Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM₁, GM₃, GD_1A and GT_1B

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.

Modulation of Growth Factor Signaling by Gangliosides: Positive Or Negative?

“…We have demonstrated an enhancement of signaling through the EGFR by the ganglioside GD1a, which stands in apparent contrast to previously published studies that demonstrate an overall inhibitory effect of gangliosides on signaling through EGFR (Alves et al, 2002;Bremer et al, 1986;Meuillet et al, 2000;Mirkin et al, 2002;Rebbaa et al, 1996;Sottocornola et al, 2003;Suarez Pestana et al, 1997) and other growth factor receptors (Farooqui et al, 1999;Hynds et al, 1995). Although it is certainly true that different gangliosides may have differing effects on signaling through the same receptor, we believe that the observed differences may be attributable in part to critical differences in methodological approaches.…”

“…This diversity has, not surprisingly, led to equally wide-ranging results, including reports delineating opposing effects of gangliosides on the same receptor-ligand pair. Inhibitory effects include inhibition of EGFR phosphorylation by GM3 (Alves et al, 2002;Bremer et al, 1986;Mirkin et al, 2002;Rebbaa et al, 1996;Suarez Pestana et al, 1997) and inhibition of PDGFR phosphorylation by GM1, GM2, GD1a, and GT1b (Farooqui et al, 1999;Hynds et al, 1995). In contrast, enhancement of growth factor receptor phosphorylation has been demonstrated for GD1a and GM1 in a number of systems including EGFR Liu et al, 2004), and FGF (Rusnati et al, 2002).…”

“…In addition, the serum content of the cell culture medium and use of cell washing to remove unbound ganglioside varied among the 11 studies, with 4 studies ,Liu et al, 2004Suarez Pestana et al, 1997)using low serum concentrations (≤2%). In eight of the studies (Alves et al, 2002;Bremer et al, 1986;Meuillet et al, 2000;Mirkin et al, 2002;Rebbaa et al, 1996;Sottocornola et al, 2003;Suarez Pestana et al, 1997) gangliosides inhibited signaling through the EGF receptor, in three studies (Bremer et al, 1986;Mirkin et al, 2002;Sottocornola et al, 2003) no ganglioside effect could be demonstrated, and in four studies Liu et al, 2004; signaling was enhanced. Although the cell lines and the gangliosides used in these studies varied, a review of the methods used reveals striking differences in the experimental conditions used.…”

“…The membrane content of tumor cells is generally much higher, and active shedding of gangliosides results in their cell-to-cell transfer followed by binding to the cell membrane. Third, gangliosides of differing carbohydrate structures are known to have differential effects on a number of cellular functions including growth factor signaling Mirkin et al, 2002). Two other characteristics of gangliosides are important when considering in vitro systems using their exogenous addition.…”

The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer

“…Thus, it is well established that G M3 inhibits cell motility and invasiveness in bladder tumors (8). Gangliosides Gt 1b , GD 1A , G M3 , and G M1 inhibit cell proliferation and epidermal growth factor receptor tyrosine phosphorylation (9,10), whereas depletion of Gt 1b and G M3 by sialidase overexpression facilitates epidermal growth factor receptor phosphorylation and cell migration (10). Oppositely, a different GSL, G b5 , strongly enhances motility of breast cancer cells (11).…”