Regulation of the lysosome by sphingolipids: Potential role in aging

Sphingolipids are a class of bioactive complex lipids that have been closely associated with aging and aging-related diseases. However, the mechanism through which sphingolipids control aging has long been a mystery. Emerging studies reveal that sphingolipids exert tight control over lysosomal homeostasis and function, as evidenced by sphingolipid-related diseases, including but not limited to lysosomal storage disorders. These diseases are defined by primary lysosomal defects and a few secondary defects such as mitochondrial dysfunction. Intriguingly, recent research indicates that the majority of these defects are also associated with aging, implying that sphingolipid-related diseases and aging may share common mechanisms. We propose that the lysosome is a pivotal hub for sphingolipid-mediated aging regulation. This review discusses the critical roles of sphingolipid metabolism in regulating various lysosomal functions, with an emphasis on how such regulation may contribute to aging and aging-related diseases.     

Sphingolipids as modulators of cancer cell death: Potential therapeutic targets

Through modifications in the fine membrane structure, cell-cell or cell-matrix interactions, and/or modulation of intracellular signaling pathways, sphingolipids can affect the tumorigenic potential of numerous cell types. Whereas ceramide and its metabolites have been described as regulators of cell growth and apoptosis, these lipids as well as other sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer treatments, and their susceptibility to non-apoptotic cell deaths. This review summarizes our current knowledge on the properties of sphingolipids in the regulation of cancer cell death and tumor development. It also provides an update on the potential perspectives of manipulating sphingolipid metabolism and using sphingolipid analogues in anticancer therapy.     

Sphingolipids as modulators of cancer cell death: potential therapeutic targets

Through modifications in the fine membrane structure, cell-cell or cell-matrix interactions, and/or modulation of intracellular signaling pathways, sphingolipids can affect the tumorigenic potential of numerous cell types. Whereas ceramide and its metabolites have been described as regulators of cell growth and apoptosis, these lipids as well as other sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer treatments, and their susceptibility to non-apoptotic cell deaths. This review summarizes our current knowledge on the properties of sphingolipids in the regulation of cancer cell death and tumor development. It also provides an update on the potential perspectives of manipulating sphingolipid metabolism and using sphingolipid analogues in anticancer therapy.     

Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus-nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.     

Cancer and sphingolipid storage disease therapy using novel synthetic analogs of sphingolipids

Sphingolipid metabolites have become recognized for their participation in cell functions and signaling events that control a wide array of cellular activities. Two main sphingolipids, ceramide and sphingosine-1-phosphate, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, angiogenesis, stress responses, protein synthesis, carbohydrate metabolism, and intracellular trafficking. Ceramide and S1P often exert opposing effects on cell survival, ceramide being pro-apoptotic and S1P generally promoting cell survival. Therefore, the conversion of one of these metabolites to the other by sphingolipid enzymes provides a vast network of regulation and provides a useful therapeutic target. Here we provide a survey of the current knowledge of the roles of sphingolipid metabolites in cancer and in lipid storage disease. We review our attempts to interfere with this network of regulation and so provide new treatments for a range of diseases. We synthesized novel analogs of sphingolipids which inhibit the hydrolysis of ceramide or its conversion to more complex sphingolipids. These analogs caused elevation of ceramide levels, leading to apoptosis of a variety of cancer cells. Administration of a synthetic analog to tumor-bearing mice resulted in reduction and even disappearance of the tumors. Therapies for sphingolipid storage diseases, such as Niemann-Pick and Gaucher diseases were achieved by two different strategies: inhibition of the biosynthesis of the substrate (substrate reduction therapy) and protection of the mutated enzyme (chaperone therapy). Sphingolipid metabolism was monitored by the use of novel fluorescent sphingolipid analogs. The results described in this review indicate that our synthetic analogs could be developed both as anticancer drugs and for the treatment of sphingolipid storage diseases.     

Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Cyclin-dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe

The cyclin-dependent kinase 1 (Cdk1), formerly called Cdc2 (or p34(Cdc2)), interacts with cyclin B1 to form an active heterodimer. The activity of Cdk1 is subjected to a complex spatiotemporary regulation, required to guarantee its scheduled contribution to the mitotic prophase and metaphase. Moreover, the activation of Cdk1 may be required for apoptosis induction in some particular pathways of cell killing. This applies to several clinically important settings, for instance to paclitaxel-induced killing of breast cancer cells, in which the ErbB2 receptor kinase can mediate apoptosis inhibition through inactivation of Cdk1. The activation of Cdk1 participates also in HIV-1-induced apoptosis, upstream of the p53-dependent mitochondrial permeabilization step. An unscheduled Cdk1 activation may contribute to neuronal apoptosis occurring in neurodegenerative diseases. Finally, the premature activation of Cdk1 can lead to mitotic catastrophe, for instance after irradiation-induced DNA damage. Thus, a cell type-specific modulation of Cdk1 might be taken advantage of for the therapeutic correction of pathogenic imbalances in apoptosis control.     

Sphingolipid regulation of ezrin,radixin, and moesin proteins family: Implications for cell dynamics

A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

Apoptosis is the essential target of selective pressure against p53, whereas loss of additional p53 functions facilitates carcinoma progression

The high frequency of p53 mutation in human cancers indicates the important role of p53 in suppressing tumorigenesis. It is well established that the p53 regulates multiple, distinct cellular functions such as cell-cycle arrest and apoptosis. Despite intensive studies, little is known about which function is essential, or if multiple pathways are required, for p53-dependent tumor suppression in vivo. Using a mouse brain carcinoma model that shows high selective pressure for p53 inactivation, we found that even partially abolishing p53-dependent apoptosis by Bax inactivation was sufficient to significantly reduce the selective pressure for p53 loss. This finding is consistent with previous reports that apoptosis is the primary p53 function selected against during Eμ-myc-induced mouse lymphoma progression. However, unlike observed in the Eμ-myc-induced lymphoma model, attenuation of apoptosis is not sufficient to phenocopy the aggressive tumor progression associated with complete loss of p53 activity. We conclude that apoptosis is the primary tumor suppressive p53 function and the ablation of additional p53 pleiotropic effects further exacerbates tumor progression.     

Sphingoid bases and phospholipase D activation

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine-l-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.     

Molecular facets of sphingolipids: Mediators of diseases

Sphingolipids constitute a biologically active lipid class that is significantly important from both structural and regulatory aspects. The manipulation of sphingolipid metabolism is currently being studied as a novel strategy for cancer therapy. The basics of this therapeutic approach lie in the regulation property of sphingolipids on cellular processes, which are important in a cell's fate, such as cell proliferation, apoptosis, cell cycle arrest, senescence, and inflammation. Furthermore, the mutations in the enzymes catalyzing some specific reactions in the sphingolipid metabolism cause mortal lysosomal storage diseases like Fabry, Gaucher, Niemann-Pick, Farber, Krabbe, and Metachromatic Leukodystrophy. Therefore, the alteration of the sphingolipid metabolic pathway determines the choice between life and death. Understanding the sphingolipid metabolism and regulation is significant for the development of new therapeutic approaches for all sphingolipid-related diseases, as well as for cancer. An important feature of the sphingolipid metabolic pathway is the compartmentalization into endoplasmic reticulum, the Golgi apparatus, lysosome and plasma membrane, and this compartmentalization makes the transport of sphingolipids critical for proper functioning. This paper focuses on the structures, metabolic pathways, localization, transport mechanisms, and diseases of sphingolipids in Saccharomyces cerevisiae and humans, and provides the latest comprehensive information on sphingolipid research.     

Activation of a p53-independent, sphingolipid-mediated cytolytic pathway in p53-negative mouse fibroblast cells treated with N-methyl-N-nitro-N-nitrosoguanidine

Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxin N-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous sphingolipids as well as their changes in endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of approximately 50% over a period of 3 h. Finally, treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells, suggesting that the two pathways do interact.     

Sphingolipids in mitochondria

Sphingolipids are bioactive lipids found in cell membranes that exert a critical role in signal transduction. In recent years, it has become apparent that sphingolipids participate in growth, senescence, differentiation and apoptosis. The anabolism and catabolism of sphingolipids occur in discrete subcellular locations and consist of a strictly regulated and interconnected network, with ceramide as the central hub. Altered sphingolipid metabolism is linked to several human diseases. Hence, an advanced knowledge of how and where sphingolipids are metabolized is of paramount importance in order to understand the role of sphingolipids in cellular functions. In this review, we provide an overview of sphingolipid metabolism. We focus on the distinct pathways of ceramide synthesis, highlighting the mitochondrial ceramide generation, transport of ceramide to mitochondria and its role in the regulation of mitochondrial-mediated apoptosis, mitophagy and implications to disease. We will discuss unanswered questions and exciting future directions. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Genome-wide RNAi identifies p53-dependent and -independent regulators of germ cell apoptosis in C. elegans

We used genome-wide RNA interference (RNAi) to identify genes that affect apoptosis in the C. elegans germ line. RNAi-mediated knockdown of 21 genes caused a moderate to strong increase in germ cell death. Genetic epistasis studies with these RNAi candidates showed that a large subset (16/21) requires p53 to activate germ cell apoptosis. Apoptosis following knockdown of the genes in the p53-dependent class also depended on a functional DNA damage response pathway, suggesting that these genes might function in DNA repair or to maintain genome integrity. As apoptotic pathways are conserved, orthologues of the worm germline apoptosis genes presented here could be involved in the maintenance of genomic stability, p53 activation, and fertility in mammals.     

p53RDL1 regulates p53-dependent apoptosis

Although a number of targets for p53 have been reported, the mechanism of p53-dependent apoptosis still remains to be elucidated. Here we report a new p53 target-gene, designated p53RDL1 (p53-regulated receptor for death and life; also termed UNC5B). The p53RDL1 gene product contains a cytoplasmic carboxy-terminal death domain that is highly homologous to rat Unc5H2, a dependence receptor involved in the regulation of apoptosis, as well as in axon guidance and migration of neural cells. We found that p53RDL1 mediated p53-dependent apoptosis. Conversely, when p53RDL1 interacted with its ligand, Netrin-1, p53-dependent apoptosis was blocked. Therefore, p53RDL1 seems to be a previously un-recognized target of p53 that may define a new pathway for p53-dependent apoptosis. We suggest that p53 might regulate the survival of damaged cells by balancing the regulation of Netrin-p53RDL1 signalling, and cell death through cleavage of p53RDL1 for apoptosis.