Glucosylceramide synthase protects glioblastoma cells against autophagic and apoptotic death induced by temozolomide and Paclitaxel.

Glioblastoma is a deadly cancer with intrinsic chemoresistance. Understanding this property will aid in therapy. Glucosylceramide synthase (GCS) is associated with resistance and poor outcome; little is known about glioblastomas. In glioblastoma cells, temozolomide and paclitaxel induce ceramide increase, which in turn promotes cytotoxicity. In drug-resistant cells, both drugs are unable to accumulate ceramide, increased expression and activity of GCS is present, and its inhibitors hinder resistance. Resistant cells exhibit cross-resistance, despite differing in marker expression, and cytotoxic mechanism. These findings suggest that GCS protects glioblastoma cells against autophagic and apoptotic death, and contributes to cell survival under chemotherapy.

Salvage pathways in glycosphingolipid metabolism.

In this review, the focus is on the role of salvage pathways in glycosphingolipid, particularly, ganglioside metabolism. Ganglioside de novo biosynthesis, that begins with the formation of ceramide and continues with the sequential glycosylation steps producing the oligosaccharide moieties, is briefly outlined in its enzymological and cell-topological aspects. Neo-synthesized gangliosides are delivered to the plasma membrane, where their oligosaccharide chains protrude toward the cell exterior. The metabolic fate of gangliosides after internalization via endocytosis is then described, illustrating: (a) the direct recycling of gangliosides to the plasma membrane through vesicles gemmated from sorting endosomes; (b) the sorting through endosomal vesicles to the Golgi apparatus where additional glycosylations may take place; and (c) the channelling to the endosomal/lysosomal system, where complete degradation occurs with formation of the individual sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) components of gangliosides. The in vivo and in vitro evidence concerning the metabolic recycling of these components is examined in detail. The notion arises that these salvage pathways, leading to the formation of gangliosides and other glycosphingolipids, sphingomyelin, glycoproteins and glycosaminoglycans, represent an important saving of energy in the cell economy and constitute a relevant event in overall ganglioside (or glycosphingolipid, in general) turnover, covering from 50% to 90% of it, depending on the cell line and stage of cell life. Sialic acid is the moiety most actively recycled for metabolic purposes, followed by sphingosine, hexosamine, galactose and fatty acid. Finally, the importance of salvage processes in controlling the active concentrations of ceramide and sphingosine, known to carry peculiar bioregulatory/signalling properties, is discussed.

Effects of sympathetic denervation on follicular distribution, oestradiol and progesterone serum levels in prepubertal hemi-ovariectomized female guinea pig.

Hemi-gonadectomy performed in prepubertal female guinea pigs is followed by a compensatory ovarian hypertrophy (COH) and compensatory ovulation (CO). Sympathetic denervation diminished the COH (left ovary: 28 +/- 1.6% versus 46 +/- 4% (control), P < 0.05; and right ovary: 21.3 +/- 3.2% versus 34.2 +/- 3.7% (control), P < 0.01) and does not modify the CO. The mean follicular diameter increased only in the right ovary of hemi-gonadectomized animals. This increase is greater in the hemi-gonadectomized-denervated groups. The mean follicular diameter measured in the right and left ovaries showed an opposite response in hemi-ovariectomized and hemi-ovariectomized-denervated animals: the diameter increased in the right ovary without modifications in the left. Present results add further support to the participation of ovarian innervation on the mechanisms, which regulate follicular development.

Effects of guanethidine administration on compensatory ovarian hypertrophy, compensatory ovulation and follicular development in the prepubertal female guinea pig.

We analyzed the participation of sympathetic ovarian innervation in the prepubertal female guinea pig on regulation of compensatory ovarian hypertrophy (COH) and compensatory ovulation at puberty. The COH of the left ovary was significantly higher that of the right one (left ovary: 41.5+/-5.2 vs. 27.5+/-5.6%, p<0.05, Kruskal-Wallis test). The sympathetic denervation induced by guanethidine administration beginnings at birth or on day 10 resulted in a significant increase of the COH by each ovary (p<0.05, Kruskal-Wallis test). Only one of the six untreated control guinea pigs sacrificed at the follicular phase ovulate. All the hemiovariectomized animals with the left ovary in situ ovulated, while only two out of five with the right ovary in situ did (100 vs. 40%: p<0.001, Kruskal-Wallis test), unlike the denervated animals, which did not ovulate. The number of corpora lutea present in the ovaries was similar among all groups of animals. These results demonstrate differences in the follicular diameter in untreated female guinea pigs and add further support to the concept of asymmetrical response of the ovaries to denervation.

Effects of functional peripheral sympathetic denervation induced by guanethidine on follicular development and ovulation of the adult female guinea pig.

The present study investigates the effects of functional sympathetic peripheral denervation induced by guanethidine sulphate (GTD) to adult female guinea pigs in the follicular (FPh) or luteal phases (LPh) on their oestrous cyclicity and ovulation. No differences were observed in oestrous cyclicity or the average number of corpora lutea present in the ovaries between the control and denervated animals. Guanethidine sulphate administration resulted in a significant decrease in ovarian norepinephrine content, higher for the left ovary than for the right one. Serum oestrogen and progesterone concentrations, the mean of follicles, and its diameter were different, depending on the oestral cycle in which the treatment was performed. These results suggest that in adult normal female guinea pigs, ovarian innervation participates in the regulation of follicular development in an inhibitory way.

GM3 ganglioside inhibits endothelin-1-mediated signal transduction in C6 glioma cells.

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study.

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.

Basic fibroblast growth factor-induced proliferation of primary astrocytes. evidence for the involvement of sphingomyelin biosynthesis.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either l-[(3)H]serine or [(3)H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

Biomodulatory role of ceramide in basic fibroblast growth factor-induced proliferation of cerebellar astrocytes in primary culture.

To evaluate the role of ceramide in glial growth, primary cultures of quiescent astrocytes from rat cerebellum were stimulated to proliferate by mitogenic doses of basic fibroblast growth factor (bFGF). Parallel to the bFGF mitogenic effect was a marked, and persistent, decrease in cellular ceramide levels. Both in vitro and in culture metabolic studies have led us to exclude both sphingomyelinase and ceramidase involvement in ceramide level variation. Instead, we found evidence of a functional connection between the decrease in ceramide levels and astrocyte proliferation. In fact, cell growth in bFGF-stimulated astrocytes was inhibited by exogenous ceramide and C2-ceramide, maximal inhibition being obtained at a ceramide concentration of 5-10 microM. Under the same conditions, the dihydroderivatives of ceramides were without effect. Following ceramide treatment, the phosphorylation of the MAP kinase isoforms ERK1/2, key components in bFGF-induced cell proliferation, was examined. The administration of antiproliferative doses of ceramide or C2-ceramide, but not of their dihydroderivatives, resulted in a significant inhibition of ERK1/2 activation. In conclusion, our data indicate that the prompt modulation of ceramide levels by bFGF is an early step associated with the signaling pathways responsible for the mitogenic activity of bFGF in astrocytes.

Cultured granule cells and astrocytes from cerebellum differ in metabolizing sphingosine.

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2-h pulse with [C3-(3)H]sphingosine at different doses (0.1-200 nmol/mg of cell protein), both cell types efficiently incorporated the long chain base; the percentage of cellular [(3)H]sphingosine over total label incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell protein and increased at higher doses. Most of the [(3)H]sphingosine taken up underwent metabolic processing by N-acylation, 1-phosphorylation, and degradation (assessed as (3)H(2)O released in the medium). The metabolic processing of exogenous sphingosine was extremely efficient in both cells, granule cells and astrocytes being able to metabolize, respectively, an amount of sphingosine up to 80- and 300-fold the cellular content of this long chain base in 2 h. At the different doses, the prevailing metabolic route of sphingosine was different. At lower doses and in a wide dose range, the major metabolic fate of sphingosine was N-acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine-1-phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine-1-phosphate.

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.

Predominance of the acylation route in the metabolic processing of exogenous sphingosine in neural and extraneural cells in culture.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10-120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

The effects of exogenous sphingosine on Neuro2a cells are strictly related to the overall capacity of cells to metabolize sphingosine.

Neuro2a cells were exposed to different doses (1-40 nmol/10(6) cells) of [C3-3H]sphingosine and the relationship between metabolism and biological effects of sphingosine was investigated. Sphingosine appeared to be rapidly taken up and metabolized. The incorporation of sphingosine was not merely dependent on its concentration but primarily on the dose per cell of administered sphingosine. At low doses, [3H]sphingosine represented a minor portion of the cellular radioactivity, and N-acylated metabolites, particularly ceramide, largely prevailed over degradation products. Concomitantly with ceramide increase, Neuro2a differentiation took place. With increasing exogenous sphingosine/doses, the acylation process reached saturation. From this point on, [3H]sphingosine started accumulating and eventually cell toxicity occurred. In conclusion, the biological effects exerted by exogenous sphingosine on Neuro2a cells are not merely dependent on the long-chain base concentration in the culture medium, but are strictly related to the cellular dose of exogenous sphingosine and to the capacity of cells to metabolize sphingosine.

Metabolic fate of exogenous sphingosine in neuroblastoma neuro2A cells. Dose-dependence and biological effects.

The possible relationship between metabolism and biological effects of sphingosine was investigated in Neuro2a cells. [C3-3H]-sphingosine, administered at different doses (80 pmol-80 nmol/mg cell protein). Amounts up to hundredfold were rapidly taken up and metabolized, the intracellular content of sphingosine being processed within 2 h. At low doses, [3H]-sphingosine represented a minor portion of the cellular radiolabel, and N-acylated metabolites, particularly ceramide, prevailed over degradation products. Neuro2a cell differentiation took place in conjunction with ceramide increase. At increasing exogenous sphingosine/cell ratio, the acylation process became saturated while sphingosine degradation increased proportionally. From this point on [3H]-sphingosine accumulated and cell toxicity occurred. In conclusion, in Neuro2a cells the biological effects exerted by exogenous sphingosine are strictly connected to the exogenous sphingosine/cell ratio and to the capacity of the cell to metabolize sphingosine.

Effects of peripheral sympathetic denervation induced by guanethidine administration on the mechanisms regulating puberty in the female guinea pig.

The effects of peripheral sympathetic denervation induced by guanethidine administration to newborn and 10-day-old female guinea pigs on puberty, ovulation and the follicular population were analysed. Peripheral sympathetic denervation beginning at birth resulted in the loss of ovarian norepinephrine content (0.95. +/- 0.1 ng/mg wet tissue in untreated control animals vs non detectable in treated animals). Guanethidine administration to newborn or 10-day-old guinea pigs advanced puberty (age of vaginal opening: 27 +/- 1.2 days (newborn), 26 +/- 1.7 (10-day-old) vs 37 +/- 0.7 (control), P < 0.001) and ovulation. The number of corpora lutea in control and denervated animals was similar (3.5 +/- 0.2 vs 3.3 +/- 0.3). The relative weight (mg/100 g body weight) of the ovaries and adrenals in the denervated animals autopsied during the late follicular phase (24-48 h after vaginal opening) increased (ovaries: 27.8 +/- 1.3, 28.9 +/- 3.0 vs 20.9 +/- 0.8, P < 0.05; adrenals 36.4 +/- 1.4, 37.0 +/- 0.8 vs 31.6 +/- 1.5, P < 0.05), while the uterine weight diminished (179 +/- 13, 149 +/- 28 vs 292 +/- 20). When the animals were killed during the late luteal phase (9-11 days after vaginal closure), the relative weight of the ovaries of newborn guanethidine-treated animals was higher than that of the control animals (21.4 +/- 1.7 vs 16.8 +/- 1.4, P < 0.05). The mean number of follicles counted in the ovaries of denervated animals was significantly higher than in control animals (1736 +/- 230 vs 969 +/- 147, P < 0.05). The mean diameter of the follicles in the untouched control ovary in animals killed in the late follicular phase was significantly larger than from animals killed in the late luteal phase (263 +/- 3.9 microns vs 248 +/- 3.0 microns, P < 0.01). The mean diameter of the follicles measured in the ovaries of denervated animals was significantly higher than in controls (animals treated from birth 274 +/- 2.0 microns vs 255 +/- 2.4, P < 0.05; animals treated from day 10, 286 +/- 2.3 microns vs 257 +/- 2.3, P < 0.05). When the mean diameter of the follicles in the left and right ovary of the untouched control was analysed, the follicular diameter in the left ovary was significantly larger than in the right ovary (309 +/- 6.0 microns vs 214 +/- 3.9, P < 0.01); the response of the left and right ovaries to sympathetic denervation was the opposite. The results obtained in the present study suggest that ovarian innervation plays a role in the regulation of follicular growth, maturation and atresia which is not related to changes in steroid secretion by the ovary, but to other regulatory mechanisms.

Involvement of a ceramide activated protein phosphatase in the differentiation of neuroblastoma Neuro2a cells.

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydro-derivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

Behaviour of nitric oxide synthase in rat cerebellar granule cells differentiating in culture.

The possible relation between nitric oxide synthase (NOS) activity and neural differentiation was investigated using primary cultures of rat cerebellar granule cells differentiating in culture. NOS activity was measured in the cytosolic and particulate fractions obtained from cell homogenate. In the experimental conditions used the optimal pH for NOS activity was about 6.4, the activity being about 3-fold higher than at pH 7.4. Cerebellar granule cell differentiation was associated with marked increases in NOS activity. In undifferentiated cells the enzyme was almost evenly distributed between the cytosolic and particulate fractions, during differentiation there was a 12-fold increase in activity in the cytosolic enzyme and a 3-fold increase in the particulate one. This indicates a marked preferential enrichment of the cytosolic enzyme during differentiation. Cerebellar granule cells produced and released NO in the culture medium; NO formation being markedly higher in differentiated cells (7-12 DIC) than in undifferentiated (2-3 DIC) ones. These data demonstrate a relationship between NOS expression and NO production and the differentiation of cerebellar granule cells, supporting the notion that NO may play a role in this process.

Sphingoid bioregulators in the differentiation of cells of neural origin.

The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation.