ganglioside--gd2 and Lung-Neoplasms

Based on the remodeling of glycosphingolipids on the human tumor cell lines with manipulation of glycosyltransferase genes, roles of sugar moieties in tumor-associated carbohydrate antigens have been analyzed. Two main topics, that is, the roles of ganglioside GD3 in human malignant melanomas and those of GD2 in small cell lung cancer (SCLC) were reported. GD3 enhances tyrosine phosphorylation of two adaptor molecules, p130Cas and paxillin, resulting in the increased cell growth and invasion in melanoma cells. GD2 also enhances the proliferation and invasion of SCLC cells. GD2 also mediates apoptosis with anti-GD2 monoclonal antibodies (mAbs) via dephosphorylation of the focal adhesion kinase. These approaches have promoted further understanding of mechanisms by which gangliosides modulate malignant properties of human cancer, and the results obtained here propose novel targets for cancer therapy.

Topics: Anoikis; Antibodies, Monoclonal; Carcinoma, Small Cell; Cell Line, Tumor; Cell Proliferation; Crk-Associated Substrate Protein; Focal Adhesion Protein-Tyrosine Kinases; Gangliosides; Glycosphingolipids; Humans; Lung Neoplasms; Melanoma; Neoplasm Invasiveness; Paxillin; Phosphorylation; Tyrosine

The present study evaluated the ability of the anti-GD2 ganglioside monoclonal antibody 3F8 to target tumor sites in patients with small-cell lung cancer (SCLC). Of 12 patients entered into the trial, ten received intravenous 3F8 labeled with 2 or 10 mCi iodine-131. The first five patients had recurrent or progressive disease after chemotherapy. Subsequent patients were studied before starting chemotherapy. Radionuclide scans were performed on days 1, 2, and 3 post-infusion and once between day 5 and day 7. Four patients underwent single-photon emission tomography (SPET) imaging. Radionuclide scans demonstrated localization to all known sites of disease, other than small brain metastases in one patient. SPET/CT scan fusion images confirmed precise localization. No significant toxicity was observed. Mean serum half-life was 64.2 h. Analysis of specimens from one patient who died of unrelated causes 6 days post-infusion confirmed the scan results. The present study demonstrates that 3F8 targets SCLC sites in patients. Further studies of anti-GD2 antibodies with higher doses of antibody and radionuclide are warranted to evaluate their role in SCLC.

Topics: Carcinoma, Small Cell; Female; Gangliosides; Humans; Iodine Radioisotopes; Lung Neoplasms; Male; Middle Aged; Pilot Projects; Radioimmunodetection; Tomography, Emission-Computed, Single-Photon

The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Bone Neoplasms; Cell Proliferation; Gangliosides; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Recurrence, Local; Osteosarcoma; Positron-Emission Tomography; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Gangliosides GD3 and GD2 are specifically expressed in neuro-ectoderm-derived tumors, and are considered to play roles in the malignant properties of those cells. We analysed effects of small interfering (si) RNAs against GD3 synthase gene on the expression of ganglioside GD2 and biological phenotypes of human lung cancer cells expressing GD2. An siRNA could suppress the mRNA level of GD3 synthase gene even by single transfection, whereas repeated transfection was required to suppress GD2 expression on the cell surface. Significant reduction in the cell growth and invasion activity was observed in both lung cancer cell lines examined, when repeatedly transfected with the siRNA twice a week. DNA ladder formation was observed after third transfection, indicating the potent induction of apoptosis. Stable transfection of an RNAi expression vector with H1 RNA promoter was also examined. Transfectant cells with the RNAi expression vector showed almost equivalent suppression of GD2 expression and tumor properties in vitro. Furthermore, the stable transfectant cells showed slower cell growth than the control cells in severe combined immunodeficiency mice. These results suggested that siRNAs and/or RNAi expression vectors to generate siRNAs are promising approach to overcome human lung cancers.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Gangliosides; Gene Expression; Gene Expression Regulation, Neoplastic; Genetic Therapy; Genetic Vectors; Humans; Lung Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sialyltransferases; Transfection

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD(2) [aGD(2)-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD(2)-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

Topics: Animals; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Doxorubicin; Female; Gangliosides; GPI-Linked Proteins; Humans; Lung Neoplasms; Mice; Mice, Nude; Mice, SCID; Myelin Proteins; Neovascularization, Pathologic; Neuroblastoma; Nogo Receptor 1; Ovarian Neoplasms; Peptide Fragments; Receptors, Cell Surface; Tissue Distribution; Xenograft Model Antitumor Assays

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.

Topics: Anoikis; Antibodies, Monoclonal; Apoptosis; Carcinoma, Small Cell; Cell Line, Tumor; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gangliosides; Humans; Integrins; Lung Neoplasms; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein-Tyrosine Kinases

Ganglioside functions in tumor metastasis were analyzed by carbohydrate remodeling of a mouse Lewis lung cancer (subline P29) by introducing beta1,4GalNAc-T cDNA. Although P29 was originally a low-metastatic subline in the s.c. injection system, it showed high potential in lung metastasis when i.v.-injected via the tail vein. Two lines of GM(2)(+) transfectants showed markedly reduced metastatic potential to the lung compared to 2 control lines. However, cell proliferation rates and expression levels of various cell adhesion molecules, e.g., integrin family members, SLe(x) and CD44, were essentially unchanged after transfection of the cDNA. Then, cell adhesion to fibronectin-coated dishes was examined, showing that GM(2) (+) transfectants attached to the plates much more slowly than controls, suggesting functional modulation of integrins with newly expressed GM(2). Phosphorylation of the FAK located at downstream of integrin molecules was markedly reduced in GM(2)(+) transfectants, suggesting that GM(2) suppressed cell adhesion signals via fibronectin-integrin interaction.

Topics: Animals; Carcinoma, Lewis Lung; Cell Membrane; DNA, Complementary; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; G(M2) Ganglioside; Gangliosides; Integrins; Lung Neoplasms; Mice; Mice, Inbred C57BL; N-Acetylgalactosaminyltransferases; Neoplasm Transplantation; Phosphorylation; Protein-Tyrosine Kinases; Skin Neoplasms; Transfection; Tumor Cells, Cultured; Tyrosine; Vitronectin

Small cell lung cancer (SCLC) cell lines specifically express ganglioside GD2, and anti-GD2 monoclonal antibodies (mAbs) caused suppression of cell growth and induced apoptosis of SCLC cells with single use. Here, enhancement of the cytotoxic effects of various anti-cancer drugs with an anti-GD2 mAb was demonstrated. The cytotoxicity of all six drugs examined was markedly enhanced, i.e. 2.4 - 7.8-fold increase of cell sensitivity in terms of IC(50). In particular, the combination of cisplatin (CDDP) with an anti-GD2 mAb resulted in prominent enhancement of cytotoxicity even in low - moderate GD2-expressing lines. The anti-GD2 mAb induced weak activation of c-Jun terminal kinase (JNK) in SCLC cells, and all anti-cancer drugs also induced its activation to various degrees. When CDDP and an anti-GD2 mAb were used together, significantly stronger JNK activation was observed corresponding to the cytotoxic effects, suggesting that synergistic phosphorylation of JNK with two reagents induced prominent apoptosis. The essential role of JNK in the induction of SCLC apoptosis with CDDP and anti-GD2 mAb was confirmed by experiments with a JNK inhibitor, curcumin. These results suggest that anti-GD2 mAbs would be very efficient in combination with anti-cancer drugs, both to achieve SCLC-specific cytotoxicity and to enhance its magnitude.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Small Cell; Cisplatin; Coloring Agents; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Flow Cytometry; Gangliosides; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Mitogen-Activated Protein Kinases; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Cells, Cultured

Expression levels of gangliosides and glycosyltransferase genes responsible for their syntheses in human lung cancer cell lines and a normal bronchial epithelial cell line were analyzed. Both non-small cell lung cancers and small cell lung cancers (SCLCs) mainly expressed G(M2) and G(M1), whereas only SCLCs expressed b-series gangliosides, such as G(D2), G(D1b), and G(T1b). Accordingly, many SCLC cell lines showed up-regulation of the G(D3) synthase gene. Consequently, we introduced G(D3) synthase cDNA into a SCLC line with low expression of b-series gangliosides and analyzed the effects of newly expressed gangliosides on tumor phenotypes. The transfectant cells expressing high levels of G(D2) and G(D3) exhibited markedly increased growth rates and strongly enhanced invasion activities. Addition of anti-G(D2) monoclonal antibodies into the culture medium of these cells resulted in the marked growth suppression of G(D2)-expressing cell lines with reduced activation levels of mitogen-activated protein kinases but not of nonexpressants, suggesting that G(D2) plays important roles in cell proliferation. Moreover, G(D2)-expressing cells treated with anti-G(D2) antibodies showed features of apoptotic cell death at 30 min after addition of antibodies, i.e., shrinkage of cytoplasm, binding of Annexin V, and staining with propidium iodide, followed by DNA fragmentation. This G(D2)-mediated apoptosis was associated with caspase-3 activation and partly inhibited by a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. The finding that anti-G(D2) antibodies suppressed the cell growth and induced apoptosis of SCLC cells strongly suggested the usefulness of G(D2) as a target for the therapy of disastrous cancer, although the precise mechanisms for apoptosis remain to be clarified.

Topics: Antibodies, Monoclonal; Apoptosis; Carbohydrate Sequence; Carcinoma, Small Cell; Cell Division; DNA, Complementary; Flow Cytometry; G(M1) Ganglioside; G(M2) Ganglioside; Gangliosides; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Molecular Sequence Data; N-Acetylgalactosaminyltransferases; Phenotype; Sialyltransferases; Transfection; Up-Regulation

Several gangliosides such as GM2, GD2, and GD3 have been thought of as target molecules for active or passive immunotherapy of human cancers because of their dominant expression on the tumor cell surface, especially in tumors of neuroectodermal origin. We established a number of mouse or rat monoclonal antibodies (mAbs) to a series of gangliosides to investigate the nature of the molecules on the cell surface. Some of those mAbs were converted to chimeric or humanized mAbs with the aim of developing immunotherapy for human cancer. It is desirable for mAbs to remain on the cell surface for a long time so that they can exert effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). We found that mAbs to GM2, GD2, and GD3 remain on the cell surface for > or =60 min after binding, while mAbs to other types of carbohydrate such as sialy Le(a) are quickly internalized. A chimeric mAb to GD3, KM871, was generated by linking cDNA sequences encoding light- and heavy-chain variable regions of mouse mAb KM641 with cDNAs encoding the constant region of human immunoglobulin gamma1 (IgG-1). KM871 bound to a variety of tumor cell lines, especially melanoma cells, including some cell lines to which R24 failed to bind. In a preclinical study, intravenous injection of KM871 markedly suppressed tumor growth and radiolabeled KM871 efficiently targeted the tumor site in a nude mouse model. This chimeric mAb is being evaluated in a phase I clinical trial in melanoma patients. The chimeric mAb KM966 and humanized mAb KM8969 to GM2 originated from a mouse IgM mAb. When human serum and human peripheral blood mononuclear cells were used as effectors in CDC and ADCC, respectively, KM966 and KM8969 killed GM2-expressing tumor cells effectively. In addition, these mAbs may induce apoptosis of a small cell lung cancer cell line cultured under conditions mimicking physiological tumor conditions.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; G(M2) Ganglioside; Gangliosides; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Recombinant Fusion Proteins; Recombinant Proteins; Tumor Cells, Cultured

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Topics: Animals; Antibodies; Base Sequence; Cell Line; DNA Primers; ErbB Receptors; Gangliosides; Humans; Immunotherapy; Immunotoxins; Interleukin-2; Killer Cells, Lymphokine-Activated; Liver Neoplasms; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mice, SCID; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Fusion Proteins; Skin Neoplasms; Survival Rate; Time Factors; Transplantation, Heterologous

The relationship between the cellular internalization of an anti-ganglioside GD2 monoclonal antibody (14.G2a) and the toxic effect of its ricin A-chain immunotoxin (14.G2a-RA) was examined on GD2-bearing M21 human melanoma and T293 small cell lung carcinoma cell lines. The capacity for ligand uptake was determined by examining the parameters that contribute to this constant, including the number of cell-surface binding sites and the internalization rate constant (ke). The maximum uptake of 14.G2a is 11-fold greater for M21 than for T293 cells, due to a 2.7-fold difference in binding sites and a 4-fold difference in the rate of antibody internalization. The capacity for ligand uptake correlates with the cytotoxic activity of the 14.G2a-RA immunotoxin against these two cell lines. Furthermore, we were able to demonstrate that the consequence of internalization of 14.G2a-RA is the intracellular release of undegraded ricin A-chain from the antibody. These studies indicate that the rate of internalization is a quantitative parameter that plays a key role in predicting the cytotoxic potency of this immunotoxin.

Topics: Antibodies, Monoclonal; Biological Transport; Carcinoma, Small Cell; Cell Survival; DNA Replication; Gangliosides; Humans; Immunotoxins; Kinetics; Lung Neoplasms; Melanoma; Ricin; Tumor Cells, Cultured

A human melanoma variant cell line was obtained from a lung metastasis that arose spontaneously after we inoculated melanoma cells sc into a nude mouse. In this model, IgG2a monoclonal antibody (MAb) ME 36.1 defining the GD2/GD3 gangliosides inhibited melanoma growth at the primary site and metastatic spread of the cells, whereas an IgG1 variant of MAb ME 36.1 inhibited lung metastasis formation only. Possible mechanisms of antitumor effects of MAb ME 36.1 are discussed.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Female; Gangliosides; Humans; Lung Neoplasms; Lymphatic Metastasis; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Transplantation, Heterologous

Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigens, Surface; Cell Adhesion; Cell Line; Extracellular Matrix; Fibronectins; Gangliosides; Humans; Laminin; Lung Neoplasms; Melanoma; Neuroblastoma; Periodic Acid