Exogenous GM1 Research Articles

Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric β-Galactosidase correctors.

Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase β-galactosidase (β-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) β-Gal and four disease-related β-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing β-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.

GM1 ganglioside protects against LPS-induced neuroinflammatory and oxidative responses by inhibiting the activation of Akt, TAK1 and NADPH oxidase in MG6 microglial cells.

GM1 is a major brain ganglioside that exerts neurotrophic, neuroprotective and antineuroinflammatory effects. The aim of this study was to obtain insights into the antineuroinflammatory mechanisms of exogenous GM1 in lipopolysaccharide (LPS)-stimulated MG6 mouse transformed microglial cell line. First, we found that GM1 prevented the LPS-induced transformation of microglia into an amoeboid-like shape. GM1 treatment inhibited LPS-induced expression of inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), and proinflammatory cytokines such as TNF-α, IL-1β and IL-6 in MG6 cells. In LPS-treated mice, GM1 also reduced striatal microglia activation and attenuated COX-2 expression. Subsequent mechanistic studies showed that GM1 suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB) and activator protein-1 (AP-1), two critical transcription factors responsible for the production of proinflammatory mediators. GM1 exhibited antineuroinflammatory properties by suppressing Akt/NF-κB signaling and the activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Furthermore, GM1 suppressed LPS-induced activation of transforming growth factor-β-activated kinase 1 (TAK1) and NADPH oxidase 2 (NOX2), upstream regulators of the IκBα/NF-κB and MAPK/AP-1 signaling pathways. GM1 also inhibited NOX-mediated reactive oxygen species (ROS) production and protected against LPS-induced MG6 cell death, suggesting an antioxidant role of GM1. In conclusion, GM1 exerts both antineuroinflammatory and antioxidative effects by inhibiting Akt, TAK1 and NOX2 activation.

Ganglioside GM3-Functionalized Reconstituted High-Density Lipoprotein (GM3-rHDL) as a Novel Nanocarrier Enhances Antiatherosclerotic Efficacy of Statins in apoE-/- C57BL/6 Mice.

Previously, we found that exogenous ganglioside GM3 had an antiatherosclerotic efficacy and that its antiatherosclerotic efficacy could be enhanced by reconstituted high-density lipoprotein (rHDL). In this study, we hypothesized that GM3-functionalized rHDL (i.e., GM3-rHDL) as a nanocarrier can promote the efficacy of traditional antiatherosclerotic drugs (e.g., statins). To test this hypothesis, lovastatin (LT) was used as a representative of statins, and LT-loaded GM3-rHDL nanoparticle (LT-GM3-rHDL or LT@GM3-rHDL; a mean size of ~142 nm) and multiple controls (e.g., GM3-rHDL without LT, LT-loaded rHDL or LT-rHDL, and other nanoparticles) were prepared. By using two different microsphere-based methods, the presences of apolipoprotein A-I (apoA-I) and/or GM3 in nanoparticles and the apoA-I-mediated macrophage-targeting ability of apoA-I/rHDL-containing nanoparticles were verified in vitro. Moreover, LT-GM3-rHDL nanoparticle had a slowly sustained LT release in vitro and the strongest inhibitory effect on the foam cell formation of macrophages (a key event of atherogenesis). After single administration of rHDL-based nanoparticles, a higher LT concentration was detected shortly in the atherosclerotic plaques of apoE-/- mice than non-rHDL-based nanoparticles, suggesting the in vivo plaque-targeting ability of apoA-I/rHDL-containing nanoparticles. Finally, among all nanoparticles LT-GM3-rHDL induced the largest decreases in the contents of blood lipids and in the areas of atherosclerotic plaques at various aortic locations in apoE-/- mice fed a high-fat diet for 12 weeks, supporting that LT-GM3-rHDL has the best in vivo antiatherosclerotic efficacy among the tested nanoparticles. Our data imply that GM3-functionalized rHDL (i.e., GM3-rHDL) can be utilized as a novel nanocarrier to enhance the efficacy of traditional antiatherosclerotic drugs (e.g., statins).

Anti-Inflammatory Effects of GM1 Ganglioside on Endotoxin-Induced Uveitis in Rats

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. Results: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing transforming growth factor-β-activated kinase 1 (TAK1) and reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Conclusions: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases.

Enhanced Anti-Atherosclerotic Efficacy of pH-Responsively Releasable Ganglioside GM3 Delivered by Reconstituted High-Density Lipoprotein

Recently, the atheroprotective role of endogenous GM3 and an atherogenesis-inhibiting effect of exogenous GM3 suggested a possibility of exogenous GM3 being recruited as an anti-atherosclerotic drug. This study seeks to endow exogenous GM3 with atherosclerotic targetability via reconstituted high-density lipoprotein (rHDL), an atherosclerotic targeting drug nanocarrier. Unloaded rHDL, rHDL loaded with exogenous GM3 at a low concentration (GM3L-rHDL), and rHDL carrying GM3 at a relatively high concentration (GM3H-rHDL) were prepared and characterized. The inhibitory effect of GM3-rHDL on lipid deposition in macrophages was confirmed, and GM3-rHDL did not affect the survival of red blood cells. In vivo experiments using ApoE−/− mice fed a high fat diet further confirmed the anti-atherosclerotic efficacy of exogenous GM3 and demonstrated that GM3 packed in HDL nanoparticles (GM3-rHDL) has an enhanced anti-atherosclerotic efficacy and a reduced effective dose of GM3. Then, the macrophage- and atherosclerotic plaque-targeting abilities of GM3-rHD, most likely via the interaction of ApoA-I on GM3-rHDL with its receptors (e.g., SR-B1) on cells, were certified via a microsphere-based method and an aortic fragment-based method, respectively. Moreover, we found that solution acidification enhanced GM3 release from GM3-rHDL nanoparticles, implying the pH-responsive GM3 release when GM3-rHDL enters the acidic atherosclerotic plaques from the neutral blood. The rHDL-mediated atherosclerotic targetability and pH-responsive GM3 release of GM3-rHDL enhanced the anti-atherosclerotic efficacy of exogenous GM3. The development of the GM3-rHDL nanoparticle may help with the application of exogenous GM3 as a clinical drug. Moreover, the data imply that the GM3-rHDL nanoparticle has the potential of being recruited as a drug nanocarrier with atherosclerotic targetability and enhanced anti-atherosclerotic efficacy.

GM1 Is Cytoprotective in GPR37-Expressing Cells and Downregulates Signaling.

G-protein-coupled receptors (GPCRs) are commonly pharmacologically modulated due to their ability to translate extracellular events to intracellular changes. Previously, studies have mostly focused on protein–protein interactions, but the focus has now expanded also to protein–lipid connections. GM1, a brain-expressed ganglioside known for neuroprotective effects, and GPR37, an orphan GPCR often reported as a potential drug target for diseases in the central nervous system, have been shown to form a complex. In this study, we looked into the functional effects. Endogenous GM1 was downregulated when stably overexpressing GPR37 in N2a cells (N2aGPR37-eGFP). However, exogenous GM1 specifically rescued N2aGPR37-eGFP from toxicity induced by the neurotoxin MPP+. The treatment did not alter transcription levels of GPR37 or the enzyme responsible for GM1 production, both potential mechanisms for the effect. However, GM1 treatment inhibited cAMP-dependent signaling from GPR37, here reported as potentially consecutively active, possibly contributing to the protective effects. We propose an interplay between GPR37 and GM1 as one of the many cytoprotective effects reported for GM1.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain

A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Lentiviral-mediated knock-down of GD3 synthase protects against MPTP-induced motor deficits and neurodegeneration

Converging evidence demonstrates an important role for gangliosides in brain function and neurodegenerative diseases. Exogenous GM1 is broadly neuroprotective, including in rodent, feline, and primate models of Parkinson’s disease, and has shown positive effects in clinical trials. We and others have shown that inhibition of the ganglioside biosynthetic enzyme GD3 synthase (GD3S) increases endogenous levels GM1 ganglioside. We recently reported that targeted deletion of St8sia1, the gene that codes for GD3S, prevents motor impairments and significantly attenuates neurodegeneration induced by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The current study investigated the effects of GD3S inhibition on the neurotoxicity and parkinsonism induced by MPTP. Mice were injected intrastriatally with a lentiviral-vector-mediated shRNA construct targeting GD3S (shGD3S) or a scrambled-sequence control (scrRNA). An MPTP regimen of 18 mg/kg x 5 days reduced tyrosine-hydroxylase-positive neurons in the substantia nigra pars compacta of scrRNA-treated mice by nearly two-thirds. In mice treated with shGD3S the MPTP-induced lesion was approximately half that size. MPTP induced bradykinesia and deficits in fine motor skills in mice treated with scrRNA. These deficits were absent in shGD3S-treated mice. These results suggest that inhibition of GD3S protects against the nigrostriatal damage, bradykinesia, and fine-motor-skill deficits associated with MPTP administration.

Chloroquine Restores Ganglioside Homeostasis and Improves Pathological and Behavioral Outcomes Post-stroke in the Rat.

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1μM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45mg/kgi.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3days post-stroke and partially prevented complex ganglioside depletion 21days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.

Disease‐modifying effects of ganglioside GM1 in Huntington's disease models

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by motor, cognitive and psychiatric problems. Previous studies indicated that levels of brain gangliosides are lower than normal in HD models and that administration of exogenous ganglioside GM1 corrects motor dysfunction in the YAC128 mouse model of HD. In this study, we provide evidence that intraventricular administration of GM1 has profound disease‐modifying effects across HD mouse models with different genetic background. GM1 administration results in decreased levels of mutant huntingtin, the protein that causes HD, and in a wide array of beneficial effects that include changes in levels of DARPP32, ferritin, Iba1 and GFAP, modulation of dopamine and serotonin metabolism, and restoration of normal levels of glutamate, GABA, L‐Ser and D‐Ser. Treatment with GM1 slows down neurodegeneration, white matter atrophy and body weight loss in R6/2 mice. Motor functions are significantly improved in R6/2 mice and restored to normal in Q140 mice, including gait abnormalities that are often resistant to treatments. Psychiatric‐like and cognitive dysfunctions are also ameliorated by GM1 administration in Q140 and YAC128 mice. The widespread benefits of GM1 administration, at molecular, cellular and behavioural levels, indicate that this ganglioside has strong therapeutic and disease‐modifying potential in HD.

Ganglioside GM3 induces cumulus cell apoptosis through inhibition of epidermal growth factor receptor-mediated PI3K/AKT signaling pathways during in vitro maturation of pig oocytes.

Gangliosides are components of the mammalian plasma membrane that help regulate receptor signaling. Ganglioside GM3, for example, plays an important role in initiating apoptosis in cancer cells; however, physiological roles for GM3 in normal processes, such as during pig oocyte maturation, are not clear. The aim of this study was to investigate the functional link between GM3 and cellular apoptosis in porcine cumulus-oocyte-complexes (COCs) during in vitro maturation. Our results indicated that denuded oocytes possess less ST3GAL5, a GM3-synthesizing enzyme, than cumulus cells or COCs after 44 hr of in vitro maturation. GM3 also affected the meiotic maturation of cultured pig oocytes, as evaluated by orcein staining. In vitro treatment of COCs with exogenous GM3 also reduced cumulus cell expansion, the proportion of meiotic maturation, and increased cumulus cell transcription of PTX3, TNFAIP6, and HAS2. Interestingly, GM3 treatment reduced the expression of Epidermal growth factor receptor (EGFR)-mediated Phosphoinositide 3-kinase/AKT signaling proteins in COCs in a concentration-dependent manner, instead increasing the abundance of pro-apoptotic factors such as AIF, activated Caspase 9, cleaved PARP1, and Caspase 3 were. Thus, GM3 might affect porcine oocyte maturation via suppression of EGFR-mediated PI3K/AKT signaling and/or induction of apoptosis during in vitro maturation.

Monosialyl Ganglioside GM3 Decreases Apolipoprotein B-100 Secretion in Liver Cells.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

Exogenous GM1 ganglioside increases accumbal BDNF levels in rats

Gangliosides are compounds that are abundant throughout the CNS, participating actively in neuroplasticity. We previously described that exogenous GM1 ganglioside pretreatment enhances the rewarding properties of cocaine, evidenced by a lower number of sessions and/or dosage necessary to induce conditioned place preference (CPP). Since GM1 pretreatment did not modify cocaine's pharmacokinetic parameters, we suspected that the increased rewarding effect found might be mediated by BDNF, a neurotrophic factor closely related to cocaine addiction. This study was performed to investigate the possibility that GM1 may induce changes in BDNF levels in the nucleus accumbens (NAc), a core structure in the brain's reward circuitry, of rats submitted to three conditioning sessions with cocaine (10mg/kg, i.p.). The results demonstrate that GM1 administration, which showed no rewarding effect by itself in the CPP, induced a significant increase of BDNF protein levels in the NAc, which may account for the increased rewarding effect of cocaine shown in the CPP paradigm.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling

Ganglioside GM3 plays a well-documented and important role in the regulation of tumor cell proliferation, invasion, and metastasis by modulating tyrosine kinase growth factor receptors. However, the effect of GM3 on the hepatocyte growth factor receptor (HGFR, cMet) has not been fully delineated. In the current study, we investigated how GM3 affects cMet signaling and HGF-stimulated cell motility and migration using three hepatic cancer cell lines of mouse (Hca/A2, Hca/16A3, and Hepa1-6). Decreasing GM3 expression with the use of P4, a specific inhibitor for ganglioside synthesis inhibited the HGF-stimulated phosphorylation of cMet and activity of PI3K/Akt signaling pathway. In contrast, the increased expression of GM3 as a result of adding exogenous GM3 enhanced the HGF-stimulated phosphorylation of cMet and activity of PI3K/Akt signaling pathway. Furthermore, HGF-stimulated cell motility and migration in vitro were inhibited by reduced expression of GM3 and enhanced by increased expression of GM3. All the observations indicate that ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling.

Ganglioside GM3 inhibits hepatoma cell motility via down‐regulating activity of EGFR and PI3K/AKT signaling pathway

Two related sublines derived from murine ascites hepatoma cell lines Hca-F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3-suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway.

Antitumor Effects of Exogenous Ganglioside GM3 on Bladder Cancer in an Orthotopic Cancer Model

To investigate the therapeutic effects of exogenous gangliosides GM3 on human bladder cancer cell lines and the severe combined immunodeficiency mouse model of orthotopic bladder cancer. Human bladder cancer cell lines YTS-1, T24, 5637, and KK47 were used in the study. Invitro cytotoxicity of GM3 was assessed using the cell counting kit-8. Cell adhesion was determined using a spreading assay. Phosphorylation of epidermal growth factor receptor was determined by Western blotting. Invivo, the orthotopic bladder cancer model was established using severe combined immunodeficiency mice and GM3 was administered intravesically by way of a transurethral catheter. GM3 inhibited the proliferation of all the bladder cancer cell lines tested. The addition of GM3 decreased cell adhesion and epidermal growth factor-dependent phosphorylation of epidermal growth factor receptor. Direct instillation of GM3 into the bladder of the orthotopic model significantly inhibited tumor growth. Our results suggest exogenous GM3 as a potential therapeutic agent for treating bladder cancer.

Effects of exogenous ganglioside GM1 on different stages of cell spreading studied by directly quantifying spreading rate

We developed novel methods to directly quantify cell spreading rate. By comparing our methods with traditional methods, we found that the enhancement effects of fetal calf serum (FCS) or the inhibitory effects of exogenous ganglioside GM1 occurred at different stages of cell spreading. GM1 mainly influenced the early and late stages of cell spreading of HUVECs. In the presence of 0.5% FCS, GM1 significantly impaired the area-based spreading rates (127.4 ± 35.7 μm(2)/h and 22.2 ± 3.8 μm(2)/h, respectively) on the early (0-0.5 h) and late (12-24 h) stages of cell spreading compared with the controls (238.1 ± 11.7 μm(2)/h and 35.4 ± 19.5 μm(2)/h, respectively), which was confirmed by the data on the GM1-induced changes in average length of actin filaments during cell spreading. The real-time observation and quantification of cold-induced de-spreading of GM1-free or GM1-treated HUVECs further confirmed that GM1 can influence cell de-spreading process having inhibitory (0-10 min) or enhancement (10-20 min or 40-50 min) effects on different stages. The methods can be recruited for investigating effects of other reagents on different stages of cell spreading.

Glycosphingolipids Regulate Ameloblastin Expression in Dental Epithelial Cells

Neurotrophin 4 (NT-4) and its receptors regulate the differentiation of ameloblasts in tooth development. Gangliosides, sialic acids that contain glycosphingolipids (GSLs), are involved in a variety of membrane-associated cell physiological functions such as ligand-receptor signal transmission. However, the expression patterns and functions of GSLs during tooth development remain unclear. In this study, we identified strong expressions of GM3 and LacCer in dental epithelium, which give rise to differentiation into enamel-secreting ameloblasts. Exogenous GM3 and LacCer in dental epithelial cells induced the expression of ameloblastin (Ambn), while it was also interesting that GM3 synergistically exerted enhancement of NT-4-mediated Ambn expression. In addition, consistently exogenous GM3 and LacCer in dental epithelial cells induced distinct activation of extracellular signal-regulated kinase 1/2 (ERK1/2), an event upstream of the expression of Ambn. Furthermore, depletion of GSLs from dental epithelial cells by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibited Ambn expression as well as phosphorylation of ERK1/2. In contrast, exogenous addition of GM3 or LacCer rescued the phosphorylation of ERK1/2 repressed by pre-treatment with D-PDMP. Taken together, these results suggest that GM3 and LacCer are essential for NT-4-mediated Ambn expression, and contribute to dental epithelial cell differentiation into ameloblasts.