Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

Inhibition of ceramide biosynthesis preserves photoreceptor structure and function in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a genetic disease causing progressive apoptotic death of photoreceptors and, ultimately, incurable blindness. Using the retinal degeneration 10 (rd10) mouse model of RP, we investigated the role of ceramide, a proapoptotic sphingolipid, in retinal degeneration. We also tested the possibility that photoreceptor loss can be slowed or blocked by interfering with the ceramide signaling pathway of apoptosis in vivo. Retinal ceramide levels increased in rd10 mice during the period of maximum photoreceptor death. Single intraocular injections of myriocin, a powerful inhibitor of serine palmitoyl-CoA transferase, the rate-limiting enzyme of ceramide biosynthesis, lowered retinal ceramide levels to normal values and rescued photoreceptors from apoptotic death. Noninvasive treatment was achieved using eye drops consisting of a suspension of solid lipid nanoparticles loaded with myriocin. Short-term noninvasive treatment lowered retinal ceramide in a manner similar to intraocular injections, indicating that nanoparticles functioned as a vector permitting transcorneal drug administration. Prolonged treatment (10-20 d) with solid lipid nanoparticles increased photoreceptor survival, preserved photoreceptor morphology, and extended the ability of the retina to respond to light as assessed by electroretinography. In conclusion, pharmacological targeting of ceramide biosynthesis slowed the progression of RP in a mouse model, and therefore may represent a therapeutic approach to treating this disease in humans. Transcorneal administration of drugs carried in solid lipid nanoparticles, as experimented in this study, may facilitate continuous, noninvasive treatment of patients with RP and other retinal pathologies.

Supplementation of creatine and ribose prevents apoptosis in ischemic cardiomyocytes.

BACKGROUND/AIMS: To alleviate ischemia-induced injury in the myocardium, a tissue that depends critically on energy-yielding processes, creatine may be used to enhance energy metabolism, whereas D-ribose may provide building blocks for ATP synthesis. We test the hypothesis that simultaneous supplementation of creatine+D-ribose protects non-irreversibly injured ischemic cardiomyocytes by reducing apoptosis. RESULTS: When H9c2 cardiomyocytes were exposed to 24-h ischemia (1% O(2) with glucose deprivation), viability was severely compromised, but administration of 2.5 mM creatine + 5 mM D-ribose alleviated the fall in viability, whereas 2.5 mM creatine or 5 mM D-ribose did not. These findings correlated with up-regulation of protein kinase B (Akt) phosphorylation. Creatine+D-ribose also blunted adenosine monophosphate-activated protein kinase (AMPK) and down-regulated apoptosis by reducing caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. CONCLUSIONS: Simultaneous administration of creatine+D-ribose confers anti-ischemic protection that was absent when treating cardiomyocytes with either creatine or D-ribose. The involved mechanisms stem from the Akt and AMPK signaling pathways. These findings may form the basis of a paradigm whereby re-energization of non-irreversibly damaged cardiomyocytes is a critical step to counteract apoptosis.

Sphingolipids in macroautophagy.

Sphingolipids are constituents of biological membranes. Ceramide and sphingosine 1-phosphate (S1P) also act as second messengers and are part of a rheostat system, in which ceramide promotes cell death and growth arrest, and S1P induces proliferation and maintains cell survival. As macroautophagy is a lysosomal catabolic mechanism involved in determining the duration of the lifetime of cells, we raised the question of its regulation by sphingolipid messengers. Using chemical and genetic methods, we have shown by GFP-LC3 staining and analysis of the degradation of long-lived proteins that both ceramide and S1P stimulate autophagy.

Disruption of retinoic acid receptor alpha reveals the growth promoter face of retinoic acid.

BACKGROUND: Retinoic acid (RA), the bioactive derivative of Vitamin A, by epigenetically controlling transcription through the RA-receptors (RARs), exerts a potent antiproliferative effect on human cells. However, a number of studies show that RA can also promote cell survival and growth. In the course of one of our studies we observed that disruption of RA-receptor alpha, RARalpha, abrogates the RA-mediated growth-inhibitory effects and unmasks the growth-promoting face of RA (Ren et al., Mol. Cell. Biol., 2005, 25:10591). The objective of this study was to investigate whether RA can differentially govern cell growth, in the presence and absence of RARalpha, through differential regulation of the "rheostat" comprising ceramide (CER), the sphingolipid with growth-inhibitory activity, and sphingosine-1-phosphate (S1P), the sphingolipid with prosurvival activity. METHODOLOGY/PRINCIPAL FINDINGS: We found that functional inhibition of endogenous RARalpha in breast cancer cells by using either RARalpha specific antagonists or a dominant negative RARalpha mutant hampers on one hand the RA-induced upregulation of neutral sphingomyelinase (nSMase)-mediated CER synthesis, and on the other hand the RA-induced downregulation of sphingosine kinase 1, SK1, pivotal for S1P synthesis. In association with RA inability to regulate the sphingolipid rheostat, cells not only survive, but also grow more in response to RA both in vitro and in vivo. By combining genetic, pharmacological and biochemical approaches, we mechanistically demonstrated that RA-induced growth is, at least in part, due to non-RAR-mediated activation of the SK1-S1P signaling. CONCLUSIONS/SIGNIFICANCE: In the presence of functional RARalpha, RA inhibits cell growth by concertedly, and inversely, modulating the CER and S1P synthetic pathways. In the absence of a functional RARalpha, RA-in a non-RAR-mediated fashion-promotes cell growth by activating the prosurvival S1P signaling. These two distinct, yet integrated processes apparently concur to the growth-promoter effects of RA.

Resveratrol sensitization of DU145 prostate cancer cells to ionizing radiation is associated to ceramide increase.

Radiotherapy is an established therapeutic modality for prostate cancer. Since it is well known that radiotherapy is limited due to its severe toxicity towards normal cells at high dose and minimal effect at low dose, the search for biological compounds that increase the sensitivity of tumors cells to radiation may improve the efficacy of therapy. Resveratrol, a natural antioxidant, was shown to inhibit carcinogenesis in animal models, and to block the process of tumor initiation and progression. The purpose of this study was to examine whether or not resveratrol can sensitize DU145, an androgen-independent human prostate cancer cell line, to ionizing radiation. We report here that DU145 cells are resistant to ionizing radiation-induced cell death, but pretreatment with resveratrol significantly enhances cell death. Resveratrol acts synergistically with ionizing radiation to inhibit cell survival in vitro. Resveratrol also potentiates ionizing radiation-induced ceramide accumulation, by promoting its de novo biosynthesis. This confirms ceramide as an effective mediator of the anticancer potential induced by resveratrol.

Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and sphingosine 1-phosphate (S1P) constitute a rheostat system in which ceramide promotes cell death and S1P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S1P, respectively.

Resveratrol impairs the formation of MDA-MB-231 multicellular tumor spheroids concomitant with ceramide accumulation.

Resveratrol exerts a drastic growth inhibitory effect on human breast cancer MDA-MB-231 cells grown both in vitro and in vivo. Here we show that resveratrol affects the aggregation properties of MDA-MB-231 cells into multicellular tumor spheroids (MCTSs), in association with induction of de novo synthesis of ceramide. After 9 days of 3D growth in the presence of resveratrol (64 microM), MDA-MB-231 cells formed significantly smaller MCTSs. Further, cells from these aggregates failed to form colonies. Addition of resveratrol (64 microM) to preformed MDA-MB-231 MCTSs caused no significant size change, consistent with lack of ceramide induction. Only some apoptotic blebs were found on the MCTSs surface. Altogether these findings indicate that resveratrol might be effective for prevention of breast cancer cell growth.

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 SK1(wt)-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.

Synthesis of a resveratrol analogue with high ceramide-mediated proapoptotic activity on human breast cancer cells.

Resveratrol, a natural product with a stilbene structure, exerts profound proapoptotic activity in human cancer cells, by triggering the accumulation of ceramide, a bioactive sphingolipid. We studied the biological effects of seven methoxylated and/or naphthalene-based resveratrol analogues and compared these compounds with resveratrol with the objective to identify an analogue with higher ceramide-mediated proapoptotic activity relative to resveratrol. Here we show that the compound with three hydroxyls and a naphthalene ring is the most effective in triggering apoptosis coupled to the induction of endogenous ceramide in human cancer cells.

Inhibitory effect of aureobasidin A on Toxoplasma gondii.

The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.

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.

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

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.

Increased biosynthesis of glycosphingolipids in congenital disorder of glycosylation Ia (CDG-Ia) fibroblasts.

Congenital disorder of glycosylation Ia (CDG-Ia) is an autosomal recessive disease, characterized by the impaired biosynthesis of the N-linked oligosaccharide chains of proteins due to a deficiency of phosphomannomutase (PMM), the enzyme converting mannose-6-phosphate into mannose-1-phosphate. We investigated the consequences of the altered N-linked glycoprotein (GP) biosynthesis on the quantity and quality of glycosphingolipids (GSLs) in fibroblasts of CDG-Ia patients. First, we found that CDG-Ia fibroblasts contain an increased amount of total GSLs when compared with normal fibroblasts. Further, we assessed by metabolic labeling of CDG-Ia fibroblasts with radioactive sugar precursors, including galactose and N-acetylmannosamine, that a diminished biosynthesis of cellular GPs is antagonized by an increased biosynthesis of GSLs. An increased GSL biosynthesis was also observed by means of radiolabeled lipid precursors including sphingosine and lactosylceramide. Notably, also the degradation of GLSs is slowed down in CDG-Ia fibroblasts. Finally, when we labeled normal human fibroblasts and CHO cells with radioactive galactose in the presence and absence of deoxymannojirimycin (dMM), an inhibitor of N-glycan processing, we found that this cellular model mimics what occurs in CDG-Ia fibroblasts. Since an inverse relationship between GP expression and GSL content does exist, we assume that increased glycosphingolipid biosynthesis is secondary to protein hypoglycosylation. Altogether, our data suggest that the cell metabolic machinery may be able to partially re-equilibrate protein hypoglycosylation with increased biosynthesis of glycosphingolipids, possibly to preserve the overall physico-chemical equilibrium of the outer layer of the plasma membrane.