| Growth factors and gangliosides are highly expressed in tumor cells, and secreted and shedded into the microenvironment of tumor cells respectively. There is a strong correlation between gangliosides shedding and tumor progression in several tumor systems. Furthermore, several experimental results have demonstrated the capacity of gangliosides to (1) suppress host immune responses, (2) promote tumor angiogenesis, and (3) induce tumor metastasis. EGF modulates epithelial cell growth by binding to EGFR, activating EGFR, transferring the growth signal from outside into the cell, and then modulating the gene expression and cell metabolism. Our previous work have also found that ganglioside GD1a enhanced EGF induced NHDF cell proliferation. Based on our previous work, in the present study techniques and approaches of cell culture, enzyme activity assay, binding assay of growth factor to the cell, SDS-PAGE, Western-blot, extraction and HPTLC purification of gangliosides, and cell membrane separation were employed to study the effects of ganglioside GD1a on EGF receptor and its down stream signaling, and its probable mechanism. To study the signal transduction of tumor angiogenesis, we focused on EGF and gangliosides induced signaling in normal human dermal fibroblasts. We first assessed EGF signaling pathway. When the fibroblasts were pre-incubated with ganglioside GD1a (NeuNAcα2-3Galβ1-3GalNAcβ1-4(NeuNAcα2-3) Galβ1-4Glcβ1-1Cer), EGF induced autophosphorylation and receptor tyrosine kinase activity of EGF receptor, and the activities of Ras and MAP kinase were enhanced in a dose-dependent manner (0~50μmol/L). At the condition of 20μmol/L GD1a pre-incubation, the EGF induced EGFR tyrosine kinase activity was increased by about 2 folds, and autophosphorylation of the receptor increased about 56%, if compared with control. Exposure of the cells to GD1a also enhanced the phosphorylation of Elk-1 by the activated MAP kinase and Ras. In the absence of serum and growth factors, GD1a could induce phosphorylation of Src kinase, Ras activation, and phosphorylation of MAP kinase and Elk-1 in a dose-dependent manner. The activation of Src kinase was confirmed by enhanced Src kinase activity. Pretreatment of the cells with AG1478, and PD98059, the enhanced effect of gangliosides on EGF-induced EGFR phosphorylation and MAP kinase activation were blocked. These results demonstrated that gangliosides GD1a could enhance EGF induced EGFR/Ras/Raf/MAKP signaling. The treatment of the cells with PP1 could block the activation of Src kinase and MAP kinase. These indicated that gangliosides could induce Src/Ras/MAKP signaling. The enhancement of GD1a was rapid (within 30 min.), and then there was no time-dependent alteration in the response of EGFR on EGF. As exogenous ganglioside 3H-GM1 and 14C -GD1a could bind to the membrane, when 5μmol/L of GD1a was used to pre-incubate, 0.15%±0.01% of exogenous GD1a was bound to the membrane, it was about 5.0±0.1×106 molecules/cell. After evaluating the initial steps of the activation, EGF binding, and EGFR dimerization, we found that GD1a enrichment on the cell membrane increased EGFR dimerization and the effective amounts of high affinity EGFR, but without increasing total receptor proteins. With 10 μmol/L of GD1a pre-incubation, the amounts of high affinity EGFR on NHDF were increased from 0.3×103/cell (control) to 2.7×103/cell, increasing about 9 folds. And the percentage of high affinity moiety of the total receptors was changed from 10% to 60% and Kd value increased from 0.12nmol/L to 0.28nmol/L. The amounts of the low affinity moiety of receptors were not significantly changed, only from 2.7×103/cell to 1.9×103/cell, and the Kd value was not clearly changed. The total EGFR on the membrane increased about 50%, from 3.0×103/cell to 4.6×103/cell. Unexpectedly, GD1a enrichment also triggered the increase of EGFRdimerization about 100% in the absence of growth factor, if compared with control. This resulted in enhancing the activity of the EGFR signal transduction cascade when E...
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