title: Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B.
abstract: The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.
authors.affiliation: Departments of Pediatrics and Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60614, USA. b-mirkin@northwestern.edu
authors.forename: B L
authors.initials: BL
authors.lastname: Mirkin
authors.forename: S H
authors.initials: SH
authors.lastname: Clark
authors.forename: C
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004285:Dogs; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005679:G(M3) Ganglioside / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D005679:G(M3) Ganglioside; D005732:Gangliosides; C021298:ganglioside, GD1a; D005677:G(M1) Ganglioside; D014443:Tyrosine; C016481:trisialoganglioside GT1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors