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Ganglioside GM3 Is Antiangiogenic in Malignant Brain Cancer

Ganglioside GM3 Is Antiangiogenic in Malignant Brain Cancer Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 961243, 8 pages doi:10.1155/2010/961243 Review Article Thomas N. Seyfried and Purna Mukherjee Department of Biology, Boston College, Chestnut Hill, MA 02467, USA Correspondence should be addressed to Thomas N. Seyfried, thomas.seyfried@bc.edu Received 17 December 2009; Revised 12 March 2010; Accepted 10 April 2010 Academic Editor: Arkadiusz Dudek Copyright © 2010 T. N. Seyfried and P. Mukherjee. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Progression of malignant brain tumors is dependent upon vascularity and is associated with altered ganglioside composition and distribution. Evidence is reviewed showing that the simple monosialoganglioside, GM3, possesses powerful antiangiogenic action against the highly vascularized CT-2A mouse astrocytoma, which primarily expresses complex gangliosides. Brain tumors expressing high levels of GM3 are generally less vascularized and grow slower than tumors that express low levels of GM3. GM3 inhibits angiogenesis through autocrine and paracrine effects on vascular endothelial growth factor (VEGF) and associated receptors. GM3 should be a clinically useful compound for managing brain tumor angiogenesis. 1. Introduction group attached to a lipophilic ceramide, consisting of a sphingosine base and a long-chain fatty acid (Figure 1). The Malignant brain cancer persists as a catastrophic illness and presence of sialic acid (N-acetylneuraminic acid, NeuAc) is the second leading cause of cancer death in children distinguishes the gangliosides from other glycosphingolipids. [1–4]. The failure to effectively manage malignant brain Gangliosides are anchored in the outer surface of plasma cancer has been due in large part to the highly invasive membranes through their ceramide moiety, which allows the nature of the disease and to the unique anatomical and head group to modulate numerous cell surface events such as metabolic environment of the brain, which prevents the growth, migration, adhesion, and signaling [12–15]. large-scale resection of tumor tissue and impedes the The structurally simple monosialoganglioside GM3 delivery of therapeutic drugs. Invasion/metastasis involves contains a single terminal sialic acid (Figure 1). N- the dissemination of tumor cells from the primary neoplasm acetylneuraminic acid is the predominant sialic acid species to surrounding tissue and distant regions. In addition, the expressed in mammalian brain gangliosides [16, 17]. In invasive cells establish a microenvironment facilitating col- contrast to N-acetylneuraminic acid, N-glycolylneuraminic onization (angiogenesis and further proliferation), resulting acid is a predominant sialic acid species expressed in in macroscopic malignant secondary tumors [5, 6]. Tumor gangliosides from nonneural tissues of most nonhuman cell invasion is correlated with tumor angiogenesis (vascu- species (rodents, bovine, etc.) [17]. As humans lack the larity), as prognosis is generally worse for brain tumors that gene for the synthesis of N-glycolylneuraminic acid [18, 19], are more vascular than for those that are less vascular [7–9]. expression of N-glycolylneuraminic acid in gangliosides of Consequently, therapies that can simultaneously target both human cells or tissues is attributed to contamination from angiogenesis and invasion could provide effective longer- exposure to nonhuman serum or from diet [17, 20, 21]. The term management of malignant brain cancer. involvement of gangliosides in angiogenesis is dependent on the intact molecules as neither asialo species nor sialic alone influence angiogenesis [22]. 2. Glycosphingolipids and Angiogenesis GM3 modulates the function of several receptors impli- Gangliosides are a family of cell surface-enriched glycosph- cated with angiogenesis to include those for the insulin- ingolipids that have long been implicated in tumorigenesis like growth factor-1 (IGF-1), basic fibroblast growth factor [10–12]. These molecules contain an oligosaccharide head (b-FGF), epidermal growth factor (EGF), platelet-derived 2 Journal of Oncology Gal Glc NH HO CH OH OH HO O O O CH C OH Ceramide O O OH CH OH HO HO HOH C 2 OH H C 3 H NeuAc Figure 1: Structure of Ganglioside GM3 (NeuAc-alpha 2→ 3Gal-beta1 → 4Glc-beta1 → 1 Ceramide) (from [14] with permission). Table 1: Association of GM3 levels to the vascularity of experimen- tal brain tumors . GM4 GM3 GM3 Ganglioside Distribution Vascularity Brain Tumors GM3 Complex Gangliosides GM2 GM2 Mouse EPEN High Low Low GM1 GM1 CBT-1 High Low Low GD3 CBT-3 High Low Low GD1a GD1a CBT-4 Low high High GT1a CT-2 Low High High GD1b CT-2A Low High High GT1b Human GQ1b U87MG Low High High All mouse brain tumors were produced from implantation of 20- Stds C V T methylcholantherene into the ventricle (EPEN), the cerebrum (CT), or the cerebellum (CBT) of C57BL/6J mice as we previously described [43]. Figure 3: High-performance thin-layer chromatographic analysis of ganglioside distribution in the CT-2A astrocytoma: Control untransfected CT-2A (C), CT-2A transfected with empty vector (GM3) Gal-Glc-Cer alone (V), and CT-2A transfected with the antisense sequence SA to the GalNAc-T gene (T). Synthesized gangliosides appear as GalNAcT double bands due to ceramide structural heterogeneity. Analysis (GM2) GalNAc-Gal-Glc-Cer of synthesized gangliosides and standards (left lane) was as we SA Gal T -II described [7]. Knockdown of the GalNAc-T gene elevated GM3 (GM1) Gal-GalNAc-Gal-Glc-Cer content, while reducing GD1a content in the antisense T cells. SA ST -IV (GD1a) Gal-GalNAc-Gal-Glc-Cer SA SA The ratio of GM3 to the proangiogenic gangliosides Figure 2: Pathway for the synthesis of ganglioside GM2 from GM3 GD3 and GD1a (GM3/GD3; GM3/GD1a) is lower in more by GalN Ac-T. GalN Ac-T adds a beta-linked N -acetylgalactosamine metastatic and aggressive tumors than that in less metastatic residue to the galactose of GM3 to form GM2, a key step required tumors [7, 33–35], suggesting that elevated expression for the synthesis of complex gangliosides, GM2, GM1, and GD1a. of complex gangliosides enhances tumor malignancy. In Antisense targeting of the GalNAc-T gene reduces GD1a content, contrast to most human glioma tumor tissues, which contain while increasing GM3 content [7]. high levels of the pro-angiogenic ganglioside GD3 [36–41], GD3 is not heavily expressed in mouse brain tumors or in most cultured human brain tumor cells [17, 42–44]. Although GM3 is also expressed in malignant human brain growth factor (PDGF), vascular endothelial growth factor tumors, we think that GM3 expression in these tumors might (VEGF), and cell adhesion molecules including the integrins serve to regulate or to counteract the pro-angiogenic action [7, 12, 23–28]. GM3 also reduces proliferation and enhances of GD3 and other complex gangliosides. apoptosis of rapidly proliferating neural stem cells [29]. Furthermore, Alessandri, Ziche, and coworkers originally found that several complex gangliosides (GM2, GM1, GD3, 3. Evidence Supporting the Anti-Angiogenic GD1a, GD1b, and GT1b) enhanced the action of angiogenic Action of GM3 in Brain Cancer inducers, whereas ganglioside GM3 was inhibitory [30–32]. These observations suggest that GM3 could have therapeutic Table 1 summarizes data from our previous studies on potential against tumor cell proliferation and angiogenesis. the association of GM3 expression with the angiogenic Journal of Oncology 3 CV T (a) VEGF VEGF 27 kDa β-actin β-actin C V T C V T 0 0 CCVVTT (b) CV T (c) Figure 4: Ganglioside shift reduces growth, VEGF gene and protein expression, and vascularity in the CT-2A astrocytoma: Control untransfected CT-2A (C), CT-2A transfected with empty vector alone (V), and CT-2A transfected with the antisense sequence to the GalNAc- T gene (T). (a) The values are expressed as mean mg wet weight± SE. : significant compared to control C and V tumors at the P< .01 level. CT-2A (n = 10), CT-2A/V (n = 12), and CT-2A/TNG (n = 14) independent tumors. (b) RT-PCR and Western blot for VEGF. Other details are in [7]. (c) Vascularity determined using factor VIII-immunostained microvessels per ×200 field (hpf, high-powered field) from tissue sections as we described [7]. VEGF/β-actin Tumor weight (mg) Vessels/hpf VEGF/β-actin 4 Journal of Oncology VEGF 1.5 0.5 HIF-1α NP-1 VEGF HIF-1α NP-1 CT-2A β-actin CT-2A/V CT-2A/TNG CV T (a) (b) Figure 5: Ganglioside shift reduces VEGF, HIF-1α, and NP-1 gene expression in CT-2A- cultured cells: Control untransfected CT-2A (C), CT-2A transfected with empty vector alone (V), and CT-2A transfected with the anti-sense sequence to (a) the GalNAc-T gene (T). VEGF (multiple splice variants: 400–600 bp), HIF-1α (365 bp), and NP-1 (551 bp) amplification products were detected in each tumor cell line. Experimental conditions are as we described [7]. Thegeneto β-actin levels are expressed as the means of three independent samples± SE. : Significant compared to control C and V cells at (b) the P< .01. Control GM3 treated Figure 6: GM3 reduces CT-2A tumor vascularity when added to the tumor microenvironment. Small fragments of the CT-2A tumor were grown in Matrigel that contained either no GM3 (control) or GM3 (40 μM). The tumor was grown in Matrigel for approximately two weeks in the flank of the syngeneic host C57BL/6 mice according to our standard procedures [7, 34]. Florid vascularization and the number and size thrombotic vessels were noticeably less in the presence than in the absence of GM3. Similar results were found in two independent experiments. properties of multiple experimental mouse and human the experimental ependymoblastoma (EPEN) tumor, which brain tumor models [17, 44, 45]. This survey shows that contains GM3 as the only ganglioside, increased vascularity brain tumors with high GM3 expression are less angiogenic (angiogenesis) [34]. An opposite effect was observed in the (vascularized) than brain tumors with low GM3 expression. highly angiogenic CT-2A astrocytoma when we upregulated GM3 expression was also correlated with greater cell-cell GM3 expression (below). These and other findings led us adhesion and slower growth [14, 44]. We later showed to conclude that the ratio of GM3 to complex gangliosides that the gene-linked knockdown of GM3 expression in (GM1, GD1a, GT1b) can influence the angiogenic properties Relative intensity Journal of Oncology 5 GD1a (40 μM) GM3+GD1a (40 μMeach) Control Figure 7: GM3 inhibits the pro-angiogenic effects of GD1a in the in vivo Matrigel assay. Matrigel alone (control) or containing GD1a or GD1a with GM3 was injected subcutaneously (s.c.) in SCID mice as we described [49]. Plugs were photographed (12.5×)onday 7after Matrigel injection. of a broad range of brain tumor types and are consistent 60]. Our most recent findings show that GM3, by itself, with previous findings on the role of GM3 in other systems markedly reduces CT-2A vascularity when grown in the [22, 29, 32, 41, 46–49]. in vivo Matrigel model (Figure 6). These findings suggest that GM3 could be applied as a drug therapy directly to the tumor site and to surrounding areas following surgical tumor resection in humans. Alternatively, GM3 4. Anti-Angiogenic Action of GM3 in could be applied in liposomes as a pharmacotherapy for the CT-2A Astrocytoma preformed tumors. Our findings in brain tumor cells are The CT-2A astrocytoma was produced following implan- also consistent with previous findings in rabbit cornea tation of the chemical carcinogen, 20-methylcholanthrene, showing that GM3 applied directly to tissue is anti- angiogenic [32]. Viewed, collectively, our findings indicate into the cerebral cortex of C57BL/6J mouse according to the procedures of Zimmerman and Arnold [44, 50]. The that GM3 has powerful anti-angiogenic action against the CT-2A tumor grows rapidly, is deficient in the phosphatase CT-2A astrocytoma when present in the microenvironment and can counteract the pro-angiogenic effects of complex and tensin homologue/tuberous sclerosis complex 2, and is highly angiogenic [7, 51, 52]. We used an antisense construct gangliosides. Further evidence for a direct anti-angiogenic role of to inhibit GalNAc-T expression in CT-2A cells as shown in (Figure 2). This caused a significant shift in ganglioside GM3 came from our recent studies with human umbilical distribution, elevating GM3 content while reducing GD1a vein endothelial cells, HUVEC. We found that GM3, by itself, significantly suppresses VEGF-induced proliferation content (Figure 3). The shift in ganglioside distribution significantly reduced and migration of HUVEC [49]. Moreover, GM3 significantly growth, VEGF gene and protein expression, and blood blocks GD1a-induced angiogenesis in the in vivo Matrigel assay (Figure 7). GD1a is a complex ganglioside associated vessel density in the orthotopically grown CT-2A tumors (Figure 4). Moreover, the shift in ganglioside distribution with enhanced angiogenesis [7, 61]. The suppression of reduced gene expression for hypoxia inducible factor 1a VEGF receptor 2 and Akt phosphorylation underlies the anti-angiogenic effect of GM3 on HUVEC (Figure 8). (HIF-1α) and the VEGF coreceptor neruropilin-1 (NP-1) in the CT-2A cultured cells (Figure 5). This is interesting Additionally, the EPEN tumor, which expresses only GM3, has few blood vessels relative to tumors that express complex as HIF-1α is a transcription factor that regulates VEGF expression through the PI-3k/Akt signaling pathway [51, 53– gangliosides [44, 45]. Consistent with our findings, Chung 55]. Viewed collectively, these data show that endogenous and coworkers recently showed that GM3 could suppress angiogenesis through the inactivation of VEGF-induced sig- upregulation of GM3 reduces growth and angiogenesis in the rapidly growing and highly vascularized CT-2A mouse naling by direct interaction with VEGFR-2 [47]. GM3 treat- astrocytoma. ment could also reduce in vivo vascularity in the Lewis lung carcinoma model [47], while van Cruijsen et al. showed that It was initially unclear, however, whether it was the elevation of GM3, the reduction of the pro-angiogenic vascularity was less and patient survival was better for non- small cell lung carcinomas that contained more GM3 than ganglioside GD1a, or the change in GM3/GD1a ratio that was responsible for the reduction in CT-2A angiogenesis. It less GM3 [62]. Hence, GM3 is anti-angiogenic through its is well documented that gangliosides are shed from tumor inhibition of the proangiogenic actions of complex ganglio- sides as well as through its direct inhibition of endothelial cell cells into the microenvironment where stromal (endothelial) cells take them up to influence tumor progression [56– growth. 6 Journal of Oncology 0.8 VEGF EBM Control GM3 0.6 pVEGFR-2 0.4 Total VEGFR-2 pAkt (Ser473) 0.2 Total Akt β-actin EBM Control GM3 (a) (b) 0.2 0.15 0.1 0.05 EBM Control GM3 (c) Figure 8: GM3 inhibits VEGFR-2 and Akt phosphorylation in HUVEC. HUVECs were incubated with GM3 (100 ng/ml) in endothelial basal medium (EBM) for 24 hours and were then stimulated with VEGF (100 ng/ml) for 5 minutes as we described [49]. (a) Cell lysates were prepared and measurement of VEGFR-2 and Akt phosphorylation over total was analyzed using Western blots [49]. 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Ganglioside GM3 Is Antiangiogenic in Malignant Brain Cancer

Journal of Oncology , Volume 2010 – Jun 20, 2010

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Hindawi Publishing Corporation
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Copyright © 2010 Thomas N. Seyfried and Purna Mukherjee. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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1687-8450
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1687-8469
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10.1155/2010/961243
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Abstract

Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 961243, 8 pages doi:10.1155/2010/961243 Review Article Thomas N. Seyfried and Purna Mukherjee Department of Biology, Boston College, Chestnut Hill, MA 02467, USA Correspondence should be addressed to Thomas N. Seyfried, thomas.seyfried@bc.edu Received 17 December 2009; Revised 12 March 2010; Accepted 10 April 2010 Academic Editor: Arkadiusz Dudek Copyright © 2010 T. N. Seyfried and P. Mukherjee. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Progression of malignant brain tumors is dependent upon vascularity and is associated with altered ganglioside composition and distribution. Evidence is reviewed showing that the simple monosialoganglioside, GM3, possesses powerful antiangiogenic action against the highly vascularized CT-2A mouse astrocytoma, which primarily expresses complex gangliosides. Brain tumors expressing high levels of GM3 are generally less vascularized and grow slower than tumors that express low levels of GM3. GM3 inhibits angiogenesis through autocrine and paracrine effects on vascular endothelial growth factor (VEGF) and associated receptors. GM3 should be a clinically useful compound for managing brain tumor angiogenesis. 1. Introduction group attached to a lipophilic ceramide, consisting of a sphingosine base and a long-chain fatty acid (Figure 1). The Malignant brain cancer persists as a catastrophic illness and presence of sialic acid (N-acetylneuraminic acid, NeuAc) is the second leading cause of cancer death in children distinguishes the gangliosides from other glycosphingolipids. [1–4]. The failure to effectively manage malignant brain Gangliosides are anchored in the outer surface of plasma cancer has been due in large part to the highly invasive membranes through their ceramide moiety, which allows the nature of the disease and to the unique anatomical and head group to modulate numerous cell surface events such as metabolic environment of the brain, which prevents the growth, migration, adhesion, and signaling [12–15]. large-scale resection of tumor tissue and impedes the The structurally simple monosialoganglioside GM3 delivery of therapeutic drugs. Invasion/metastasis involves contains a single terminal sialic acid (Figure 1). N- the dissemination of tumor cells from the primary neoplasm acetylneuraminic acid is the predominant sialic acid species to surrounding tissue and distant regions. In addition, the expressed in mammalian brain gangliosides [16, 17]. In invasive cells establish a microenvironment facilitating col- contrast to N-acetylneuraminic acid, N-glycolylneuraminic onization (angiogenesis and further proliferation), resulting acid is a predominant sialic acid species expressed in in macroscopic malignant secondary tumors [5, 6]. Tumor gangliosides from nonneural tissues of most nonhuman cell invasion is correlated with tumor angiogenesis (vascu- species (rodents, bovine, etc.) [17]. As humans lack the larity), as prognosis is generally worse for brain tumors that gene for the synthesis of N-glycolylneuraminic acid [18, 19], are more vascular than for those that are less vascular [7–9]. expression of N-glycolylneuraminic acid in gangliosides of Consequently, therapies that can simultaneously target both human cells or tissues is attributed to contamination from angiogenesis and invasion could provide effective longer- exposure to nonhuman serum or from diet [17, 20, 21]. The term management of malignant brain cancer. involvement of gangliosides in angiogenesis is dependent on the intact molecules as neither asialo species nor sialic alone influence angiogenesis [22]. 2. Glycosphingolipids and Angiogenesis GM3 modulates the function of several receptors impli- Gangliosides are a family of cell surface-enriched glycosph- cated with angiogenesis to include those for the insulin- ingolipids that have long been implicated in tumorigenesis like growth factor-1 (IGF-1), basic fibroblast growth factor [10–12]. These molecules contain an oligosaccharide head (b-FGF), epidermal growth factor (EGF), platelet-derived 2 Journal of Oncology Gal Glc NH HO CH OH OH HO O O O CH C OH Ceramide O O OH CH OH HO HO HOH C 2 OH H C 3 H NeuAc Figure 1: Structure of Ganglioside GM3 (NeuAc-alpha 2→ 3Gal-beta1 → 4Glc-beta1 → 1 Ceramide) (from [14] with permission). Table 1: Association of GM3 levels to the vascularity of experimen- tal brain tumors . GM4 GM3 GM3 Ganglioside Distribution Vascularity Brain Tumors GM3 Complex Gangliosides GM2 GM2 Mouse EPEN High Low Low GM1 GM1 CBT-1 High Low Low GD3 CBT-3 High Low Low GD1a GD1a CBT-4 Low high High GT1a CT-2 Low High High GD1b CT-2A Low High High GT1b Human GQ1b U87MG Low High High All mouse brain tumors were produced from implantation of 20- Stds C V T methylcholantherene into the ventricle (EPEN), the cerebrum (CT), or the cerebellum (CBT) of C57BL/6J mice as we previously described [43]. Figure 3: High-performance thin-layer chromatographic analysis of ganglioside distribution in the CT-2A astrocytoma: Control untransfected CT-2A (C), CT-2A transfected with empty vector (GM3) Gal-Glc-Cer alone (V), and CT-2A transfected with the antisense sequence SA to the GalNAc-T gene (T). Synthesized gangliosides appear as GalNAcT double bands due to ceramide structural heterogeneity. Analysis (GM2) GalNAc-Gal-Glc-Cer of synthesized gangliosides and standards (left lane) was as we SA Gal T -II described [7]. Knockdown of the GalNAc-T gene elevated GM3 (GM1) Gal-GalNAc-Gal-Glc-Cer content, while reducing GD1a content in the antisense T cells. SA ST -IV (GD1a) Gal-GalNAc-Gal-Glc-Cer SA SA The ratio of GM3 to the proangiogenic gangliosides Figure 2: Pathway for the synthesis of ganglioside GM2 from GM3 GD3 and GD1a (GM3/GD3; GM3/GD1a) is lower in more by GalN Ac-T. GalN Ac-T adds a beta-linked N -acetylgalactosamine metastatic and aggressive tumors than that in less metastatic residue to the galactose of GM3 to form GM2, a key step required tumors [7, 33–35], suggesting that elevated expression for the synthesis of complex gangliosides, GM2, GM1, and GD1a. of complex gangliosides enhances tumor malignancy. In Antisense targeting of the GalNAc-T gene reduces GD1a content, contrast to most human glioma tumor tissues, which contain while increasing GM3 content [7]. high levels of the pro-angiogenic ganglioside GD3 [36–41], GD3 is not heavily expressed in mouse brain tumors or in most cultured human brain tumor cells [17, 42–44]. Although GM3 is also expressed in malignant human brain growth factor (PDGF), vascular endothelial growth factor tumors, we think that GM3 expression in these tumors might (VEGF), and cell adhesion molecules including the integrins serve to regulate or to counteract the pro-angiogenic action [7, 12, 23–28]. GM3 also reduces proliferation and enhances of GD3 and other complex gangliosides. apoptosis of rapidly proliferating neural stem cells [29]. Furthermore, Alessandri, Ziche, and coworkers originally found that several complex gangliosides (GM2, GM1, GD3, 3. Evidence Supporting the Anti-Angiogenic GD1a, GD1b, and GT1b) enhanced the action of angiogenic Action of GM3 in Brain Cancer inducers, whereas ganglioside GM3 was inhibitory [30–32]. These observations suggest that GM3 could have therapeutic Table 1 summarizes data from our previous studies on potential against tumor cell proliferation and angiogenesis. the association of GM3 expression with the angiogenic Journal of Oncology 3 CV T (a) VEGF VEGF 27 kDa β-actin β-actin C V T C V T 0 0 CCVVTT (b) CV T (c) Figure 4: Ganglioside shift reduces growth, VEGF gene and protein expression, and vascularity in the CT-2A astrocytoma: Control untransfected CT-2A (C), CT-2A transfected with empty vector alone (V), and CT-2A transfected with the antisense sequence to the GalNAc- T gene (T). (a) The values are expressed as mean mg wet weight± SE. : significant compared to control C and V tumors at the P< .01 level. CT-2A (n = 10), CT-2A/V (n = 12), and CT-2A/TNG (n = 14) independent tumors. (b) RT-PCR and Western blot for VEGF. Other details are in [7]. (c) Vascularity determined using factor VIII-immunostained microvessels per ×200 field (hpf, high-powered field) from tissue sections as we described [7]. VEGF/β-actin Tumor weight (mg) Vessels/hpf VEGF/β-actin 4 Journal of Oncology VEGF 1.5 0.5 HIF-1α NP-1 VEGF HIF-1α NP-1 CT-2A β-actin CT-2A/V CT-2A/TNG CV T (a) (b) Figure 5: Ganglioside shift reduces VEGF, HIF-1α, and NP-1 gene expression in CT-2A- cultured cells: Control untransfected CT-2A (C), CT-2A transfected with empty vector alone (V), and CT-2A transfected with the anti-sense sequence to (a) the GalNAc-T gene (T). VEGF (multiple splice variants: 400–600 bp), HIF-1α (365 bp), and NP-1 (551 bp) amplification products were detected in each tumor cell line. Experimental conditions are as we described [7]. Thegeneto β-actin levels are expressed as the means of three independent samples± SE. : Significant compared to control C and V cells at (b) the P< .01. Control GM3 treated Figure 6: GM3 reduces CT-2A tumor vascularity when added to the tumor microenvironment. Small fragments of the CT-2A tumor were grown in Matrigel that contained either no GM3 (control) or GM3 (40 μM). The tumor was grown in Matrigel for approximately two weeks in the flank of the syngeneic host C57BL/6 mice according to our standard procedures [7, 34]. Florid vascularization and the number and size thrombotic vessels were noticeably less in the presence than in the absence of GM3. Similar results were found in two independent experiments. properties of multiple experimental mouse and human the experimental ependymoblastoma (EPEN) tumor, which brain tumor models [17, 44, 45]. This survey shows that contains GM3 as the only ganglioside, increased vascularity brain tumors with high GM3 expression are less angiogenic (angiogenesis) [34]. An opposite effect was observed in the (vascularized) than brain tumors with low GM3 expression. highly angiogenic CT-2A astrocytoma when we upregulated GM3 expression was also correlated with greater cell-cell GM3 expression (below). These and other findings led us adhesion and slower growth [14, 44]. We later showed to conclude that the ratio of GM3 to complex gangliosides that the gene-linked knockdown of GM3 expression in (GM1, GD1a, GT1b) can influence the angiogenic properties Relative intensity Journal of Oncology 5 GD1a (40 μM) GM3+GD1a (40 μMeach) Control Figure 7: GM3 inhibits the pro-angiogenic effects of GD1a in the in vivo Matrigel assay. Matrigel alone (control) or containing GD1a or GD1a with GM3 was injected subcutaneously (s.c.) in SCID mice as we described [49]. Plugs were photographed (12.5×)onday 7after Matrigel injection. of a broad range of brain tumor types and are consistent 60]. Our most recent findings show that GM3, by itself, with previous findings on the role of GM3 in other systems markedly reduces CT-2A vascularity when grown in the [22, 29, 32, 41, 46–49]. in vivo Matrigel model (Figure 6). These findings suggest that GM3 could be applied as a drug therapy directly to the tumor site and to surrounding areas following surgical tumor resection in humans. Alternatively, GM3 4. Anti-Angiogenic Action of GM3 in could be applied in liposomes as a pharmacotherapy for the CT-2A Astrocytoma preformed tumors. Our findings in brain tumor cells are The CT-2A astrocytoma was produced following implan- also consistent with previous findings in rabbit cornea tation of the chemical carcinogen, 20-methylcholanthrene, showing that GM3 applied directly to tissue is anti- angiogenic [32]. Viewed, collectively, our findings indicate into the cerebral cortex of C57BL/6J mouse according to the procedures of Zimmerman and Arnold [44, 50]. The that GM3 has powerful anti-angiogenic action against the CT-2A tumor grows rapidly, is deficient in the phosphatase CT-2A astrocytoma when present in the microenvironment and can counteract the pro-angiogenic effects of complex and tensin homologue/tuberous sclerosis complex 2, and is highly angiogenic [7, 51, 52]. We used an antisense construct gangliosides. Further evidence for a direct anti-angiogenic role of to inhibit GalNAc-T expression in CT-2A cells as shown in (Figure 2). This caused a significant shift in ganglioside GM3 came from our recent studies with human umbilical distribution, elevating GM3 content while reducing GD1a vein endothelial cells, HUVEC. We found that GM3, by itself, significantly suppresses VEGF-induced proliferation content (Figure 3). The shift in ganglioside distribution significantly reduced and migration of HUVEC [49]. Moreover, GM3 significantly growth, VEGF gene and protein expression, and blood blocks GD1a-induced angiogenesis in the in vivo Matrigel assay (Figure 7). GD1a is a complex ganglioside associated vessel density in the orthotopically grown CT-2A tumors (Figure 4). Moreover, the shift in ganglioside distribution with enhanced angiogenesis [7, 61]. The suppression of reduced gene expression for hypoxia inducible factor 1a VEGF receptor 2 and Akt phosphorylation underlies the anti-angiogenic effect of GM3 on HUVEC (Figure 8). (HIF-1α) and the VEGF coreceptor neruropilin-1 (NP-1) in the CT-2A cultured cells (Figure 5). This is interesting Additionally, the EPEN tumor, which expresses only GM3, has few blood vessels relative to tumors that express complex as HIF-1α is a transcription factor that regulates VEGF expression through the PI-3k/Akt signaling pathway [51, 53– gangliosides [44, 45]. Consistent with our findings, Chung 55]. Viewed collectively, these data show that endogenous and coworkers recently showed that GM3 could suppress angiogenesis through the inactivation of VEGF-induced sig- upregulation of GM3 reduces growth and angiogenesis in the rapidly growing and highly vascularized CT-2A mouse naling by direct interaction with VEGFR-2 [47]. GM3 treat- astrocytoma. ment could also reduce in vivo vascularity in the Lewis lung carcinoma model [47], while van Cruijsen et al. showed that It was initially unclear, however, whether it was the elevation of GM3, the reduction of the pro-angiogenic vascularity was less and patient survival was better for non- small cell lung carcinomas that contained more GM3 than ganglioside GD1a, or the change in GM3/GD1a ratio that was responsible for the reduction in CT-2A angiogenesis. It less GM3 [62]. Hence, GM3 is anti-angiogenic through its is well documented that gangliosides are shed from tumor inhibition of the proangiogenic actions of complex ganglio- sides as well as through its direct inhibition of endothelial cell cells into the microenvironment where stromal (endothelial) cells take them up to influence tumor progression [56– growth. 6 Journal of Oncology 0.8 VEGF EBM Control GM3 0.6 pVEGFR-2 0.4 Total VEGFR-2 pAkt (Ser473) 0.2 Total Akt β-actin EBM Control GM3 (a) (b) 0.2 0.15 0.1 0.05 EBM Control GM3 (c) Figure 8: GM3 inhibits VEGFR-2 and Akt phosphorylation in HUVEC. HUVECs were incubated with GM3 (100 ng/ml) in endothelial basal medium (EBM) for 24 hours and were then stimulated with VEGF (100 ng/ml) for 5 minutes as we described [49]. (a) Cell lysates were prepared and measurement of VEGFR-2 and Akt phosphorylation over total was analyzed using Western blots [49]. 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