomized 1:1 to racotumomab-alum (5 immunizations every 2 weeks and re-immunizations every 4 weeks) or placebo. Treatment was administered beyond progressive disease, until severe performance status worsening or toxicity. At progression, only five patients per group received further anticancer therapy. The primary endpoint was overall survival (OS).. One-hundred and seventy-six patients were randomized to racotumomab-alum (n = 87) and placebo (n = 89). Median OS was 8.23 and 6.80 months, respectively [HR, 0.63; 95% confidence interval (CI), 0.46-0.87; P = 0.004]. Median progression-free survival (PFS) in vaccinated patients was 5.33 versus 3.90 months for placebo (HR, 0.73; 95% CI 0.53-0.99; P = 0.039). The most common adverse events in the racotumomab-alum arm were burning and pain at the injection site, bone pain, and asthenia. A high antibody response of IgM and IgG isotype against the NeuGcGM3 ganglioside was obtained. Hyperimmune sera were able to specifically recognize and kill the NeuGcGM3-expressing L1210 cell line. Patients who developed anti-NeuGcGM3 antibodies capable to bind and kill ≥30% L1210 cells showed longer median survival times.. Switch maintenance with racotumomab-alum is an effective and a well-tolerated treatment option for patients with advanced NSCLC.

Topics: Adjuvants, Immunologic; Alum Compounds; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Double-Blind Method; Female; G(M3) Ganglioside; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Maintenance Chemotherapy; Male; Middle Aged; Neoplasm Staging; Placebos; Proportional Hazards Models; Treatment Outcome

1E10 is an anti-idiotype murine monoclonal antibody (Ab2 MAb) specific to an Ab1 MAb which reacts with NeuGc-containing gangliosides, sulfatides and with antigens expressed in some human tumors. Preparations containing this Ab2 were capable to induce a strong anti-metastatic effect in tumor-bearing mice. We conducted a Phase I clinical trial to evaluate the toxicity and humoral immune response elicited by 1E10 vaccine in patients with small cell lung cancer (SCLC). Eligible patients were those who after received chemotherapy and/or radiotherapy had partial or complete response to treatment. Patients received four biweekly injections with 2 mg of aluminum hydroxide-precipitated 1E10 MAb, then other six doses at 28-day intervals, and later the patients who maintained a good performance status were reimmunized. Six patients with limited-stage disease and three with extensive-stage disease were enrolled in the study. Most of the patients who received at least four doses of 1E10 vaccine developed strong specific antibody responses against 1E10 MAb and NeuGc-GM3 ganglioside. Antibodies able to react with lung carcinoma tissue sections were detected in sera from vaccinated patients. A prolonged survival was observed in several patients treated with the anti-idiotype vaccine. No evidence of serious adverse effects was found.

Topics: Aged; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Binding Sites, Antibody; Cancer Vaccines; Carcinoma, Small Cell; Female; G(M3) Ganglioside; Humans; Immunohistochemistry; Immunotherapy, Active; Lung Neoplasms; Male; Mice; Middle Aged; Treatment Outcome

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody-Dependent Cell Cytotoxicity; Apoptosis; Carcinoma, Non-Small-Cell Lung; G(M3) Ganglioside; Humans; Immunotherapy, Active; Lung Neoplasms; Tumor Cells, Cultured

Gangliosides are glycosphingolipids found on the cell surface. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. N-glycolylneuraminic acid (NeuGc)-containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal human tissues. Our aim was to evaluate the presence of NeuGc-containing gangliosides including GM3 (NeuGc) and assess their relationship with the prognosis of non-small-cell lung cancer (NSCLC). NeuGc-containing ganglioside expression in NSCLC tissues was analyzed immunohistochemically using the mouse monoclonal antibody GMR8, which is specific for gangliosides with NeuGc alpha 2,3Gal-terminal structures. On the basis of NeuGc-containing ganglioside expression, we performed survival analysis. We also investigated the differences in the effects of GM3 (N-acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc-containing gangliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc-containing ganglioside expression had a low overall survival rate and a significantly low progression-free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). In conclusion, NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc-containing ganglioside expression is associated with patient survival. The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc) on the inhibition of EGFR tyrosine kinase might contribute to improvement in the prognosis of NSCLC patients.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Female; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; N-Acetylneuraminic Acid; Neuraminic Acids; Phosphorylation; Prognosis; Survival Rate

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

Topics: Animals; Antibodies, Neoplasm; Cancer Vaccines; Carcinoma, Lewis Lung; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Dogs; G(M3) Ganglioside; Horses; Humans; Immunoglobulin G; Immunoglobulin Idiotypes; Immunoglobulin M; Leukemia L1210; Lung Neoplasms; Mice; Mice, Inbred BALB C; Plasmacytoma

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

The target concept means not only an aberrant expression of a particular molecule in tumour tissues but also evidence of a clear therapeutic advantage, as a consequence of immune-intervention, in an antigen-positive relevant tumour model. Since we reported the presence of NGcGM3 ganglioside in human breast tumours years ago and though Phase I clinical trials of a ganglioside containing vaccine have been conducted, a definitive direct validation of this peculiar molecule as target for cancer immunotherapy has remained unperformed.. Two animal models were used: leghorn chickens and C57BL/6 mice. The murine 3LL-D122 cell line, the derived subcutaneous tumours and metastatic lung lesions were processed for gangliosides identification. Active immunotherapy experiments in the 3LL-D122 spontaneous lung metastasis model were performed with NGcGM3/VSSP vaccine prepared by conjugation of NGcGM3 with the outer membrane proteins of Neisseria meningitides.. The 3LL-D122 Lewis lung carcinoma results were consistent with an increased expression of NGcGM3 from primary tumours to metastatic lesions, as observed in human breast cancer samples. Both vaccines, prepared with synthetic or natural-source-derived ganglioside, showed similar anti-tumour and immunogenicity profiles. Finally, a clear involvement of NK1.1(+) cells and CD8(+) T cells in the anti-metastatic effect elicited by the vaccine was manifested.. While 'proof of concept' Phase II and III clinical trials with the NGcGM3/VSSP vaccine in cancer patients are currently ongoing these results reasonably sustain the validation of this peculiar ganglioside as a novel target for cancer immunotherapy.

Topics: Animals; Bacterial Outer Membrane Proteins; Cancer Vaccines; Carcinoma, Lewis Lung; Erythrocytes; Female; Flow Cytometry; G(M3) Ganglioside; Horses; Humans; Immunohistochemistry; Immunotherapy; Lung; Lung Neoplasms; Mass Spectrometry; Mice; Mice, Inbred C57BL; Neisseria meningitidis; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG(TL)) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG(TL) was confirmed in SDS-PAGE. IgG(TL) revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG(TL) showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG(TL), the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcalpha(2-->3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG(TL) could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG(TL). Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG(TL) antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG(TL) to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.

Topics: alpha-Fetoproteins; Animals; Apoptosis; Bronchoalveolar Lavage Fluid; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; G(M3) Ganglioside; Gangliosides; Humans; Immunoglobulin G; Lung Diseases, Interstitial; Lung Neoplasms; Maackia; Sarcoidosis

Tumor immune escape and angiogenesis contribute to tumor progression, and gangliosides and activation of signal transducer and activator of transcription (STAT)-3 are implicated in these processes. As both are considered as novel therapeutic targets, we assessed the possible association of ganglioside GM3 expression and STAT3 activation with suppression of dendritic cell (DC) activation and angiogenesis in non-small cell lung cancer (NSCLC).. Immunohistochemistry was performed on a tissue array to determine N-glycolyl GM3 (GM3) and phosphorylated STAT3 (pSTAT3) expression in 176 primary NSCLC resections. Median values of GM3 and pSTAT3 expression were used as cut off. Microvessel density (MVD) was determined by CD34 staining and morphology. CD1a and CD83 were used to determine infiltrating immature and mature dendritic cells, respectively.. 94% and 71% of the NSCLC samples expressed GM3 and nuclear pSTAT3, respectively. Median overall survival was 40.0 months. Both low GM3 expression and high pSTAT3 expression were associated with a worse survival, which reached near significance for GM3 (P = 0.08). Microvessel density (MVD), determined by CD34 staining and morphology, was lower in NSCLC samples with high GM3 expression. CD1a+ cells (immature DCs) were more frequent in NSCLC tissues as compared to peritumoral lung tissue, while CD83+ cells (mature DCs) were more frequent in peritumoral lung tissue. CD83+ DCs were less frequent in NSCLC tissues with high GM3 expression.. GM3 and pSTAT3 are widely expressed in NSCLC. Based on CD83 expression, GM3, but not pSTAT3, appeared to be involved in tumor-induced DC suppression. pSTAT3 expression was not associated with MVD, while GM3 might play an anti-angiogenic role.

Topics: Carcinoma, Non-Small-Cell Lung; Dendritic Cells; Female; G(M3) Ganglioside; Humans; Immunohistochemistry; Lung Neoplasms; Male; Microvessels; Middle Aged; Neovascularization, Pathologic; STAT3 Transcription Factor; Tissue Array Analysis

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Specificity; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Dogs; Dose-Response Relationship, Immunologic; G(M3) Ganglioside; Horses; Humans; Lung Neoplasms; Tumor Cells, Cultured

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

The migration of B16LuF1 cells, B16-melanoma cells of lower metastatic potential to lung was enhanced through artificial basement membrane in presence of gangliosides of B16LuF1 cells as well as gangliosides of B16-melanoma cells of higher metastatic potential to lung, namely, B16LuF5 and B16LuF10 cells. The same concentration (50 microM) of gangliosides of B16LuF1, B16LuF5 and B16LuF10 cells gradually increased the migration of B16LuF1 cells through basement membrane. Moreover, B16LuF10 cell gangliosides modified the migratory effect of laminin and fibronectin on B16LuF1 cells. Laminin alone increased migration of B16LuF1 cells whereas fibronectin alone decreased migration of the same cells. When B16LuF10 cell gangliosides were used in combination with fibronectin, gangliosides removed the migration inhibitory effect of fibronectin resulting in net enhancing effect. Gangliosides in association with laminin also increased the enhancing effect of laminin on migration of B16LuF1 cells. Thus, gangliosides showed additive enhancing effect when used in combination with laminin. However, effect of individual gangliosides were different. Out of six gangliosides isolated from B16LuF10 cells only two gangliosides corresponding to standard gangliosides GM2 and GM3 enhanced migration of B16LuF1 cells. The migration of B16LuF1 cells in presence of each of the remaining four gangliosides corresponding to GT1b, GD1b, GD1a and GM1 was not altered and was comparable to that of untreated control. Thus, gangliosides of B16 melanoma cells alone or in combination with laminin or fibronectin enhanced migration of B16 melanoma cells through artificial basement membrane, suggesting possible role of tumor gangliosides during invasion of metastatic tumor cells through basement membrane of the surrounding tissues in vivo.

Topics: Animals; Basement Membrane; Cell Line, Tumor; Cell Movement; Chromatography, Thin Layer; Densitometry; Fibronectins; G(M2) Ganglioside; G(M3) Ganglioside; Laminin; Lung Neoplasms; Melanoma, Experimental; Membranes, Artificial; Mice

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

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

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Topics: Animals; Antigens, CD; Cell Movement; Cytosol; DNA, Complementary; G(M3) Ganglioside; Genetic Vectors; Lactosylceramides; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Neuraminidase; Transfection

Differential cell- and immuno-biological properties of two murine melanoma B16 variants, B16-F1 and F10, were investigated. Studies focused on the expression of proto-oncogene c-fos, sensitivities to LAK cells and/or IL-2, and modulation of the expression of ganglioside components after treatment with IL-2. Proto-oncogene c-fos was found to be highly expressed in F10 lines by an in situ hybridization technique and also in F10 lung metastatic nests by immunofluorescent staining with anti-c-fos antibody. F1 melanomas were more sensitive to local injection of IL-2. F10 melanomas hardly responded to IL-2 treatment, but successive injections of a combination of LAK cells and IL-2 did cause prolongation of survival rates, even of F10 melanoma-burdened mice. A major component of gangliosides of both F1 and F10 melanomas was GM3. Production of GM3 in F10 melanomas treated with IL-2 for 4 days increased, and, if the treatment was continued for 7 days, minor components of gangliosides, such as GM2, GM1, and GD1a, appeared only in F1 melanomas, while the increase of production of GM3 disappeared in both melanomas. These experimental results may provide clues for additional mechanisms which allow these two murine melanoma variants to show different implantation and metastasis rates.

Topics: Animals; Female; Fluorescent Antibody Technique; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Gene Expression Regulation, Neoplastic; Genes, fos; In Situ Hybridization; Interleukin-2; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Survival Rate; Tumor Cells, Cultured

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

In view of the evidence that cell expression of gangliosides in several tumors is positively involved in the metastatic phenotype, Lewis lung carcinoma (3LL) cell line, expressing GM3 as the major ganglioside, was analysed for the cell surface expression of GM3. An indirect immunofluorescence assay, using a M2590 monoclonal antibody recognizing GM3, was used for this purpose. Since the parental 3LL cells consist of heterogenous subpopulations differing in the degrees of GM3 expression, we have developed clones of this cell line with different degrees of metastatic potentials by using an in vitro non-selective procedure in order to investigate whether the expression of GM3 is associated with metastatic potential. The degree of cell surface expression of GM3 among the clones correlated well with their total cellular content of this ganglioside. However, we were unable to confirm the report of increased level of GM3 in high metastatic 3LL clones, nor did a decreased level correlate with weak metastatic ability. In our recent work, an inhibitor of glucosylceramide synthase, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), was found to decrease the levels of all cellular glucosphingolipids and cause the accumulation of the precursors of glucosylceramide. The present study does not, however, rule out the possible involvement of this lipid family in metastatic dissemination, since treatment of 3LL cells with D-PDMP resulted in significant inhibition of their experimental metastatic potential. Clones expressing very low GM3 grew slowly in culture dishes, suggesting that GM3 may have a regulatory role in cell proliferation. The low metastatic clones expressed high levels of H-2Kb antigen, while the expression of the same antigen on the high metastatic clones was relatively low, confirming the previous observation of this tumor system. Moreover, a clone showing the lowest tumorigenic potency revealed both a high cell surface expression of H-2Kb and a high H-2Kb/H-2Db ratio.

Topics: Animals; Cell Division; Clone Cells; Female; Flow Cytometry; Fluorescent Antibody Technique; G(M3) Ganglioside; H-2 Antigens; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Tumor Cells, Cultured

The patterns of acidic and neutral glycosphingolipids (GSLs) were examined in a syngeneic tumour system in Balb/c mice consisting of closely related cell lines with different colonisation potentials directed to the murine lungs (in vivo selected highly metastatic sublines of L1-fibrosarcoma cells and their WGA-resistant mutants with low metastatic potential). GSLs were analysed by high-performance thin-layer chromatography and structurally identified by fast atom bombardment mass spectrometry combined with compositional analyses and exo-glycosidase digestion. The results suggest that highly metastatic sublines L1-LM and L1-LM12 derived by in vivo selection from mouse fibrosarcoma cells (cell line L1) exhibit a drastic increase of polar ganglioside expression and a restriction to globo-series GSLs. Contrasting with this the low metastatic mutant cells (L1-LM13WGA) express a reduced portion of acidic GSLs and exhibit a shift to less polar ganglioside components. Total cellular and plasma membrane-integrated GSLs were demonstrated to exhibit largely identical patterns. Concomitant with a significant decrease in LacCer expression a substantial reduction of GM2 and a complete lack of GM3 expression can be assigned to the highly metastatic sublines of L1-cells. On the other hand, the more polar gangliosides GM1a and, to an even greater extent, GD1a (exceeding 70% of total gangliosides) accumulate on L1-LM and their clonal sublines. The shift to acidic GSLs of higher polarity is less pronounced on the low metastatic WGA-resistant mutant cells (L1-LM13WGA) showing a preponderance of GM1a. The portion of GD1a within the fractions of acidic GSLs does not correspond to the cellular activities of CMP-NeuAc/GM1 (alpha 2-3) sialyltransferase measured for high and low metastatic cell variants. Total sialic acid content of the various cell lines differs, but is not associated with the metastatic potential. Gangliosides on L1-cells exhibit a significant substitution of N-glycolyl for N-acetylneuraminic acid (13%) compared to their metastatic sublines and to mutant cells (less than 1%). A conversion of surface exposed GD1a to GM1a on membranes of metastatic cells by in situ treatment with Vibrio cholerae sialidase is associated with a significant reduction of tumour cell colonisation directed to the murine lungs.

Topics: Animals; Autoradiography; Cell Line, Transformed; Chromatography, High Pressure Liquid; Fibrosarcoma; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Glycosphingolipids; Lung Neoplasms; Male; Membrane Lipids; Mice; Tumor Cells, Cultured

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

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