Riccardo Ghidoni

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Abstract: Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro‐2a clone, in monolayer culture. GM1 bound to neuro‐2a cells in three distinct forms, one removable by treatment with serum‐containing solutions, one serum‐resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell‐associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10−6M); the trypsin‐labile and ‐stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro‐2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite‐bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10−4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite‐bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin‐labile and ‐stable forms of cell‐associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, <1:20,000–1:50,000. Therefore the neuritogenic effect can be attributed to ganglioside itself. The results obtained suggest that under the experimental conditions used the stimulation of neuro‐2a cell differentiation by GM1 is related to changes of the plasma membrane properties following association of exogenous GM1 molecules. This would facilitate the spontaneous process of differentiation, or enhance cell responsiveness to differentiating factors present in the serum.

Regulation of Autophagy by Sphingosine Kinase 1 and Its Role in Cell Survival during Nutrient Starvation

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

Resveratrol induces growth inhibition and apoptosis in metastatic breast cancer cells via de novo ceramide signaling

Resveratrol (3,4‘,5‐trans‐trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol can induce growth inhibition and apoptosis in MDA‐MB‐231, a highly invasive and metastatic breast cancer cell line, in concomitance with a dramatic endogenous increase of growth inhibitory/proapoptotic ceramide. We found that accumulation of ceramide derives from both de novo ceramide synthesis and sphingomyelin hydrolysis. More specifically we demonstrated that ceramide accumulation induced by resveratrol can be traced to the activation of serine palmitoyltransferase (SPT), the key enzyme of de novo ceramide biosynthetic pathway, and neutral sphingomyelinase (nSMase), a main enzyme involved in the sphingomyelin/ceramide pathway. However, by using specific inhibitors of SPT, myriocin and L‐ cycloserine, and nSMase, gluthatione and manumycin, we found that only the SPT inhibitors could counteract the biological effects induced by resveratrol. Thus, resveratrol seems to exert its growth inhibitory/apoptotic effect on the metastatic breast cancer cell line MDA‐MB‐231 by activating the de novo ceramide synthesis pathway.

Interactions of GM1 Ganglioside with Crude Rat Brain Neuronal Membranes

The binding of GM1, ganglioside to crude preparations of rat brain neuronal membranes was studied, the following results being obtained: (a) the binding process followed a biphasic kinetics, which displayed a break at 0.07–0.08 X 10−6m GM1, concentration; (b) the features of the binding process at GM1, concentrations below the break and, over the break, above 10‐6m appeared to be different. Below the break the process proceeded slowly and brought a stable and irreversible association of GM1, molecules to the membranes. Over 10‐6m the process was much more rapid and caused GM1, molecules to interact in such a way that they were releasable by washing and could exchange with newly added free ganglioside; (c) the two binding processes displayed the characteristics of a saturation phenomenon; (d) in both cases, GM1, taken up was freely available to galactose oxidase, indicating that the oligosaccharide chains protrude from the membrane surface. We postulate that GM1, occurs, below and above the break, in different physical forms, each of them having a different mechanism of interaction with the membrane. Above 10‐6m GM1, interacts as micelles, and the basis of the micelle‐membrane inter action is a fusion process. Below the break, in the 10−8–10−7m range, the binding is the result of hydrophobic interactions between sites on the membrane and the hydrophobic portion of individual ganglioside molecules, most likely in the monomeric form. Toffano G. et al. Interactions of GM1, ganglioside with crude rat brain neuronal membranes. J. Neurochem.35, 861–866 (1980).

Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides

The influence of different gangliosides (GM1, GD1a, GT1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5- and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from GM1 to GD1a and GT1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca2+ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, therefore returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface--between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions--which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca2+ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.

Recognition by two-dimensional thin-layer chromatography and densitometric quantification of alkali-labile gangliosides from the brain of different animals.

A simple method for recognition and quantification of alkali-labile gangliosides is described. The method was worked out using authentic alkali-labile gangliosides in pure form (9-O-Ac-GT1b; 9-O-Ac-GQ 1b; lactone form of GD 1b) and applied to ganglioside mixtures from the brain of mouse, rat, rabbit, pig, and pigeon. The method consists of two-dimensional thin-layer chromatography on silica gel high-performance thin-layer chromatography plates employing the same solvent, chloroform/methanol/0.2% aqueous CaCl2, 50/40/10, for both runs. Prior to the second run the plate is exposed at room temperature for 5 h to ammonia vapors in order to split alkali-labile linkages. At the end of chromatography alkali-stable gangliosides appear lined along a diagonal starting from the origin; the spots corresponding to alkali-labile gangliosides lie out of the diagonal and can be individually detected and quantified on the basis of their sialic acid content. Up to 15 different spots, corresponding to as many alkali-labile gangliosides, can be recognized by this procedure.

Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells

Resveratrol has both apoptosis and autophagy-promoting activities in different cancer cells. Dihydroceramide is the immediate precursor of the apoptotic mediator ceramide in the de novo sphingolipid synthesis pathway. Here we demonstrate that resveratrol induces autophagy in HGC-27 cells, with no sign of cell death. Autophagy occurs after an increase in dihydroceramides by inhibition of dihydroceramide desaturase. The effects of resveratrol are mimicked by a dihydroceramide desaturase inhibitor. These results demonstrate that resveratrol-induced autophagy occurs with a rise in intracellular dihydroceramide levels as the result of inhibition of dihydroceramide desaturases activity and that dihydroceramide accumulation is responsible for autophagy promotion.

Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways

Insulin‐like growth factor binding protein (IGFBP)‐3 has both growth‐inhibiting and growth‐promoting effects at the cellular level. The cytotoxic action of several anticancer drugs is linked to increased ceramide generation through sphingomyelin hydrolysis or de novo biosynthesis. Herein, we investigated the role of IGFBP‐3 on apoptosis of human umbilical vein endothelial cells (HUVEC) and its relationship with ceramide levels. We report that IGFBP‐3 exerts dual effects on HUVEC, potentiating doxorubicin‐induced apoptosis but enhancing survival in serum‐starved conditions. Ceramide was increased by IGFBP‐3 in the presence of doxorubicin and decreased when IGFBP‐3 was added alone to cells cultured in serum‐free medium. The protection exerted by the ceramide synthase inhibitor fumonisin B1 over doxorubicin‐induced apoptosis was enhanced by IGFBP‐3 with concomitant reduction of ceramide levels. IGFBP‐3 alone activated sphingosine kinase (SK) and increased SK1 mRNA; the SK inhibitor N,N‐dimethylsphingosine (DMS) blocked IGFBP‐3 antiapoptotic effect. Moreover, IGFBP‐3 increased IGF‐I mRNA and dramatically enhanced IGF‐I release. IGF‐I receptor (IGF‐IR) and its downstream signaling pathways Akt and ERK were phosphorylated by IGFBP‐3, whereas inhibition of IGF‐IR phosphorylation with tyrphostin AG1024 suppressed the antiapopoptic effect of IGFBP‐3. Finally, IGFBP‐3 increased endothelial cell motility in all experimental conditions. These findings provide evidence that IGFBP‐3 differentially regulates endothelial cell apoptosis by involvement of the sphingolipid signaling pathways. Moreover, the survival effect of IGFBP‐3 seems to be mediated by the IGF‐IR.

Normal-phase high-performance liquid chromatographic separation of non-derivatized ganglioside mixtures

A new analytical and semi-preparative high-performance liquid chromatographic method for the separation of a brain ganglioside mixture into individual components is described. Gangliosides were applied to a LiChrosorb-NH2 column and eluted with the solvent system acetonitrile-phosphate buffer at different volume ratios and ionic strengths. The elution profile was monitored by flow-through detection of UV absorbance at 215 nm. The separation of mono- to polysialogangliosides was performed in one step in a total elution time lower than 90 min and with high reproducibility.

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. GM1 and GD1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris--HCl buffer (pH 6.8) at 37 degrees C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 A argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c0) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius RH, and also contained information on the polydispersity of the sample. We find co less than 1 x 10(-6) M for both GM1 and GD1a, M = 532000 +/- 50000 and RH = 63.9 +/- 2 A for GM1, and M = 417000 +/- 40000 and RH = 59.5 +/- 2 A for GD1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range, and, in particular of Ca2+, did not influence significantly the values of co and the main features of the micelle.