g(m3)-ganglioside and Urinary-Bladder-Neoplasms

To investigate the therapeutic effects of exogenous gangliosides GM3 on human bladder cancer cell lines and the severe combined immunodeficiency mouse model of orthotopic bladder cancer.. Human bladder cancer cell lines YTS-1, T24, 5637, and KK47 were used in the study. In vitro cytotoxicity of GM3 was assessed using the cell counting kit-8. Cell adhesion was determined using a spreading assay. Phosphorylation of epidermal growth factor receptor was determined by Western blotting. In vivo, the orthotopic bladder cancer model was established using severe combined immunodeficiency mice and GM3 was administered intravesically by way of a transurethral catheter.. GM3 inhibited the proliferation of all the bladder cancer cell lines tested. The addition of GM3 decreased cell adhesion and epidermal growth factor-dependent phosphorylation of epidermal growth factor receptor. Direct instillation of GM3 into the bladder of the orthotopic model significantly inhibited tumor growth.. Our results suggest exogenous GM3 as a potential therapeutic agent for treating bladder cancer.

Topics: Administration, Intravesical; Animals; Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; ErbB Receptors; Female; G(M3) Ganglioside; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Urinary Bladder Neoplasms

Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through "cross-talk" of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway.

Topics: Animals; Cell Line; Cell Movement; CHO Cells; Cricetinae; Cricetulus; Dimerization; G(M2) Ganglioside; G(M3) Ganglioside; Kangai-1 Protein; Nanospheres; Proto-Oncogene Proteins c-met; Signal Transduction; Silicon Dioxide; Spectrometry, Mass, Electrospray Ionization; Urinary Bladder; Urinary Bladder Neoplasms

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

Topics: Antigens, CD; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Chromatography, Thin Layer; CSK Tyrosine-Protein Kinase; Electrophoresis, Polyacrylamide Gel; G(M3) Ganglioside; Gangliosides; Humans; Integrin alpha3; Integrin alpha3beta1; Integrins; Kangai-1 Protein; Ligands; Membrane Glycoproteins; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Phosphotransferases; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; RNA Interference; src-Family Kinases; Synapses; Tetraspanin 29; Urinary Bladder Neoplasms

We demonstrated previously (S. Kawamura et al., Int. J. Cancer, 94: 343-347, 2001) that large amounts of ganglioside G(M3) accumulate in superficial bladder tumor, compared with invasive bladder tumors and that exogenous G(M3) inhibits the invasive potential of bladder tumor cells. To apply the G(M3) overexpression system to bladder tumor therapy, direct evidence for the important role of G(M3) in bladder tumor invasion must be obtained through transfer of the gene responsible for G(M3) overexpression. To determine the most appropriate cancer cell line for elucidating the antitumor effect of ganglioside G(M3) overexpression, the present study examined glycolipid composition, enzyme activity, and mRNA expression of the glycosyltransferases responsible for G(M3) synthesis in the bladder tumor cell lines KK-47, J82, MGH-UI, YTS-1, and MBT-2. A murine bladder carcinoma cell line (MBT-2) was transfected with a G(M3) synthase [(lactosylceramide alpha2,3-N-acetyl sialic acid transferase); sialyltransferase-I; SAT-I] cDNA, because this line does not naturally express G(M3). Stable transfectants (MBT-2-SAT-I) that overexpressed G(M3) were characterized by a reduced potential for cell proliferation, motility, invasion, and xenograft tumor growth, and an increase in the number of apoptotic cells. In the proportion of synthetic S phase, cells did not differ between MBT-2-SAT-I and mock-transfectant cells. These results suggest that the decreased proliferative potential related to G(M3) overexpression was attributable to the increased number of apoptotic cells. Although details of the mechanism of apoptosis remain unclear, the overexpression of G(M3) by gene transfer of SAT-I may present a novel therapeutic modality.

Topics: Animals; Apoptosis; Blotting, Northern; Carcinoma, Transitional Cell; Chromatography, Thin Layer; G(M3) Ganglioside; Glycosyltransferases; Humans; Mice; S Phase; Sialyltransferases; Transfection; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We reported previously that non-invasive bladder cancer expresses high level of GM3 ganglioside, whereas invasive tumors have low levels. Since glycosphingolipid synthesis in Golgi is modified greatly by a macrocyclic lactone isolated from fungi, brefeldin A (BFA), we studied effects of BFA on expression of glycosphingolipids and on invasiveness of bladder cancer cell lines. Only GM3 synthesis in invasive tumors was greatly enhanced upon treatment with BFA; synthesis of other glycosphingolipids with lacto-series type 2 or globo-series structure in both invasive and non-invasive tumors was not changed. Invasiveness of bladder cancer cells was greatly decreased in association with the great increase of GM3 synthesis induced by BFA treatment. Level of sialyl-Lex expressed in invasive cell line YTS1, which provides the adhesive property of the cells to E-selectin, was unchanged upon BFA treatment. All the bladder cancer cell lines, regardless of invasiveness, highly express tetraspanin CD9. GM3 has been implicated as a co-factor of CD9 in control of tumor cell motility. Down-regulation of CD9 is associated with metastatic properties of tumor cells and survival of patients with colonic cancer. Therefore, enhanced synthesis of GM3 induced by BFA, causing decrease of invasiveness in bladder cancer, is ascribable to the capability of GM3 to interconnect integrin with CD9, in analogy to colonic cancer and perhaps many other types of cancer.

Topics: Antibodies, Monoclonal; Antifungal Agents; Brefeldin A; Cell Adhesion; Cell Division; Chromatography, Thin Layer; E-Selectin; Flow Cytometry; G(M3) Ganglioside; Glycosphingolipids; Humans; Interleukins; Neoplasm Invasiveness; Oligosaccharides; Sialyl Lewis X Antigen; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Glycolipids were extracted from primary bladder tumors of 14 patients and 2 normal counterparts. Their expression pattern was assessed by thin-layer chromatography (TLC). The most remarkable change was massive accumulation of GM3 in superficial bladder tumors compared with invasive tumors. This change was also confirmed by immunohistochemistry using anti-GM3 monoclonal antibody. The activities of glycosyltransferases responsible for GM3 synthesis (GM3 synthase, Gb3 synthase and GD3 synthase) were consistent with upregulated expression of GM3 in superficial tumors. It was suggested that the marked GM3 accumulation in superficial tumors was caused not only by upregulated GM3 synthase but also by downregulated activities of Gb3 and GD3 synthase. Histopathologic examination revealed an inverse correlation of the amount of GM3 expressed with invasive potential. Exogenously supplemented GM3 suppressed invasion potential in human bladder tumor cell lines (T-24, KK-47). These results indicate that the amount of GM3 expressed may serve as an indicator of the invasion potential of bladder tumor. Furthermore, new antiinvasion therapeutics may be possible by administration of GM3.

Topics: Aged; Antibodies, Monoclonal; Cell Division; Cell Movement; Chromatography, Thin Layer; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Female; G(M3) Ganglioside; Glycolipids; Glycosyltransferases; Humans; Immunohistochemistry; Kinetics; Male; Middle Aged; Models, Biological; Mucous Membrane; Neoplasm Invasiveness; Tumor Cells, Cultured; Up-Regulation; Urinary Bladder Neoplasms