Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.—Mukherjee, P., A. C. Faber, L. M. Shelton, R. C. Baek, T. C. Chiles, and T. N. Seyfried. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a. J. Lipid Res. 2008. 49: 929–938. Supplementary key words glycosphingolipid & human umbilical endothelial cell migration & matrigel plug assay & growth factor receptor & phosphorylated Akt Gangliosides are a family of sialic acid-containing glycosphingolipids that are enriched in the outer surface of plasma membranes and have long been associated with tumor malignancy and metastasis (1–3). These molecules contain an oligosaccharide head group that is attached to a lipophilic ceramide, consisting of a sphingosine base and a long-chain fatty acid. Gangliosides can be shed from the surface of tumor cells into the microenvironment, where they can influence tumor host cell interactions to include angiogenesis (1, 4–13). Ganglioside GM3, a simple monosialoganglioside (NeuAca2Y3Galb1Y4Glcb1Y 1¶-ceramide), modulates cell adhesion, proliferation, and differentiation (2, 5, 12, 14). The antiproliferative and proapoptotic effects of GM3 were observed in glioma cells grown both in vivo and in vitro (15–17). In contrast to GM3, complex gangliosides like GM2, GM1, GD1a, GD1b, GT1b, and GD3, which contain longer oligosaccharide chains than that of GM3, enhance tumor cell proliferation, invasion, and metastasis (1, 3, 14, 18, 19). Increased tumorigenic effects of complex gangliosides were observed in a variety of tumor cells, including bladder, lymphoma, glioma, neuroblastoma, and melanoma (7, 11, 14, 20–22). Specific inhibitors of ganglioside biosynthesis also reduced tumor growth (23–25), whereas gene-linked shifts in ganglioside distribution changed tumor growth and angiogenesis in vivo (4, 8, 12). Endothelial cell signaling is important in cancerassociated vascularity (angiogenesis). The proliferation and migration of endothelial cells in response to growth factors is one of the major determinants of tumor growth and progression. Dysregulation of the balance between proangiogenic and antiangiogenic factors contributes to Manuscript received 3 January 2008 and in revised form 15 February 2008. Published, JLR Papers in Press, February 20, 2008. DOI 10.1194/jlr.R800006-JLR200 Abbreviations: bFGF, basic fibroblast growth factor; EBM, endothelial basal medium; EGFR, epidermal growth factor receptor; EGM2, endothelial growth medium; HPTLC, high-performance thin-layer chromatography; HUVEC, human umbilical vein endothelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NB-DNJ, N-butyldeoxynojirimycin; VEGF, vascular endothelial growth factor; VEGFR-2, vascular endothelial growth factor receptor 2. 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright D 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 49, 2008 929 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom the abnormal vasculature in tumors. The targeting of tumor endothelial cells, therefore, is considered important for managing tumor growth (1, 8, 26). Vascular endothelial cells are responsive to a number of proangiogenic growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which promote endothelial cell survival, growth, and migration (18, 27, 28). Interestingly, complex gangliosides enhance the response of endothelial cells to the proangiogenic action of bFGF and VEGF (4, 13, 18, 29). GD1a enrichment of endothelial cells enhanced VEGF receptor dimerization, autophosphorylation, and downstream signaling pathways for endothelial cell proliferation and migration (13). The involvement of gangliosides in angiogenesis is dependent on the intact molecules, as neither asialo species nor sialic acid alone influences angiogenesis (30). In contrast to the enhancing effects of complex gangliosides on angiogenesis, GM3 reduces endothelial cell proliferation and migration (5, 8, 12, 31). Little is known, however, about the molecular mechanism by which GM3 inhibits angiogenesis. The interactions of gangliosides with cell surface receptor molecules in tumor cells as well as in endothelial cells may be critical for the tumor-induced progression of the microenvironment (8, 13, 14). The inhibitory effects of GM3 on the epidermal growth factor receptor (EGFR) tyrosine kinase are well studied (32–34). Yoon et al. (34) showed that GM3 inhibits the EGFR tyrosine kinase through interactions with N-acetylglucosamine residues on the glycan units of the receptor. In contrast to the information available on the influence of GM3 on the EGFR, little is known about the influence of GM3 on other growth factor receptors, including the vascular endothelial growth factor receptor (VEGFR). VEGFR-2 or KDR is phosphorylated upon VEGF stimulation, which induces the phosphoinositide-3 kinase/Akt pathway, resulting in enhanced endothelial proliferation and migration (35, 36). In this study, we examined the influence of the exogenous addition of GM3 on the proliferation of human umbilical vein endothelial cells (HUVECs) in the presence of VEGF and the VEGF enhancer, GD1a. We show that GM3 suppresses angiogenesis both in vitro and in vivo. We also found that GM3 enrichment of HUVECs inhibits migration toward VEGF as a chemoattractant. Additionally, we found that GM3 reduced VEGFR-2 phosphorylation and downstream Akt signaling in HUVECs, suggesting a mechanism by which GM3 reduces endothelial cell proliferation and migration. Moreover, pharmacological depletion of endogenous GM3 significantly increased HUVEC proliferation and VEGFR-2 and Akt phosphorylation. MATERIALS AND METHODS