Mirkin B L, Clark S H, Zhang C
Departments of Pediatrics and Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60614, USA.
Cell Prolif. 2002 Apr;35(2):105-15. doi: 10.1046/j.1365-2184.2002.00228.x.
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.
在N - myc基因扩增的人神经母细胞瘤细胞系NBL - W中，测定了神经节苷脂GT1B、GD1A、GM3和GM1对细胞增殖及表皮生长因子受体（EGFR）磷酸化的抑制作用。通过将NBL - W细胞与GT1B、GD1A、GM3或GM1的递增浓度（5 - 1000微摩尔）孵育4天，从浓度 - 反应回归曲线估计每种神经节苷脂的IC50。使用四氮唑染料的比色法和分光光度分析法定量测定细胞增殖，通过蛋白质免疫印迹的光密度测定法测定EGFR磷酸化。除GM1外，所有检测的神经节苷脂均以浓度依赖性方式抑制NBL - W细胞增殖。神经节苷脂GT1B [分子量（MW）2129]、GM3（MW 1236）和GD1A（MW 1838）的IC50分别为（平均值±标准误）117±26、255±29和425±44微摩尔。相比之下，GM1（MW 1547）的IC50无法确定。用浓度范围为0.1至1000 ng/ml的表皮生长因子（EGF）孵育NBL - W细胞，细胞增殖速率逐渐增加，但在浓度高于10 ng/ml时达到平台期。然而，EGF浓度从1至100 ng/ml时，EGFR酪氨酸磷酸化呈递增刺激。每种神经节苷脂对EGF诱导的EGFR磷酸化的抑制作用不同，其各自的抑制效力如下：EGFR磷酸化[曲线下面积（+ EGF）/曲线下面积（ - EGF）]：对照（未添加神经节苷脂）= 8.2；GM1 = 8.3；GD1A = 6.7；GM3 = 4.87，GT1B = 4.09。该比值越低，神经节苷脂的抑制活性越强。具有末端N - 乙酰神经氨酸部分以及分别与内部半乳糖连接的一个和两个N - 乙酰神经氨酸残基的神经节苷脂GD1A和GT1B，均抑制细胞增殖和EGFR磷酸化。然而，GD1A是更有效的细胞增殖抑制剂，而GT1B对EGFR磷酸化最有效。仅具有末端N - 乙酰神经氨酸的GM3几乎等效地抑制细胞增殖和EGFR磷酸化。这些数据表明，神经节苷脂作为NBL - W神经母细胞瘤细胞增殖和EGFR酪氨酸磷酸化抑制剂的效力不同，并且膜糖鞘脂差异表达的扰动可能在调节神经母细胞瘤生长中起作用。
