Role of tumor-associated gangliosides in cancer progression

Biochimie 85 (2003) 455–463 www.elsevier.com/locate/biochi

Original article

Role of tumor-associated gangliosides in cancer progression > S. Birklé a, G. Zeng b, L. Gao b, R.K. Yu b, J. Aubry c,* a École Nationale Vétérinaire, Nantes, France Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA c INSERM U463, Institut de Biologie & Faculté de Pharmacie, 9, quai Moncousu, 44093 Nantes cedex 0, France b

Received 14 November 2002; accepted 23 December 2002

Abstract Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted. © 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. Keywords: Ganglioside; Tumorigenesis; Adhesion; Angiogenesis; Immunomodulation

1. Introduction Accumulating evidence indicates that cellular function and phenotype are highly influenced by gangliosides. Gangliosides are acidic glycosphingolipids that are characterized by the presence of at least one sialic acid linked to their oligosaccharide chain. They are present on the outer leaflet of plasma cell membranes of all types of tissues [1], with their hydrophobic ceramide backbone anchored in the membrane

Abbreviations: RGDS, l-arginyl-glycyl-l-aspartyl-serine; ICAM, intercellular adhesion molecule; VCAM, vascular cellular adhesion molecule; VEGF, vascular endothelial growth factor; Siglec, sialic acid/immunoglobulin/lectin (Glycobiology 8 (1998) v); PKC, protein kinase; IL-2, interleukin-2; IL-4, interleukin-4; IL-8, interleukin-8; IL-10, interleukin-10; INF-c, interferon-c; Th1, T-helper cell type 1; Th2, T-helper cell type 2; CD4, cluster differentiation molecule 4; MHC, major histocompatibility complex; TNFa, tumor necrosis factor a; NK cell, natural killer cell. > The ganglioside nomenclature is that of Svennerholm (J. Neurochem. 10 (1963) 613-623) in accordance with the International Union of Pure and Applied Chemistry (www.iupac.org/). * Corresponding author. Tel.: +33-240084747; fax: +33-240356697. E-mail address: [email protected] (J. Aubry).

and the hydrophilic carbohydrate residue projected into the extracellular environment. Gangliosides have been shown to have crucial regulatory roles in the normal physiological process, such as embryogenesis [2], as well as in pathological conditions, including tumor onset and progression [3]. Many recent studies indicate that tumor-associated gangliosides are a result of initial oncogenic transformation and play a key role in the induction of invasion and metastasis. This role of tumor-associated gangliosides in promoting tumor cell invasion and metastasis is of crucial importance in current cancer research; however, it is more difficult to understand the defined functional concepts of gangliosides in cancer than the functional role of proteins and their genes in defining cancer cell phenotypes. The concept of gangliosidedependent promotion of tumor progression has been developed in conjunction with clinicopathological studies that have shown that there are some ganglioside species with relatively simple structures that show very restricted expression in normal tissues and markedly enhanced expression in a particular malignant tumor. GD3 (Fig. 1), which has been identified as a melanoma-specific antigen, is an example of such a ganglioside [4]. All primary melanoma tissues as well

© 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 3 0 0 - 9 0 8 4 ( 0 3 ) 0 0 0 0 6 - 3

456

S. Birklé et al. / Biochimie 85 (2003) 455–463

Fig. 1. Structure of ganglioside GD3. Cer, ceramide, N-acylsphingosine; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; Neu5Ac, sialic acid, N-acetyl-neuraminic acid.

as established melanoma cell lines contain high amounts of GD3 as a major ganglioside component [5]. In contrast, human melanocytes, the normal counterpart of melanoma cells, expressed no or minimal levels of GD3 [6]. Furthermore, highly metastatic cells show an increase in ganglioside content and express more GD3 or complex gangliosides than poorly metastatic cells [7,8]. These findings suggest that GD3 might play an important role in the transformation of melanocytes into melanomas and also in the maintenance of malignant characteristics in melanoma cells. Little is known about the mechanisms through which tumor-associated gangliosides induce invasive and metastatic phenotypes of tumor cells. Glycosphingolipids, including gangliosides, have been shown to be major components in some types of microdomains associated with various functional membrane proteins involved in cell adhesion and cell signaling at the cell–extracellular matrix interface [9]. Studies have shown that microdomains have different physical properties and specialized functions. The potential roles of gangliosides and the structural variety of glycoclusters in microdomains have been recently reviewed [9]. Gangliosides are also actively shed from the tumor to their microenvironment in the form of micelles, monomers, and membrane vesicles [10,11]. Shed gangliosides are able to bind and interact with a wide variety of proteins, including signaling molecules present in the tumor microenvironment [12]. Furthermore, because shed gangliosides can be incorporated into the membrane of neighboring host cells, it is possible that the shed gangliosides modulate tumor–host cell interactions [13,14]. The expression mechanism of these gangliosides in terms of the status of their respective glycosyltransferase genes has been extensively studied [15]. They are biosynthesized by the sequential action of a series of specific glycosyltransferases and sialyltransferases (Fig. 2). The first such proposal was

given by Yu and Ando [16]. Fig. 2 is derived from this paper. To date, a large number of glycosyltransferase and sialyltransferase genes have been cloned and characterized [17], and studies of these genes have shown that various expression patterns of gangliosides are determined basically by a combination of activated glycosyltransferase genes [15]. Ganglioside biosynthesis takes place in the Golgi apparatus and endoplasmic reticulum [18,19], where glucosylceramide is glycosylated by sequential addition of galactose, sialic acid, and N-acetylgalactosamine [20]. The availability of these transferase genes and information about their structure and function enable further analysis of the implication of the tumor-associated gangliosides in malignant phenotypes of cancer cells. The development of cell lines with defects in distinct biosynthetic pathways marks a significant advance in understanding the function of tumor-associated gangliosides. This review will focus on recent reports that have revealed a large amount of information about the role of gangliosides in tumorigenesis, tumor progression and tumor metastasis. 2. Gangliosides are involved in tumorigenesis Several experimental approaches can be used to study the function of endogenous gangliosides. One is to assess the effect of eliminating molecules. Early studies in this direction included pharmacological inhibition of ganglioside synthesis using an inhibitor of glucosylceramide synthase, the key enzyme for glycosphingolipid synthesis, to block endogenous cellular ganglioside production. The first such molecule was d-threo-1-phenyl-2-decanoylamino-3-morpholino1-propanol (PDMP) [21,22]. Recently, an improved, more potent inhibitor d-1-threo-1-phenyl-2-hexadecanoylamino3-pyrrodilino-1-propanol–HCl (PPPP) was characterized [23]. PPPP, like PDMP, does not cause ceramide accu-

S. Birklé et al. / Biochimie 85 (2003) 455–463

457

Fig. 2. Pathway for the ganglioside biosynthesis [16]. Cer, ceramide, N-acylsphingosine; Lac-Cer, lactosyl ceramide; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; SA, sialic acid; ST, sialyltransferase; Gal T, galactosyltransferase; GalNAc T, N-acetyl-galactosaminyltransferase; Glc T, glucosyltransferase. Further additions of sialic acids occur in some cell types and species, yielding polysialogangliosides (not shown).

mulation [23] since ceramide [24] and its metabolic products, such as sphingosine-1-phosphate [25], may act as second messengers and affect cell growth. Treatment with PPPP resulted in a significant reduction of the cell ganglioside content of murine melanoma MEB4 cells [26]. Although it was not cytotoxic to the cells and did not inhibit cell proliferation in vitro, reduction of the ganglioside content in MEB4 cells markedly reduced the ability of the cells to form tumors and metastases in mice. Also, a rapid reversal of the inhibition of ganglioside synthesis occurred when PPPP was removed from the culture medium, and the ganglioside content recovered to the original level within 3 d. One of the disadvantages of this approach is that it requires the continu-

ing presence of an exogenous substance for effective inhibition. Furthermore, blocking ganglioside biosynthesis with a glucosylceramide synthase inhibitor may result in a complex pattern of interference with cellular events, thereby making it difficult to identify the lipid species involved. The recent development in many laboratories of specific monoclonal antibodies directed against the carbohydrate moieties of gangliosides has provided new insights into the biological functions of the molecules exposed on the cell surface. For instance, monoclonal antibodies reacting with ganglioside GM2 were shown to induce necrosis in vitro in spheroid cultures of a GM2-rich human glioma cell line [27]; anti-GD3 monoclonal antibodies were demonstrated to in-

458

S. Birklé et al. / Biochimie 85 (2003) 455–463

hibit the growth of human melanoma cells both in vitro [28] and in vivo [29]; and monoclonal antibodies against ganglioside GD2 were shown to induce apoptosis in small cell lung cancer cells [30]. The use of monoclonal antibodies against tumor-associated gangliosides has provided indirect evidence that tumor-associated gangliosides should have an important function in tumorigenesis. Alternative approaches to modifying ganglioside synthesis that have been explored include antisense oligodeoxynucleotide administration [31], sialidase gene transfection [32], gene transfer [30,33], and suppression of gene expression in vivo using an antisense strategy [34,35]. Treatment of the human promyelocytic leukemia cell line HL-60 with an antisense oligodeoxynucleotide to GM2-synthase downregulates the synthesis of GM2 and the more complex ganglioside GM1 by approximately 50% [31]. The specificity of the effect was suggested by a concomitant increase in GM3 content, without a change in the total cellular ganglioside content. The drawback of antisense oligodeoxynucleotide administration is that oligodeoxynucleotides must be constantly present in the cell culture to achieve sustained inhibition of ganglioside synthesis. This is the same disadvantage as that of standard pharmacological inhibition. Antisense transfection, on the other hand, could provide a way to alter the kinetics of glycosphingolipid synthesis permanently. In the first published study of alteration of ganglioside metabolism using this technique, the GD3 level in rat F11 hybrid neuroblastoma cells was reduced by stable transfection with an antisense vector to GD3-synthase [34]. This was associated with reduced cell migration in vitro and reduced metastatic potential in a nude mouse model [36]. A similar reduction in GD3 and its O-acetylated derivative was also achieved in hamster melanoma cells, resulting in a marked decrease in tumor cell proliferation [37,38]. These observations provide direct evidence that ganglioside GD3 plays an important role in the regulation of tumor cell growth. The reduction of total cellular gangliosides by antisense transfection has been achieved using an antisense transfection vector targeting glucosylceramide synthase in the MEB4 murine melanoma cells [35]. The reduction of tumor formation associated with the inhibition of glucosylceramide synthesis by antisense transfection showed a reduction of tumor cell glycosphingolipids associated with significantly decreased tumor incidence. Consistent with these observations, a decrease in ganglioside content by transfection with a sialidase cDNA suppresses pulmonary metastasis in murine melanoma cells [39]. Since no naturally occurring ganglioside-specific O-acetyltransferase cDNA has been isolated to date [40–42], the Myxoinfluenzae C-virus O-acetylesterase cDNA was transfected into hamster melanoma AbC-1 cells to elucidate the role of O-acetyl-GD3 in oncogenic transformation of melanocytes [37]. Down-regulation of O-acetyl-GD3 expression by the influenza C-virus O-acetylesterase cDNA induced dendricity in hamster melanoma cells with a reduced proliferation rate. On the other hand, the induction of GD2 expression in human small cell lung cancer cell lines by

GD3-synthase cDNA was associated with a markedly increased growth rate in vitro [30], and the neosynthesis of complex ganglioside by the N-acetylgalactosaminyl transferase cDNA in an experimental mouse brain tumor enhanced the tumor growth in vivo [43]. Taken together, these studies strongly suggest that tumor-associated gangliosides play an important role in enhancing cellular proliferation signals. However, the mechanisms that link the interaction between specific gangliosides and tumor proliferation remain unknown.

3. Gangliosides as adhesion molecules and mediators of metastasis During the last decade, several studies have established the importance of carbohydrate–carbohydrate interactions as the basis for cell adhesion and cell recognition. Although the greatest interest has been focused on glycoprotein–glycoprotein interactions, glycolipids may also participate in these carbohydrate–carbohydrate relationships and they have attracted increasing attention as possible key modulators. Some ganglioside antigens, which are highly expressed in specific human cancers, have been identified as adhesion molecules that may promote tumor cell metastasis in various ways, as evidenced recently. Early studies showed that disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to various extracellular matrix proteins, including laminin, fibronectin, collagen, and vitronectin [44]. The discovery that gangliosides inhibit cell attachment and spreading on a fibronectin matrix [45] led investigators to consider gangliosides as cell receptors for fibronectin before integrin a5b1 was identified [46,47]. Although these early studies showed that the terminal sialic acid residues of gangliosides were critical for the inhibition effect, the mechanism of inhibition was unclear. More than 10 years later, Paller et al. [48] demonstrated that the inhibition of migration and adhesion is completely inhibited by l-arginylglycyl-l-aspartyl-serine (RGDS) peptide, suggesting that gangliosides may abrogate the interaction between integrin a5b1 and fibronectin. Using a recombinant and affinitypurified form of integrin a5b1 and a novel binding technique, Wang et al. [49] provided evidence that GT1b and GD3 bind directly to the extracellular domain of the a5 subunit through carbohydrate–carbohydrate interactions, thereby inhibiting the interaction of a5b1 with fibronectin. It has been suggested that membrane gangliosides act as “cofactors” that form complexes with receptors and facilitate or block functions by ganglioside–receptor interactions, other than those between GT1b/GD3 and a5b1. For example, it was shown that GD3 and GD2 co-immunoprecipitate with avb3 [50]. Given the structural similarities between the a5b1 and the avb3 integrins, it is likely that the ganglioside–avb3 interaction similarly involves carbohydrate–carbohydrate recognition of the extracellular N-terminal region of av.

S. Birklé et al. / Biochimie 85 (2003) 455–463

Another possibility for ganglioside involvement in the promotion of metastasis has been evidenced in a renal cell carcinoma system in vitro. The aggregation of renal carcinoma tumor cells with peripheral blood mononuclear cells was shown to be mediated by sialic acid binding proteins named Siglecs (sialic acid/immunoglobulin/lectin) [51], which are expressed on various types of blood cells, including natural killer (NK) cells as demonstrated recently [52]. One member of this family, Siglec7, was demonstrated to interact specifically with disialogangliosides having an extended globo-series core, or a lacto type 1 chain core. A renal carcinoma cell line, TOS-1, expressing disialosylgalactosylgloboside was reported to bind to peripheral blood mononuclear cells and red blood cells that express Siglec7 and form large clumps under physiological conditions in vitro. From these observations, it is tempting to speculate that renal cell carcinoma metastasis may be mediated by tumor cell aggregation with peripheral blood mononuclear cells and other types of blood cells, and that such clumps may cause embolisms of microvasculature. Tumor cell aggregates may include platelets that are activated by tumor cells to release a factor or factors. These latter in turn activate endothelial cells to elicit intercellular adhesion molecules (ICAMs), vascular cellular adhesion molecules (VCAMs), or E- or P-selectins, which initiate tumor cell adhesion or invasion [53,54]. Some Siglecs have the characteristic to bind preferentially to sialyl 2→6 Gal as originally observed for CD22 and Siglec6 [55]. A similar preferential binding to sialyl 2→6, Gal was also found for Siglec7 [52]. Interestingly, GD3, GD2, and GT1b share the same sialyl 2→6 GalNAc moiety. They are highly expressed in many types of tumors [3] and are capable of binding to Siglec7 [52]. Thus, a possibility is opened that disialo epitopes may also promote metastasis through a mechanism involving microembolisms similar to that discussed above. However, the biological significance of Siglecdependent binding of tumor cells to target cell still has to be elucidated. Gangliosides may also promote tumor cell metastasis through another mechanism involving glycosphingolipid– glycosphingolipid interactions between counterpart microdomains organized with signal transducers, as demonstrated in a typical, well-documented example. The adhesion of B16 mouse melanoma cells to mouse endothelial SPE-1 cells is based on the interaction between GM3 on the B16 tumor cells and Lac-Cer and Gb4 on the endothelial cells, whereby both adhesion and motility are strongly enhanced [56]. Gg3, a strong ligand of GM3, is suggested as being expressed in mouse lung microvascular endothelial cells [54]; so the Gg3–GM3 interaction may also account for B16 metastasis to the lung. Liposomes containing Gg3 or GM3 inhibit this metastasis [56]. These results demonstrated that GM3dependent adhesion is coupled with the activation of c-Src, RhoA, and focal adhesion kinase [57], which may promote B16 cell invasiveness. The correlation observed in B16 mouse melanoma cells is strikingly different from the situation in colorectal [58] and

459

bladder cancer [59] cell lines, where GM3 and CD9 or CD82 are co-expressed. In these cells, ganglioside GM3 inhibits cell motility and invasiveness when complexed with CD9 or CD82 [58], which are antimetastatic membrane proteins, collectively termed tetraspanin [60,61]. In various colorectal [58] and bladder cancer [59] cell lines, GM3 and CD9 are co-expressed, and inhibit Matrigel- or laminin-5-dependent cell motility through an a3 integrin/CD9/GM3 complex in glycolipid-enriched microdomains [62]. Thus, the enhanced expression of an enzyme that changes the GM3 content in these cells may enhance malignancy through reduction of the motility inhibitory effect of GM3/CD9, as evidenced recently by Kakugawa et al. [63]. However, there are several possibilities for ganglioside involvement in the promotion of metastasis as demonstrated recently. A different ganglioside monosialo-Gb5 in a complex with CD9 in breast cancer MCF7 cells was shown to strongly enhance motility/invasiveness [64]. 4. Gangliosides and angiogenesis Tumors shed glycosphingolipids, mainly gangliosides, into the microenvironment, in greater quantities than do healthy tissues. This leads to elevated levels, up to 10 µM, of tumor-associated gangliosides in the serum [65]. Changes in the amounts of gangliosides in serum can influence the rate of tumor growth through an undetermined mechanism [3]. The potential importance of gangliosides in tumor cell growth has been suggested by demonstrating the reduction of experimental tumor growth and angiogenesis through regulation of vascular endothelial growth factor (VEGF) expression [43]. Several different experimental approaches have been designed to investigate the function of gangliosides in tumor angiogenesis. Exogenous gangliosides are often added to cell cultures to investigate their effect on cell behavior. For example, the addition of exogenous GD3 to culture medium stimulates VEGF release from glioma cell lines [66]. The ratio of GM3 to GD3 influences the proliferation and migration of microvascular endothelial cells in the rabbit cornea model of angiogenesis [67]. GM3 inhibits basic fibroblast growth factor-induced angiogenesis in a dose-dependent manner [67]. However, the antiangiogenic effect of GM3 is counteracted by more complex gangliosides, such as GD3 [67]. The mechanisms by which gangliosides modulate the biological activity of growth factors are not fully elucidated. Although complex gangliosides are not angiogenic by themselves, they are proangiogenic, since they act synergistically with angiogenesis inducers, for example, prostaglandin E1, basic growth factor, and VEGF [68]. Experimental evidence indicates that exogenous gangliosides are incorporated into the plasma membrane and may affect the activity of tyrosine kinase receptors and intracellular signaling [12]. However, the diversified conditions under which the gangliosides are added to cell cultures cause different incorporations into the cell membrane, making comparisons difficult. Conflicting

460

S. Birklé et al. / Biochimie 85 (2003) 455–463

results may therefore depend on the free or cell-associated status of the ganglioside and reflect different mechanisms of its action. For instance, it has been shown that gangliosides form micelles in an aqueous solution, and that these can interact with protein to form lipoprotein complexes and sequester the growth factor [12]. In contrast, cell membraneincorporated glycolipids may regulate the biological activity of growth factors in the absence of free gangliosides by affecting the activity of tyrosine kinase receptors and intracellular signaling [12]. Furthermore, it should be emphasized that the doses of exogenous gangliosides used in most of these studies were much more than the physiological concentrations of endogenous gangliosides. Therefore, the results from these studies could only be considered as indirect evidence that gangliosides modulate tumor angiogenesis by modulating growth factor signaling.

nesis. Accumulation of the precursor GM3 may contribute, at least in part, to the reduction of angiogenesis since the addition of exogenous GD3 could not reverse these effects. These data are in line with a similar study reported by Manfredi et al. [43]. They demonstrated that induction of the biosynthesis of the complex gangliosides GM2, GM1, and GD1a enhanced VEGF expression and stimulated vascularization in vivo in an ependymoblastoma cell line by transfection of the cells with the GM2-synthase cDNA. Although it remains to be elucidated as to whether the inhibition of GD3-synthase in F-11 cells has any secondary effects on angiogenesis, it is likely that the effects of gangliosides on cellular events may be due to multiple gangliosides.

Studies of the effects of exogenous gangliosides have identified some of their pharmacological effects, but have not addressed the physiological role of endogenous gangliosides in growth factor signaling. To answer this question, genetically altered cell lines that either overexpress or lack one or more specific gangliosides have been engineered. As discussed above, experiments were carried out by suppression of GD3-synthase gene expression in F-11 tumor cells by an antisense strategy to define the biological role of ganglioside GD3 in tumorigenesis [34]. The cells exhibited markedly reduced rates of tumor growth [34] and metastasis [36] in vivo and formed small, minimally vascularized tumors exhibiting extensive necrosis [69]. In vivo Matrigel assays revealed reduced vascularization and low hemoglobin content. The reduced angiogenesis in the antisense xenografts was correlated with a decrease in VEGF production. These results demonstrate that decreased levels of GD3 in F-11 tumor cells result in minimal angiogenesis of the tumors through down-regulation of VEGF, indicating an important role of GD3 in tumor angiogenesis. The hypothesis that ganglioside GD3 is involved in tumor angiogenesis in vivo was therefore substantiated by the GD3-depleted F-11 tumor cells. However, the mechanisms that link the interactions between specific gangliosides and angiogenesis or VEGF expression remain unknown. F-11 cells express three major gangliosides: GM3, GD3, and OAc-GD3, which constitute 37%, 27% and 18%, respectively, of the total gangliosides. The inhibition of expression of the GD3-synthase gene resulted in a great decrease in the amounts of GD3 and OAcGD3, accompanied with an increase in GM3, the precursor of GD3. The effect of OAc-GD3 on angiogenesis was excluded by characterization of another subclone of the F-11 cells, in which OAc-GD3 was completely destroyed [33]. Although reduced angiogenesis and VEGF production can primarily be accounted for by the reduction of the concentration of GD3, it should be noted that there was a significant accumulation of GM3 in the antisense-transfected xenografts. GM3 has been described as a modulator of growth factor receptors, such as fibroblast and epidermal growth factor receptors, which in turn may have effects on angioge-

Interactions between tumor cells and the cells of the immune system appear to be critical for tumor growth. The hypothesis that gangliosides may be active in the suppression of the antitumor immune response is supported by studies demonstrating that tumor cells synthesize and shed gangliosides into their microenvironments [70] and that these shed gangliosides directly bind to target cells in vitro [71]. Coupled with these observations, many studies have shown that exogenous or tumor-derived gangliosides inhibit multiple steps in the cellular immune response in vitro. For instance, tumor-derived gangliosides inhibit the activity of several immune cells, including helper T cells [72,73], natural killer cell cytotoxicity [74], and antigen- and mitogenstimulated T and B cells [75–78]. Gangliosides have also been shown to block the production of TNF a as well as antigen presentation by human monocytes [79,80]. They cause down-regulation of constitutive and IL-8 inducible expression of major histocompatibility complex (MHC) class I and II molecules on astrocytes [81] and inhibit the generation of functionally active dendritic cells [82]. The inhibitory effects of tumor-derived gangliosides have also been well demonstrated in vivo as evidenced in a syngeneic animal model (FBL-3 erythroleu- kemia cells, C57Bl/6 mice, and highly purified FLB-3 cell gangliosides) [83]. The immunosuppressive activity of the gangliosides has been demonstrated to be quantitatively affected by their molecular structure. Variations in either the carbohydrate or the ceramide structure can lead to various degrees of activity. For example, gangliosides with a terminal sialic acid linked to a compact neutral oligosaccharide had the greatest immunosuppressive activity [78]. Other studies indicated that tumor gangliosides were frequently more immunosuppressive than corresponding normal human brain gangliosides of an identical carbohydrate structure. These differences were inversely related to the length of the ceramide fatty acyl chain [84]. The mechanisms of immunosuppression caused by tumor shed gangliosides are most likely multiple and remain to be fully elucidated. Some of the known mechanisms include direct binding to a cytokine, such as IL-2, thus preventing its

5. Gangliosides as immunomodulators

S. Birklé et al. / Biochimie 85 (2003) 455–463

interaction with IL-2 receptors [85–87] and thereby inhibiting T cell proliferation. In addition, as discussed above, gangliosides may physiologically function to coordinate the activation of multiple receptors. They modify the binding activity of individual receptors as well as receptor-specific signal transduction pathways as evidenced recently in mutant mice T cells lacking complex gangliosides (GA1, GM1, GD1b) [88]. Within the immune system of these GM2/GD2 synthase gene-disrupted mutant mice, the T cells showed defects in IL-2 transduction [88]. Other studies indicated that exogenous GM3 induces down-modulation of CD4 molecules in human T lymphocytes, with a CD4 redistribution on the cell surface, and clustering and internalization via endocytic pits and vesicles followed by intracellular degradation [89]. The recent finding that GM3-enriched domains on the plasma membrane are associated with the integral membrane glycoprotein CD4 and its non-covalently linked Src family tyrosine kinase p56lck provides insights into the role of gangliosides in the function of T lymphocytes. These observations support the suggestion that GM3 may elicit CD4·p56lck dissociation via protein kinase (PKC) -d activation and serine phosphorylation [90]. Most recently, it has been shown that exposure of mouse splenocytes to gangliosides results in reduced gene transcription of the T-helper cell type 1 (Th1) -associated cytokines, IL-2, and INF-c, while leaving gene transcription of the Th2-associated cytokines, IL-4, and IL-10 unaffected [87]. Others have reported a ganglioside-induced increase in T cell IL-10 production [91]. Together, these findings suggest that shed tumor gangliosides may shift the balance of the antitumor immune response from the normally predominant Th1 immune response toward the Th2 response, possibly leading to a reduction in the cellular antitumor immune response, which is critical for tumor elimination. Taken together, these observations strongly suggest that the immunosuppressive effects of gangliosides may explain, at least in part, immunosuppression in the cancer patient [92]. To date, attempts to enhance tumor cell recognition and elimination only had limited success. This may, in part, be due to the ability of tumor cells to escape immune recognition by the shedding of tumor gangliosides into the tumor microenvironment. Thus, a potentially therapeutic enhancement of the immune response could be negated by a particular immunosuppressive activity of the tumor. Further understanding and elucidation of the mechanisms by which gangliosides promote cancer progression could be useful in the development of antitumor drugs. Inhibitors of the different processes described above could be realistic targets for the development of antitumoral reagents in the future.

Ligue Contre le Cancer, and the Groupement des Entreprises Françaises dans la Lutte contre le Cancer.

References [1] [2]

[3]

[4]

[5]

[6]

[7]

[8]

[9] [10] [11]

[12]

[13] [14] [15] [16] [17] [18]

Acknowledgements Work in the laboratory of the authors was supported by National Institute of Health grants NS 11853, American Cancer Society grant IRG-105, and US Public Health Service grant NS11853-24. SB was also supported by a fellowship from the Fondation pour la Recherche Médicale, the

461

[19]

[20]

H. Wiegandt, Gangliosides, in: H. Wiegandt (Ed.), Glycolipids, Elsevier Science Publishers, Amsterdam, 1985, pp. 199–260. T. Yamashita, R. Wada, T. Sasaki, C. Deng, U. Bierfreund, K. Sandhoff, R.L. Proia, A vital role for glycosphingolipid synthesis during development and differentiation, Proc. Natl. Acad. Sci. USA 96 (1999) 9142–9147. S. Hakomori, Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism, Cancer Res. 56 (1996) 5309–5318. C.S. Pukel, K.O. Lloyd, L.R. Travassos, W.G. Dippold, H.F. Oettgen, L.J. Old, GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody, J. Exp. Med. 155 (1982) 1133–1147. K. Furukowa, Gangliosides in melanoma, in: K. Furukowa, K.O. Lloyd (Eds.), Gangliosides in Melanoma, Springer-Verlag, Heildeberg, 1990, pp. 15–30. J.M. Carubia, R.K. Yu, L.J. Macala, J.M. Kirkwood, J.M. Varga, Gangliosides of normal and neoplastic human melanocytes, Biochem. Biophys. Res. Commun. 120 (1984) 500–504. M.H. Ravindranath, T. Tsuchida, D.L. Morton, R.F. Irie, Ganglioside GM3:GD3 ratio as an index for the management of melanoma, Cancer 67 (1991) 3029–3035. A. Merzak, S. Koochekpour, G.J. Pilkington, Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro, Neurosci. Lett. 177 (1994) 44–46. S.I. Hakomori, Inaugural article: the glycosynapse, Proc. Natl. Acad. Sci. USA 99 (2002) 225–232. Y. Kong, R. Li, S. Ladisch, Natural forms of shed tumor gangliosides, Biochim. Biophys. Acta 1394 (1998) 43–56. V. Dolo, R. Li, M. Dillinger, S. Flati, J. Manel, B.J. Taylor, A. Pavan, S. Ladisch, Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles, Biochim. Biophys. Acta 1486 (2000) 265–274. M. Rusnati, E. Tanghetti, C. Urbinati, G. Tulipano, S. Marchesini, M. Ziche, M. Presta, Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures, Mol. Biol. Cell 10 (1999) 313–327. F. Chang, R. Li, S. Ladisch, Shedding of gangliosides by human medulloblastoma cells, Exp. Cell Res. 234 (1997) 341–346. R.X. Li, S. Ladisch, Shedding of human neuroblastoma gangliosides, Biochim. Biophys. Acta 1083 (1991) 57–64. T. Kolter, R.L. Proia, K. Sandhoff, Combinatorial ganglioside biosynthesis, J. Biol. Chem. 277 (2002) 25859–25862. R.K. Yu, S. Ando, Structures of some new complex gangliosides of fish brain, Adv. Exp. Med. Biol. 125 (1980) 33–45. K.O. Lloyd, K. Furukawa, Biosynthesis and functions of gangliosides: recent advances, Glycoconj. J. 15 (1998) 627–636. E. Bieberich, T. Tencomnao, D. Kapitonov, R.K. Yu, Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells, J. Neurochem. 74 (2000) 2359–2364. E. Bieberich, S. MacKinnon, J. Silva, D.D. Li, T. Tencomnao, L. Irwin, D. Kapitonov, R.K.Yu, Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases, Biochemistry 41 (2002) 11479–11487. G. van Echten, K. Sandhoff, Ganglioside metabolism. Enzymology, topology, and regulation, J. Biol. Chem. 268 (1993) 5341–5344.

462

S. Birklé et al. / Biochimie 85 (2003) 455–463

[21] J. Inokuchi, M. Jimbo, K. Momosaki, H. Shimeno, A. Nagamatsu, N.S. Radin, Inhibition of experimental metastasis of murine Lewis lung carcinoma by an inhibitor of glucosylceramide synthase and its possible mechanism of action, Cancer Res. 50 (1990) 6731–6737. [22] R. Li, S. Ladisch, Abrogation of shedding of immunosuppressive neuroblastoma gangliosides, Cancer Res. 56 (1996) 4602–4605. [23] L. Lee, A. Abe, J.A. Shayman, Improved inhibitors of glucosylceramide synthase, J. Biol. Chem. 274 (1999) 14662–14669. [24] Y.A. Hannun, Functions of ceramide in coordinating cellular responses to stress, Science 274 (1996) 1855–1859. [25] O. Cuvillier, G. Pirianov, B. Kleuser, P.G. Vanek, O.A. Coso, S. Gutkind, S. Spiegel, Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate, Nature 381 (1996) 800–803. [26] W. Deng, R. Li, S. Ladisch, Influence of cellular ganglioside depletion on tumor formation, J. Natl. Cancer Inst. 92 (2000) 912–917. [27] R. Bjerkvig, O. Engebraaten, O.D. Laerum, P. Fredman, L. Svennerholm, F.D. Vrionis, C.J. Wikstrand, D.D. Bigner, Anti-GM2 monoclonal antibodies induce necrosis in GM2-rich cultures of a human glioma cell line, Cancer Res. 51 (1991) 4643–4648. [28] W.G. Dippold, A. Knuth, K.H. Meyer zum Buschenfelde, Inhibition of human melanoma cell growth in vitro by monoclonal anti-GD3ganglioside antibody, Cancer Res. 44 (1984) 806–810. [29] A.N. Houghton, D. Mintzer, C. Cordon-Cardo, S. Welt, B. Fliegel, S. Vadhan, E. Carswell, M.R. Melamed, H.F. Oettgen, L.J. Old, Mouse monoclonal IgG3 antibody detecting GD3 ganglioside: a phase I trial in patients with malignant melanoma, Proc. Natl. Acad. Sci. USA 82 (1985) 1242–1246. [30] S. Yoshida, S. Fukumoto, H. Kawaguchi, S. Sato, R. Ueda, K. Furukawa, G. Ganglioside, (D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis, Cancer Res. 51 (2001) 4244–4252. [31] G. Zeng, T. Ariga, X.B. Gu, R.K. Yu, Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation, Proc. Natl. Acad. Sci. USA 92 (1995) 8670–8674. [32] E.J. Meuillet, R. Kroes, H. Yamamoto, T.G. Warner, J. Ferrari, B. Mania-Farnell, D. George, A. Rebbaa, J.R. Moskal, E.G. Bremer, Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431, Cancer Res. 59 (1999) 234–240. [33] T. Ariga, G.M. Blaine, H. Yoshino, G. Dawson, T. Kanda, G.C. Zeng, T. Kasama, Y. Kushi, R.K. Yu, Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3, Biochemistry 34 (1995) 11500–11507. [34] G. Zeng, D.D. Li, L. Gao, S. Birkle, E. Bieberich, A. Tokuda, R.K. Yu, Alteration of ganglioside composition by stable transfection with antisense vectors against GD3-synthase gene expression, Biochemistry 38 (1999) 8762–8769. [35] W. Deng, R. Li, M. Guerrera, Y. Liu, S. Ladisch, Transfection of glucosylceramide synthase antisense inhibits mouse melanoma formation, Glycobiology 12 (2002) 145–152. [36] G. Zeng, L. Gao, R.K. Yu, Reduced cell migration, tumor growth and experimental metastasis of rat F-11 cells whose expression of GD3synthase is suppressed, Int. J. Cancer 88 (2000) 53–57. [37] S. Birkle, S. Ren, A. Slominski, G. Zeng, L. Gao, R.K. Yu, Downregulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis, J. Neurochem. 72 (1999) 954–961. [38] S. Birkle, L. Gao, G. Zeng, R.K. Yu, Down-regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells: effects on cellular growth, melanogenesis, and dendricity, J. Neurochem. 74 (2000) 547–554.

[39] S. Tokuyama, S. Moriya, S. Taniguchi, A. Yasui, J. Miyazaki, S. Orikasa, T. Miyagi, Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA, Int. J. Cancer 73 (1997) 410–415. [40] K. Ogura, K. Nara, Y. Watanabe, K. Kohno, T. Tai, Y. Sanai, Cloning and expression of cDNA for O-acetylation of GD3 ganglioside, Biochem. Biophys. Res. Commun. 225 (1996) 932–938. [41] A. Kanamori, J. Nakayama, M.N. Fukuda, W.B. Stallcup, K. Sasaki, M. Fukuda, Y. Hirabayashi, Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: a putative acetyl-CoA transporter, Proc. Natl. Acad. Sci. USA 94 (1997) 2897–2902. [42] W.X. Shi, R. Chammas, A. Varki, Induction of sialic acid 9-Oacetylation by diverse gene products: implications for the expression cloning of sialic acid O-acetyltransferases, Glycobiology 8 (1998) 199–205. [43] M.G. Manfredi, S. Lim, K.P. Claffey, T.N. Seyfried, Gangliosides influence angiogenesis in an experimental mouse brain tumor, Cancer Res. 59 (1999) 5392–5397. [44] D.A. Cheresh, M.D. Pierschbacher, M.A. Herzig, K. Mujoo, Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins, J. Cell Biol. 102 (1986) 688–696. [45] H.K. Kleinman, G.R. Martin, P.H. Fishman, Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen, Proc. Natl. Acad. Sci. USA 76 (1979) 3367–3371. [46] K.M. Yamada, D.W. Kennedy, G.R. Grotendorst, T. Momoi, Glycolipids: receptors for fibronectin?, J. Cell Physiol. 109 (1981) 343–351. [47] R.M. Perkins, S. Kellie, B. Patel, D.R. Critchley, Gangliosides as receptors for fibronectin? Comparison of cell spreading on a ganglioside-specific ligand with that on fibronectin, Exp. Cell Res. 141 (1982) 231–243. [48] A.S. Paller, S.L. Arnsmeier, J.D. Chen, D.T. Woodley, Ganglioside GT1b inhibits keratinocyte adhesion and migration on a fibronectin matrix, J. Invest. Dermatol. 105 (1995) 237–242. [49] X. Wang, P. Sun, A. Al-Qamari, T. Tai, I. Kawashima, A.S. Paller, Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function, J. Biol. Chem. 276 (2001) 8436–8444. [50] D.A. Cheresh, R. Pytela, M.D. Pierschbacher, F.G. Klier, E. Ruoslahti, R.A. Reisfeld, An Arg–Gly–Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2, J. Cell Biol. 105 (1987) 1163–1173. [51] P.R. Crocker, E.A. Clark, M. Filbin, S. Gordon, Y. Jones, J.H. Kehrl, S. Kelm, N. Le Douarin, L. Powell, J. Roder, R.L. Schnaar, D.C. Sgroi, K. Stamenkovic, R. Schauer, M. Schachner, T.K. van den Berg, P.A. van der Merwe, S.M. Watt, A. Varki, Siglecs: a family of sialic-acid binding lectins, Glycobiology 8 (1998) V. [52] A. Ito, K. Handa, D.A. Withers, M. Satoh, S. Hakomori, Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression, FEBS Lett. 504 (2001) 82–86. [53] A.M. Khatib, M. Kontogiannea, L. Fallavollita, B. Jamison, S. Meterissian, P. Brodt, Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells, Cancer Res. 59 (1999) 1356–1361. [54] N. Kojima, M. Shiota, Y. Sadahira, K. Handa, S. Hakomori, Cell adhesion in a dynamic flow system as compared to static system. Glycosphingolipid–glycosphingolipid interaction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B16 melanoma cells to non-activated endothelial cells, J. Biol. Chem. 267 (1992) 17264–17270. [55] D. Sgroi, A. Varki, S. Braesch-Andersen, I. Stamenkovic, CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acidbinding lectin, J. Biol. Chem. 268 (1993) 7011–7018.

S. Birklé et al. / Biochimie 85 (2003) 455–463 [56] N. Kojima, S. Hakomori, Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid–glycolipid interaction, J. Biol. Chem. 266 (1991) 17552–17558. [57] S. Yamamura, K. Handa, S. Hakomori, A close association of GM3 with c-Src and Rho in GM3-enriched microdomains at the B16 melanoma cell surface membrane: a preliminary note, Biochem. Biophys. Res. Commun. 236 (1997) 218–222. [58] M. Ono, K. Handa, S. Sonnino, D.A. Withers, H. Nagai, S. Hakomori, GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy, Biochemistry 40 (2001) 6414–6421. [59] M. Satoh, A. Ito, H. Nojiri, K. Handa, K. Numahata, C. Ohyama, S. Saito, S. Hoshi, S.I. Hakomori, Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells, Int. J. Oncol. 19 (2001) 723–731. [60] J.F. Cajot, I. Sordat, T. Silvestre, B. Sordat, Differential display cloning identifies motility-related protein (MRP1/CD9) as highly expressed in primary compared to metastatic human colon carcinoma cells, Cancer Res. 57 (1997) 2593–2597. [61] M. Adachi, T. Taki, Y. Ieki, C.L. Huang, M. Higashiyama, M. Miyake, Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer, Cancer Res. 56 (1996) 1751–1755. [62] Y. Kawakami, K. Kawakami, W.F. Steelant, M. Ono, R.C. Baek, K. Handa, D.A. Withers, S. Hakomori, Tetraspanin CD9 is a “proteolipid”, and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5dependent cell motility, J. Biol. Chem. 277 (2002) 34349–34358. [63] Y. Kakugawa, T. Wada, K. Yamaguchi, H. Yamanami, K. Ouchi, I. Sato, T. Miyagi, Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression, Proc. Natl. Acad. Sci. USA 99 (2002) 10718–10723. [64] W.F. Steelant, Y. Kawakami, A. Ito, K. Handa, E.A. Bruyneel, M. Mareel, S. Hakomori, Monosialyl-Gb5 organized with Src and FAK in GEM on human breast carcinoma MCF-7 cells defines their invasive properties, FEBS Lett. 53 (2002) 93–98. [65] L.A. Valentino, S. Ladisch, Localization of shed human tumor gangliosides: association with serum lipoproteins, Cancer Res. 52 (1992) 810–814. [66] S. Koochekpour, A. Merzak, G.J. Pilkington, Vascular endothelial growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells in vitro, Cancer Lett. 102 (1996) 209–215. [67] M. Ziche, L. Morbidelli, G. Alessandri, P.M. Gullino, Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio, Lab. Invest. 67 (1992) 711–715. [68] M. Ziche, G. Alessandri, P.M. Gullino, Gangliosides promote the angiogenic response, Lab. Invest. 61 (1989) 629–634. [69] G. Zeng, L. Gao, S. Birkle, R.K. Yu, Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production, Cancer Res. 60 (2000) 6670–6676. [70] P.H. Black, Shedding from normal and cancer-cell surfaces, N. Engl. J. Med. 303 (1980) 1415–1416. [71] R. Olshefski, S. Ladisch, Intercellular transfer of shed tumor cell gangliosides, FEBS Lett. 386 (1996) 11–14. [72] H. Offner, T. Thieme, A.A. Vandenbark, Gangliosides induce selective modulation of CD4 from helper T lymphocytes, J. Immunol. 139 (1987) 3295–3305.

463

[73] J.W. Chu, F.J. Sharom, Gangliosides interact with interleukin-4 and inhibit interleukin-4-stimulated helper T-cell proliferation, Immunology 84 (1995) 396–403. [74] G. Grayson, S. Ladisch, Immunosuppression by human gangliosides II. Carbohydrate structure and inhibition of human NK activity, Cell Immunol. 139 (1992) 18–29. [75] E.V. Dyatlovitskaya, L.D. Bergelson, Glycosphingolipids and antitumor immunity, Biochim. Biophys. Acta 907 (1987) 125–143. [76] C. Dumontet, A. Rebbaa, J. Bienvenu, J. Portoukalian, Inhibition of immune cell proliferation and cytokine production by lipoproteinbound gangliosides, Cancer Immunol. Immunother. 38 (1994) 311–316. [77] F.J. Sharom, A.L. Chiu, T.E. Ross, Gangliosides and glycophorin inhibit T-lymphocyte activation, Biochem. Cell Biol. 68 (1990) 735–744. [78] S. Ladisch, H. Becker, L. Ulsh, Immunosuppression by human gangliosides: I. Relationship of carbohydrate structure to the inhibition of T cell responses, Biochim. Biophys. Acta 1125 (1992) 180–188. [79] S. Ladisch, L. Ulsh, B. Gillard, C. Wong, Modulation of the immune response by gangliosides. Inhibition of adherent monocyte accessory function in vitro, J. Clin. Invest. 74 (1984) 2074–2081. [80] H.W. Ziegler-Heitbrock, E. Kafferlein, J.G. Haas, N. Meyer, M. Strobel, C. Weber, D. Flieger, Gangliosides suppress tumor necrosis factor production in human monocytes, J. Immunol. 148 (1992) 1753–1758. [81] P.T. Massa, Specific suppression of major histocompatibility complex class I and class II genes in astrocytes by brain-enriched gangliosides, J. Exp. Med. 178 (1993) 1357–1363. [82] G.V. Shurin, M.R. Shurin, S. Bykovskaia, J. Shogan, M.T. Lotze, E.M. Barksdale Jr, Neuroblastoma-derived gangliosides inhibit dendritic cell generation and function, Cancer Res. 61 (2001) 363–369. [83] R. McKallip, R. Li, S. Ladisch, Tumor gangliosides inhibit the tumorspecific immune response, J. Immunol. 163 (1999) 3718–3726. [84] S. Ladisch, R. Li, E. Olson, Ceramide structure predicts tumor ganglioside immunosuppressive activity, Proc. Natl. Acad. Sci. USA 91 (1994) 1974–1978. [85] J.W. Chu, F.J. Sharom, Interleukin-2 binds to gangliosides in micelles and lipid bilayers, Biochim. Biophys. Acta 1028 (1990) 205–214. [86] P. Lu, F.J. Sharom, Gangliosides are potent immunosuppressors of IL-2-mediated T-cell proliferation in a low protein environment, Immunology 86 (1995) 356–363. [87] D.N. Irani, Brain-derived gangliosides induce cell cycle arrest in a murine T cell line, J. Neuroimmunol. 87 (1998) 11–16. [88] J. Zhao, K. Furukawa, S. Fukumoto, M. Okada, R. Furugen, H. Miyazaki, K. Takamiya, S. Aizawa, H. Shiku, T. Matsuyama, Attenuation of interleukin 2 signal in the spleen cells of complex ganglioside-lacking mice, J. Biol. Chem. 274 (1999) 13744–13747. [89] M. Sorice, A. Pavan, R. Misasi, T. Sansolini, T. Garofalo, L. Lenti, G.M. Pontieri, L. Frati, M.R. Torrisi, Monosialoganglioside GM3 induces CD4 internalization in human peripheral blood T lymphocytes, Scand. J. Immunol. 41 (1995) 148–156. [90] T. Garofalo, M. Sorice, R. Misasi, B. Cinque, M. Giammatteo, G.M. Pontieri, M.G. Cifone, A. Pavan, A novel mechanism of CD4 down-modulation induced by monosialoganglioside GM3. Involvement of serine phosphorylation and protein kinase C delta translocation, J. Biol. Chem. 273 (1998) 35153–35160. [91] N. Kanda, Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells, Biochem. Biophys. Res. Commun. 256 (1999) 41–44. [92] G. Floutsis, L. Ulsh, S. Ladisch, Immunosuppressive activity of human neuroblastoma tumor gangliosides, Int. J. Cancer 43 (1989) 6–9.