Os mastócitos são células multifuncionais do sistema imunológico que participam em diversos processos biológicos. As funções dos mastócitos estão diretamente relacionados com a sua ativação e, subsequente, liberação de mediadores químicos. Os eventos iniciais da ativação dos mastócitos e da transdução de sinais ocorrem em microdomínios lipídicos (lipid rafts) da membrana plasmática. Os gangliosídeos derivados do GD1b são constituintes dos lipid rafts de mastócitos de roedores. O intercruzamento destes gangliosídeos pelo mAb AA4, resulta na formação de agregados (caps) na superfície celular e promove uma ativação parcial dos mastócitos, sem que ocorra a desgranulação. A ativação é semelhante a observada quando os FcRIs são intercruzados por antígenos multivalentes ligados a IgEs, mas neste caso ocorre a desgranulação. O presente estudo tem como objetivo caracterizar o papel dos gangliosídeos derivados do GD1b na liberação de mediadores de mastócitos da linhagem RBL-2H3. O intercruzamento dos gangliosídeos derivados do GD1b resulta na ativação dos fatores de transcrição NFAT e NFB e esta ativação é mediada pela proteína quinase Syk. A ativação destes fatores de transcrição resulta na liberação de mediadores neo-sintetizados, tais como: TNF-, interleucina (IL)-4. Por outro lado, o intercruzamento dos gangliosídeos derivados de GD1b não induz a liberação dos mediadores neoformados como o leucotrieno B4 (LTB4) e o leucotrieno C4 (LTC4). A agregação dos gangliosídeos derivados do GD1b resulta na desorganização dos lipid rafts e na redistribuição de seus componentes, como demostrado pela análise proteômica. Estes dados mostraram proteínas capazes de desencadear uma ativação parcial dos mastócitos e proteínas reguladoras negativas da desgranulação estão up reguladas, enquanto que proteínas críticas para a transdução do sinal estão down reguladas. Os resultados obtidos neste trabalho demonstram que os gangliosídeos derivados do GD1b desempenham papel crucial na integridade dos lipid rafts modulando a ativação e liberação de mediadores de mastócitos. / Mast cells are immunoregulatory cells that participate in diverse biological events. The action of mast cells is directly related to their activation and subsequent mediator release. Early signal transduction events occur in lipid rafts in the plasma membrane. GD1b-derived gangliosides are known constituents of lipid rafts in rodent mast cells. The cross-linking of these gangliosides by mAb AA4 results in a partial activation of mast cells similar to that observed when FcRIs are cross-linked, but does not result in the mast cell degranulation. With time, the gangliosides bound to mAb AA4 cap on the cell surface. The present study aims to characterize the role of the rodent mast cell specific gangliosides derived from GD1b in mediator release from RBL-2H3 mast cells. Cross-linking the GD1b-derived gangliosides activated the transcription factors NFAT and NFB and this activation was mediated by Syk. The activation of theses transcription factors by cross-linked GD1b-derived gangliosides results in the release of the neo-synthesized mediators TNF- and interleukin (IL)-4. However, cross-linking GD1b-derived gangliosides did not stimulate release of the newly formed mediators leukotriene B4 (LTB4) and leukotriene C4 (LTC4). Capping of GD1b-derived gangliosides disorganized lipid rafts and resulted in a redistribution of lipid raft components. Proteomic analysis showed that proteins that trigger mast cell activation and negative regulatory proteins of degranulation are up regulated, whereas proteins critical for signal transduction are down regulated in mast cells where the gangliosides are capped. The results of this work demonstrate that the mast cell-specific GD1b-derived gangliosides are crucial in maintaining the functional integrity of the lipid rafts and modulate cell activation and subsequent mediator release from mast cells.

Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T16:26:14Z (GMT). No. of bitstreams: 1 Ferro_KarlaPriscilaVieira_M.pdf: 1249697 bytes, checksum: a31e2763981008c52bfdee0373185f3f (MD5) Previous issue date: 2007 / Resumo: A linhagem de camundongos NOD (non obese diabetic) desenvolve espontaneamente diabetes mellitus tipo 1 (DM-1) com marcante similaridade ao observado em humanos, que se estabelece entre 12ª e 24ª semana de vida, com presença de autoanticorpos específicos contra antígenos pancreáticos. Grande parte das células encontradas são linfócitos T CD4+ e T CD8+ e, embora células NK, linfócitos B, células dendríticas e macrófagos também possam ser identificados nas lesões, o desenvolvimento da doença é primariamente dependente de linfócitos T CD4+ e CD8+ auto-reativos. A diferenciação e funcionamento de células ß são regulados por uma variedade de hormônios e fatores de crescimento, incluindo Nerve Growth Factor (NGF). Sabe-se que células-ß pancreáticas expressam receptores funcionais para NGF e esta neurotrofina induz modificações morfológicas e fisiológicas, incluindo estimulação da secreção de insulina. Estudos de terapias para o DM-1 baseadas na intervenção sobre o sistema imunológico revelam que estas podem ser estratégias promissoras para impedir a instalação e/ou evolução da doença. Neste contexto, investigamos os efeitos da administração de GM1 sobre a incidência do DM-1 e insulite em camundongos NOD, expressão de citocinas, NGF e seu receptor de alta afinidade TrkA. Nossos resultados sugerem que administração de GM1 na dose de 100mg/kg/dia em camundongos NOD fêmeas a partir da 4ª semana de vida é capaz de diminuir o índice de infiltrado inflamatório e conseqüentemente a expressão do diabetes, modulando negativamente o infiltrado inflamatório bem como a expressão gênica de citocinas pró-inflamatórias (IL-12, IFN-?, TNF-a e IL-1ß), além de aumentar a expressão gênica e protéica de NGF e TrkA, que pode atuar como regulador de sobrevivência da célula ß de maneira a inibir a apoptose desta célula / Abstract: The strain of NOD mice (non obese diabetic) spontaneously develops diabetes mellitus type 1 (DM-1) with strong similarity to the observed in humans. In this model, the diabetes manifestation occurs among 12th and 24th weeks of life, with presence of pancreas-specific autoantibodies. Great part of the cells are CD4+ and CD8+T cells, and even so NK cells, lymphocytes B, dendritics cells and macrophages also can be identified in the injuries, the development of the disease is essentially dependent of autoreactive CD4+ and CD8+ T cells. It was demonstrated that ? - pancreatic cells express NGF functional receptors and that this neurotrophin induces morphological and physiological modifications in pancreatic ? cells, including stimulation in insulin secretion. The inquiries of therapies for the DM-1 based on the intervention on the immune system disclose that these can be promising strategies to hinder the installation and/or evolution of the disease. In this context, we investigate the effect of GM1 administration on the incidence of DM-1 and insulitis in NOD mice, cytokines expression, NGF and its high affinity receptor TrkA. Our results suggest that administration of GM1 in the dose of 100mg/kg/dia in female NOD mice from 4ª week of life are capable to reduce the index of inflammatory infiltrated and consequently the expression of diabetes, down-modulating the inflammatory infiltrated as well as the gene expression of pro-inflammatory cytokines (IL-12, IFN-?, TNF-? and IL-1?), besides increasing the gene and protein expression of NGF and TrkA, that can act as regulating of ß cell - survival in way to inhibit apoptosis of this cell / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:15:46Z (GMT). No. of bitstreams: 1 Silva_LuisGuilhermeStivanin_M.pdf: 3878753 bytes, checksum: bb8270f964b31680efc65e277f167c0a (MD5) Previous issue date: 2012 / Resumo: Empregando as linhagens de camundongos NOD (non-obese diabetic) de desenvolvimento espontâneo do diabetes mellitus tipo 1 e BALB/c como linhagem controle, administrou-se exogenamente gangliosídeo GM1, mistura de gangliosídeos (GGs) (GM1 21%, GD1a 40%, GD1b 16%, GT1b 19%) e solução salina (0,9% NaCl) estéril da 4ª à 28ª semana de vida. Os efeitos da administração dos gangliosídeos sobre a frequência da manifestação do diabetes, índice de insulite, imunofenotipificação e atividade apoptótica de células presentes em ilhotas pancreáticas de NOD foram verificados por meio de análise glicêmica semanal, técnica colorimétrica com Eosina-Hematoxilina, imunofluorescência e TUNEL. A expressão gênica e os níveis séricos de insulina, além das expressões celular protéica e gênica dos fatores de regeneração GLP-1, PDX-1 e Ngn3 nos tecidos pancreáticos de BALB/c e NOD foram analisados por meio de ELISA, imunofluorescência e RT-PCR em tempo real. Após 28 semanas de tratamento, pôde-se verificar que os animais tratados com GM1 reduziram o diabetes de 70% observado nos animais controle salina, para 38%. Os animais tratados com GGs não apresentaram diabetes. O índice de insulite estava diminuído nos animais tratados com GM1 (p=0.09), GGs (p=0.004) e salina não-diabético (ND) (p=0.02) em relação ao grupo salina diabético (DM). O número de células apoptóticas nas ilhotas dos grupos NOD salina ND e NOD DM estava aumentado em relação aos grupos tratados com GM1 e GGs. Os níveis de insulina sérica estavam aumentados nos grupos BALB/c GGs (p=0.01) e BALB/c GM1 (p=0.03) em relação ao grupo BALB/c salina e nos grupos NOD GGs (p=0.008) e NOD salina ND (p=0.01) em relação ao grupo diabético. Por outro lado, os níveis de expressão gênica de insulina no grupo NOD GM1 (p=0.02) estavam aumentados em relação ao grupo salina. Quanto às expressões protéicas de GLP-1, PDX-1 e Ngn3, em ilhotas pancreáticas e tecido acinar, verificamos aumento no grupo NOD tratado com GGs. O conjunto dos resultados demonstra que os gangliosídeos diminuem a manifestação do diabetes espontâneo na linhagem NOD. Hipotetizamos que uma das propriedades dos gangliosídeos estudados é a de estimular a expressão de GLP-1, PDX-1 e Ngn3 em células do tecido acinar e em células da linhagem endócrina nas ilhotas pancreáticas dos animais tratados seja NOD ou BALB/c. Desta forma abrem-se novas frentes de estudos das propriedades antiinflamatórias e possivelmente regenerativas dos gangliosídeos / Abstract: In the present study we evaluate the properties of GM1 and GGs (21% GM1, 40% GD1a 16% GD1b, 19%GT1b) in NOD (non-obese diabetic) mice and BALB/c as a control lineage. Animals of both lineages were treated with GM1, GGs or saline from 4th to the 28th weeks of life. The ganglioside-treated NOD mice demonstrated a decrease in insulitis compared with saline-treated mice: 70% of saline control animals, 38% of GM1 group and 0% of GGs group. GLP-1 gene expression was increased in GM1-treated BALB/c and in GGs-treated NOD groups in comparison to the saline groups. Insulin gene expression was increased only in the GM1-treated NOD group. Serum insulin levels were increased in ganglioside-treated BALB/c and NOD groups. In the islets, the cell co-labeling of Insulin/GLP1 and somatostatin/GLP1 was increased in NOD and BALB/c gangliosides-treated mice compared to saline-treated mice. PDX-1 and Ngn3 protein expression were increased in pancreatic islets and exocrine tissues of GGs treated NOD mice in comparison to the saline treated group. Results suggest that gangliosides have modulatory properties, decreasing the insulitis score, maintaining of insulin levels, increasing GLP-1 protein expression in ? and ? pancreatic cells and retarding diabetes onset in NOD mice. Similarly to observation in neural tissue, the gangliosides studied could contribute to islets survival. We hypothesize that ganglioside play a role stimulating growth factor expression GLP-1, PDX-1 and Ngn3 in the cells from acinar tissue and islets cells / Mestrado / Clinica Medica / Mestre em Clinica Medica

Please read the abstract in the section 05summary, of this document / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17α− ethinylestradiol induced cholestasis, but the increase of b-series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatis induced bile duct ligation (HO-1 activator- hemine, HO-1 inhibitor- Sn-mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals, but a decrease of some a- and b- series gangliosides was observed. In group with activated HO-1 total sialic acid increased, but the composition of gangliosides...

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17ethinylestradiol induced cholestasis, but the increase of b- series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatic rats (HO-1 activator- hemine, HO- 1 inhibitor- Sn- mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals but without significant changes in gangliosides composition. Lipid sialic acid and gangliosides were not changed in animals with hemine activated HO-1. Expression of mRNA of key...

1 Abstract In this thesis I have focused on the role of lipids in the pathogenesis of liver diseases, specifically on cholestasis and non-alcoholic fatty liver disease (NAFLD). The first major aim was to clarify the changes in liver ganglioside metabolism in various types of cholestasis and to elucidate the role of heme oxygenase-1 (HMOX1) and associated oxidative stress. The second objective was to determine the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) administration on NAFLD development in a rodent dietary model of NAFLD and in patients with metabolic syndrome and NAFLD. Our results suggest that increased ganglioside biosynthesis and their re-distribution might represent a general protective mechanism of hepatocytes under cholestatic conditions (both estrogen-induced and obstructive aetiology). These changes are closely related to oxidative stress and might protect hepatocytes against deleterious effect of accumulated bile acids. The lack of HMOX1 activity and subsequent oxidative stress potentiate pathological changes in the liver and resulted in tissue-specific modulation of synthesis and re-distribution of gangliosides (in vivo and in vitro). Contrary to it, HMOX1 activation has an opposite effect and may represent a general hepatoprotective mechanism. We have proven that observed changes...

Les lysosomes sont importants pour la survie et la fonction des cellules du système nerveux central et en particulier des neurones. Le mécanisme de la reformation des lysosomes est crucial pour maintenir une quantité adéquate de lysosomes fonctionnels dans les cellules. La spatacsine, qui joue un rôle dans le ce mécanisme est impliquée dans la paraplégie spastique de type SPG11 ; une maladie caractérisée par des troubles moteurs et cognitifs sévères. L’utilisation de modèles cellulaires de cette pathologie permet d’étudier les mécanismes physiopathologiques à l’origine d’altérations de la reformation des lysosomes. J’ai montré que la perte de fonction de la spatacsine est responsable de l’accumulation de lipides dans les lysosomes. Ces accumulations sont constituées de gangliosides et de cholestérol et sont présentes dans les autolysosomes perturbant leur recyclage en lysosomes, notamment en empêchant le recrutement de protéines impliquées dans le mécanisme. Les accumulations de gangliosides rendent les neurones à l’exposition au glutamate ce qui suggère que ces altérations pourraient avoir un rôle dans la neurodégénérescence. J’ai aussi montré que l’absence de spatacsine provoque une dérégulation de l’import de Ca2+ extracellulaire par le « store-operated calcium entry » ce qui conduit à altération de l’homéostasie calcique. L’inhibition de l’import de calcium par le SOCE permet de réduire les accumulations de lipides et de rétablir partiellement le recyclage des lysosomes. Ainsi, l’absence de spatacsine induit une altération de l’homéostasie calcique qui participe à l’accumulation de lipides dans le système lysosomal ce qui est délétère pour la survie des neurones. / Lysosomal dysfunctions are involved in a large number of neurodegenerative diseases highlightingthe crucial function of lysosomes in neuron survival and function. The mechanism of lysosomereformation from autolysosomes allow cells to maintain the ool of functional lysosomes.Disruptions of this rocess are involved in athologies affecting the central nervous system. Inparticular, spatacsin that lays a role in lysosome recycling is implicated in hereditary spasticparaplegia type SPG11, a severe disease characterized by motors and cognitive alterations. Thispathology is caused by loss of function mutations in SPG11, encoding spatacsin. The study ofSPG11 cellular models gives the opportunity to decipher the hysiopathological mechanismsunderlying lysosomal reformation disruptions.During my thesis, I showed that loss of spatacsin function induces lipid accumulation in lysosomesand articularly in autolysosomes both in fibroblasts and neurons from Spg11-/- mice. Gangliosidesand cholesterol are among lipids that accumulate in autolysosomes impairing lysosomal membranerecycling by disrupting the recruitment of keys roteins. Neurons with ganglioside accumulationsare more sensitive to glutamate induced neuronal death, suggesting that these accumulations areinvolved in neurodegeneration. These results could be of great importance since accumulations ofgangliosides in lysosomes arise in many diseases.I also showed that loss of spatacsin disrupts extracellular calcium import by the store-operatedcalcium entry (SOCE) leading to an increase in cytosolic calcium levels. Lysosomal calcium contentis also increased in Spg11-/- cells and calcium release from lysosome by TRPML1 is reduced.Inhibiting SOCE and stimulating lysosomal calcium release by TRPML1 reduced lipidsaccumulations in lysosomes and artially restored lysosome reformation.Our data suggest that absence of spatacsin is responsible for a disruption of calcium homeostasisthat contributes to lipid accumulation in autolysosomes, disturbing reformation of lysosomes fromautolysosomes. Inhibiting gangliosides synthesis could be used as a therapeutic strategy. However,understanding how loss of function of spatacsin alters these cellular athways will allow thedevelopment of targeted therapeutic approaches.

The goal of this thesis was to develop a high-throughput shotgun-MS lipidomics method to qualitatively and quantitively analyze the major mammalian brain ganglioside classes: GM1, GD1, GT1 and GQ1. As a starting point for the method to be developed, a modified ganglioside extraction method from Svennerholm and Ladisch was used (Svennerholm and Fredman, 1980; Ladisch and Gillard, 1985). The efficiencies and the impact of different extraction procedures to the overall performance were evaluated with a software called OptiVal™. The evaluation showed that the most important steps of the protocol are the salt concentration of the water phase during the 2-phase extraction, and 10 mM NaCl yielded the best sensitivity. Also, the number of washing steps with water during reverse solid phase extraction using C18 resin has a significant effect. The next step was to find suitable standards for quantification of the individual ganglioside classes. Since deuterated and alike ganglioside standards were commercially not available, we initially used a deuterated PE standard with limited success. A collaboration with the Ludger Johannes lab provided us with modified C17-ganglioside standards. The term “modified” describes the enzymatic exchange of the fatty acid in the hydrophobic tail by a 17-carbon atom long fatty acid. Since odd numbered fatty acids occur very rarely in nature, it is possible to use the measured intensity of the modified ceramide headgroup of 35:1 (Sphingosine C18:1 + Fatty Acid C17:0) to quantify natural gangliosides. Ideally, we would need to have a fitting modified C17-ganglioside standard for each class to be quantified. Since first only GM1 as a modified standard was available, it was necessary to determine response factors (RFs) for the ganglioside classes GD1, GT1 and GQ1. RFs were assessed empirically by titrating a variety of equimolar concentrations of the modified C17-GM1 standard versus wildtype standards of the other ganglioside classes. After establishment of the RFs it was possible to determine the limits of detection (LOD) and quantification (LOQ) for the ganglioside classes GD1, GT1 and GQ1 - with regard to the modified C17-GM1 standard. When the modified C17-standards for GD1 and GT1 became available, I was able to find out whether the correct internal standards are superior to the proxy method via response factors. The results clearly showed that the use of a correct class standards is preferable. For GQ1 no modified C17-standard was obtainable, therefore this class still has to be quantified via RFs. Experiments showed that the modified C17-GT1 standard is best suited for that. Another major goal was to integrate the ganglioside method into the general lipid analysis workflow of the high-throughput shotgun mass spectrometry platform that we were using. To achieve these goals adjustments on the evaluated (=old) protocol had to be done. These adjustments included changes in the extraction steps from the Svennerholm & Ladisch more into the direction of a Bligh & Dyer based extraction method. This meant abandoning the 2-phase extraction step as well as the chloroform/methanol/water (C/M/W) 4:8:3 extraction, in favor of a C/M 10:1 followed by a C/M 2:1 extraction of 150 mM ammonium-bicarbonate water solution. The goal behind this was to enable a combination of the global lipidome extraction (Surma et al., 2015) with the ganglioside extraction. Another important improvement was scaling up the extraction process. The use of standard single solid phase extraction (SPE) cartridges was limiting the extraction throughput to only 24 samples at a time, therefore the single SPE cartridges were replaced with the 96-well SPE SOLA™ plates. To process the SOLA™ plates it was necessary to establish the usage of a vacuum manifold. Combined, these changes lowered the overall process time of the protocol from nearly two working days to one working day, without significant loss of sensitivity regarding the measured sample concentrations. This was assessed by performing the mouse brain tissue titration experiment, with all three modified C17-ganglioside class standards GM1, GD1 and GT1. Finally, the established method was applied to investigate the difference in ganglioside levels in the cerebellum compared to the brain hemispheres in mice of different age. First the C/M 10:1 and 2:1 extraction was done for the analysis of all non-ganglioside lipids in the sample. The leftover water phase was then loaded onto the SOLA™ plates and processed with the new protocol. The results matched the given goals - to establish a protocol to measure and quantify the four major brain ganglioside classes in combination with the global lipidomics in a high-throughput manner - and thus were a success. To the best of our knowledge, this was the first time such a broad lipidomic measurement has been performed, hence no other studies exist to which the outcome could be compared.

Dissialogangliosídios, como GD3 e derivados são marcadores da progressão de melanomas. Para avaliar as possíveis funções desta molécula, transfectamos células de melanócitos com o gene da enzima ST8Sia I, que converte GM3 em GD3. Mostramos que GD3 não interfere na capacidade proliferativa dessas células, porém a expressão de GD3 mostrou-se associada à sobrevivência celular. Melanomas adquirem autonomia quanto às vias dependentes do fator de crescimento de fibroblastos (FGF-1 e -2). A expressão de GD3 não interfere na resposta proliferativa a estes fatores, porém GD3 e outros glicoesfingolipídios de membrana modulam a resposta migratória induzida por FGF-2. A expressão de GD3 sensibiliza as células à morte celular induzida por diferentes quimioterápicos, como cisplatina e vimblastina; porém, torna as células resistentes ao tratamento com temozolamida. A sensibilização ao tratamento com vimblastina, mas não às outras drogas, depende da presença de GD3, como observado por ensaios de depleção metabólica / Disialoganglioside GD3 and its derivatives are melanoma progression markers. To evaluate the possible roles of these molecules along melanoma progression, we have transfected the GD3 synthase gene (ST8Sia I) in a melanocyte cell line. Accumulation of GD3 did not confer any proliferative advantage to melanocytes. However, GD3 expression was associated with cell survival. The autonomic growth of melanomas is in part related to a constitutive activation of fibroblast growth factor dependent pathways. GD3 expression did not alter the proliferative response to either FGF-1 or FGF-2. However, GD3 and other membrane glycospingolipids modulate the motogenic activity of FGF-2. GD3 expression sensitizes melanocytes to chemotherapeutic agent-induced cell death, as cisplatin and vimblastin. On the other hand, GD3 turned melanocytes more resistant to temozolomide. Chemosensitization to vimblastin, but not to the other drugs, was dependent on the presence of GD3 within the cells, as shown by metabolic depletion of glycosphingolipids