Ceramide Signaling Research Articles

Activated PKR inhibits pancreatic β-cell proliferation through sumoylation-dependent stabilization of P53.

Double-stranded RNA-dependent protein kinase (PKR) is intimately involved in type 2 diabetes due to its role in insulin resistance in peripheral tissues and anti-proliferative effect on pancreatic β-cells. Activated PKR was found to inhibit β-cell proliferation, partially through accumulation of P53. However the molecular mechanisms underlying PKR-dependent upregulation of P53 remain unknown. The results of the present study showed that PKR can be specifically activated in PKR overexpressing β-cells by a low dosage of the previously synthesized compound 1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride (BEPP), and this led to upregulation of P53 through sumoylation-dependent stability. Activated PKR was found to interact with sumo-conjugating enzyme Ubc9, and P53 sumoylation relies on a PKR-Ubc9 protein-protein interaction. Additionally, a ceramide signal was needed for PKR activation to be triggered by glucolipotoxicity and TNFα stimulation, and stabilization of P53 required endogenous ceramide accumulation. Glucolipotoxicity and pro-inflammatory cytokines therefore promote the sumoylation-dependent stability of P53 via the ceramide/PKR/Ubc9 signalling pathway that is involved in pancreatic β-cell proliferation inhibition in the development of type 2 diabetes.

Induction of apoptosis in prostate cancer cells by the novel ceramidase inhibitor ceranib-2.

Ceramidases are key enzymes that decrease ceramide levels in cells. A reduction in ceramide concentration impairs ceramide signalling, and results in apoptosis resistance in cancer cells. This study investigates the potential for ceranib-2, a novel ceramidase inhibitor, to affect the survival and/or promote apoptosis of prostate cancer cells (LNCaP and DU145) in vitro. Cell viability was determined using MTT, and apoptosis assessed via flow cytometry. We examined structural changes with both confocal and transmission electron microscopes. Ceranib-2 concentrations of 0.1, 1, 5, 10, 25 and 50μM were applied to LNCaP and DU145 cell lines. The corresponding reduction in LNCaP cell viability (against the control) was 84%, 80%, 64%, 56%, 40% and 15% after 24h, and 81%, 74%, 60%, 55%, 27% and 11% after 48h. For DU145 cells, viability was reduced to 84%, 82%, 63%, 50%, 41% and 18% after 24h, and 64%, 42%, 30%, 20%, 8% and 5% after 48h. Following treatment with 25 and 50μM ceranib-2, the respective observed rates of early apoptosis in LNCaP cells were 23% and 36% after 24h and 27% and 58% after 48h. The morphological and ultrastructural signs of apoptosis detected were fragmented nuclei, chromatin condensations and cytoskeleton laceration. The inhibitory effects of ceranib-2 on prostate cancer cell survival are dose and time dependent. For LNCaP cells, ceranib-2 toxicity was predominately apoptotic in nature, while for DU145 cells, cell death may be related to non-apoptotic mechanisms.

Ceramide signalling impinges on Sit4p and Hog1p to promote mitochondrial fission and mitophagy in Isc1p-deficient cells

Mitochondria function as the powerhouses of the cell for energy conversion through the oxidative phosphorylation process. Accumulation of dysfunctional mitochondria promotes a bioenergetic crisis and cell death by apoptosis. Yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit mitochondrial dysfunction and shortened lifespan associated with the accumulation of specific ceramide species and activation of the PP2A-like protein phosphatase Sit4p and of the Hog1p kinase. Here, we show that isc1Δ cells display hyperactivation of mitophagy that is suppressed by downregulating Sit4p, Hog1p or the TORC1–Sch9p pathway. Notably, isc1Δ cells also have high levels of Dnm1p associated with unbalanced mitochondrial fission, leading to mitochondrial fragmentation, and DNM1 deletion suppressed the oxidative stress sensitivity and shortened lifespan of isc1Δ cells. Moreover, Isc1p and Dnm1p physically interact, suggesting a possible regulatory role for Isc1p in mitochondrial dynamics. Overall, our work demonstrates that Isc1p-mediated ceramide signalling regulates mitophagy and mitochondrial dynamics in yeast with impact on mitochondrial function and lifespan. Since ceramides have been implicated in ageing and diseases associated with mitochondrial dysfunction, our findings suggest that therapeutic strategies targeting ceramide signalling may improve mitochondrial function and human healthspan.

Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts.

Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin-ceramide mixtures in excess water as a function of temperature (30-70 °C) and pressure (1-200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (P(β)') below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.

Ceramide-induced apoptosis in renal tubular cells: a role of mitochondria and sphingosine-1-phoshate.

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed.

Novel mechanisms of action of classical chemotherapeutic agents on sphingolipid pathways.

The prevailing mechanisms of action of traditional chemotherapeutic agents have been challenged by sphingolipid cancer research. Many studies have shown that ceramide generation in response to cytotoxic agents is central to tumor cell death. Ceramide can be generated either via hydrolysis of cell-membrane sphingomyelin by sphingomyelinases, hydrolysis of cerebrosides, or via de novo synthesis by ceramide synthases. Ceramide can act as a second messenger for apoptosis, senescence or autophagy. Inherent or acquired alterations in the sphingolipid pathway can account for resistance to the classic chemotherapeutic agents. In particular, it has been shown that activation of the acid ceramidase can lead to the formation of sphingosine 1-phosphate, which then antagonizes ceramide signaling by initiating a pro-survival signaling pathway. Furthermore, ceramide glycosylation catalyzed by glucosylceramide synthase converts ceramide to glucosylceramide, thus eliminating ceramide and consequently protecting cancer cells from apoptosis. In this review, we describe the effects of some of the most commonly used chemotherapeutic agents on ceramide generation, with a particular emphasis on strategies used to enhance the efficacy of these agents.

PDE5 inhibitors enhance celecoxib killing in multiple tumor types.

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer.

Activation of Mitochondrial Apoptosis and Regulation of Ceramide Signalling by COX-2 Inhibitors in Colon Cancer

Bcl-2 family of proteins is implicated in the malignant tumors including colorectal cancer. Activation of Bcl-2 inhibits the pro-apoptotic proteins (Bax and Bad) and regulates many biological processes such as apoptosis, cell proliferation and cell growth. As the mitochondrial enzymes are involved in sphingolipid metabolism, it can regulate ceramide formation and in turn mitochondria play a central role for the regulation of ceramide induced apoptosis. Bcl-2/ BclxL activates sphingosine kinases (SKs), resulting in the accumulation of S1P (sphingosine-1-phosphate), thereby reducing apoptosis. In the present study, the anti-neoplastic effects have been observed of Etoricoxib and Celecoxib, two COX-2 selective non-steroidal anti-inflammatory drugs (NSAIDs), and Diclofenac, a preferential COX-2 inhibitory NSAIDs, in the early stage of colon cancer in rats. These NSAIDs regress the expressions of Bcl-2 and SK-1 and promote apoptosis. Gross morphological analysis revealed the occurrence of raised mucosal lesions called MPL or multiple plaque lesions, which were maximum in the 1, 2-Dimethylhydrazine (DMH) treated group and their number regressed with the co-administration of the NSAIDs. An abnormal histo-architecture like hyperplasia and dysplasia were evident in the carcinogenic group, which were reduced with NSAIDs co-administration.

Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes.

Our understanding of the functions of ceramide signaling has advanced tremendously over the past decade. In this review, we focus on the roles and regulation of neutral sphingomyelinase 2 (nSMase2), an enzyme that generates the bioactive lipid ceramide through the hydrolysis of the membrane lipid sphingomyelin. A large body of work has now implicated nSMase2 in a diverse set of cellular functions, physiological processes, and disease pathologies. We discuss different aspects of this enzyme's regulation from transcriptional, post-translational, and biochemical. Furthermore, we highlight nSMase2 involvement in cellular processes including inflammatory signaling, exosome generation, cell growth, and apoptosis, which in turn play important roles in pathologies such as cancer metastasis, Alzheimer's disease, and other organ systems disorders. Lastly, we examine avenues where targeted nSMase2-inhibition may be clinically beneficial in disease scenarios.

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma.

A Neurotoxic Glycerophosphocholine Impacts PtdIns-4, 5-Bisphosphate and TORC2 Signaling by Altering Ceramide Biosynthesis in Yeast

Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.

Acute and chronic immunomodulatory changes in rat liver after fetal and perinatal asphyxia.

Hypoxic-ischemic encephalopathy (HIE) caused by fetal and perinatal asphyxia is an important cause of mortality in the neonatal period. Not only will asphyxia affect the brain but also other organs such as the liver and kidneys. Interestingly, it has been shown that liver damage is proportional to the severity of the asphyctic insult, implying an association between liver impairment and HIE. Accordingly, we investigated in an established rat model the acute and chronic hepatic response to both fetal (FA) and perinatal asphyxia (PA). In addition, we assessed whether fetal asphyctic preconditioning (PC) would have any beneficial effect on the liver. Inflammation, ceramide signaling and hepatocellular damage were analyzed in the livers of newborn and adult rats at several short- and long-term time points after both FA and PA. We found that although FA induced an acute inflammatory response, apoptotic mRNA levels and oxidative DNA damage were decreased at 96 h post FA. Whereas increased IL-6 and IL-10 mRNA levels were observed after PA, the combination of FA and PA (PC) attenuated the inflammatory response. Moreover, 6 h after PA anti-apoptotic genes were downregulated and associated with less lipid peroxidation, while preconditioned animals were comparable to controls. In summary, asphyctic PC seems to have an acute protective effect on the liver by modulating the inflammatory, apoptotic and anti-oxidative response. More insight into the hepatic response to asphyxia is necessary, as disturbed hepatic function is associated with metabolic diseases in later life.

Chapter Five - The Retrograde Response: A Conserved Compensatory Reaction to Damage from Within and from Without

The retrograde response was discovered in Saccharomyces cerevisiae as a signaling pathway from the mitochondrion to the nucleus that triggers an array of gene regulatory changes in the latter. The activation of the retrograde response compensates for the deficits associated with aging, and thus it extends yeast replicative life span. The retrograde response is activated by the progressive decline in mitochondrial membrane potential during aging that is the result of increasing mitochondrial dysfunction. The ensuing metabolic adaptations and stress resistance can only delay the inevitable demise of the yeast cell. The retrograde response is embedded in a network of signal transduction pathways that impinge upon virtually every aspect of cell physiology. Thus, its manifestations are complicated. Many of these pathways have been implicated in life span regulation quite independently of the retrograde response. Together, they operate in a delicate balance in promoting longevity. The retrograde response is closely aligned with cell quality control, often performing when quality control is not sufficient to assure longevity. Among the key pathways related to this aspect of retrograde signaling are target of rapamycin and ceramide signaling. The retrograde response can also be found in other organisms, including Caenorhabditis elegans, Drosophila melanogaster, mouse, and human, where it exhibits an ever-increasing complexity that may be corralled by the transcription factor NFκB. The retrograde response may have evolved as a cytoprotective mechanism that senses and defends the organism from pathogens and environmental toxins.

Distinct signaling roles of ceramide species in yeast revealed through systematic perturbation and systems biology analyses.

Ceramide, the central molecule of sphingolipid metabolism, is an important bioactive molecule that participates in various cellular regulatory events and that has been implicated in disease. Deciphering ceramide signaling is challenging because multiple ceramide species exist, and many of them may have distinct functions. We applied systems biology and molecular approaches to perturb ceramide metabolism in the yeast Saccharomyces cerevisiae and inferred causal relationships between ceramide species and their potential targets by combining lipidomic, genomic, and transcriptomic analyses. We found that during heat stress, distinct metabolic mechanisms controlled the abundance of different groups of ceramide species and provided experimental support for the importance of the dihydroceramidase Ydc1 in mediating the decrease in dihydroceramides during heat stress. Additionally, distinct groups of ceramide species, with different N-acyl chains and hydroxylations, regulated different sets of functionally related genes, indicating that the structural complexity of these lipids produces functional diversity. The transcriptional modules that we identified provide a resource to begin to dissect the specific functions of ceramides.

Effects and mechanisms of ceramide signaling pathway in gastrin-inhibited cell apoptosis of large intestinal cancer

Objective To investigate the effects and mechanisms of ceramide signaling pathway in gastrin-inhibited cell apoptosis of large intestinal cancer.Methods Gastrin group and proglumide group were divided into five different densities with concentration gradient of gastrin and proglumide being 6.25,12.50,25.00,50.00,100.00 mg/L and 8.00,16.00,32.00,64.00,128.00 mg/L,respectively.The changes of proliferation of the HT-29 cells were detected by methyl thiazol tetrazolium (MTT) assay,and the optimal concentrations of gastrin and proglumide were determined.The changes of apoptosis rate of HT-29 cells were detected by using flow cytometry.The mRNA expression levels of gastrin receptor/cholecystokinin-B receptor (CCK-BR),ceramide,p38,B lymphocytes/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) were detected by using reverse transcriptase-polymerase chain reaction (RT-PCR).The protein expression levels of bcl-2,bax and ceramide,and p38 phosphorylation levels were detected by using Western blotting.Results Gastrin could promote the proliferation of HT-29 cells,and the optimal concentration was 25 mg/L (F =31.36,P ＜ 0.05).Proglumide could significantly inhibit the proliferation of HT-29 cells stimulated by gastrin,and the optimal concentration was 32 mg/L (F =24.31,P ＜ 0.05).The apoptosis rate of the gastrin group was significantly lower than in the control group and the gastrin + proglumid group (q =4.23,4.06,P ＜0.05).The mRNA and protein expression of bax and the levels of phosphorylated ceramide protein and phosphorylated p38 protein in the gastrin group were significantly lower than in the control group,the proglumid group,and the gastrin + proglumid group (q =5.50,6.00,7.50; 6.50,7.00,8.50; 8.00,8.50,10.00; 4.60,4.90,6.53,P＜0.05).On the contrary,the mRNA and protein expression of bcl-2 in the gastrin group was significantly higher than in the control group,the proglumid group,and the gastrin + proglumid group (q =4.95,4.24,7.07; 7.07,6.36,7.78,P ＜ 0.05).Conclusion Gastrin could inhibit the apoptosis of HT-29 cells by down-regulate the phosphorylation levels of ceramide and p38 protein,thus up-regulate bcl-2 and down-regulate bax through the ceramide signaling transduction pathway,while the effect can be restrained by gastrin receptor antagonist proglumide. Key words: Colorectal cancer; Gastrin ; Ceramide ; p38 ; B lymphocytes/leukemia-2 ; Bcl-2 associated X protein

Critical appraisal of the potential use of cannabinoids in cancer management

Cannabinoids have been attracting a great deal of interest as potential anticancer agents. Originally derived from the plant Cannabis sativa, there are now a number of endo-, phyto- and synthetic cannabinoids available. This review summarizes the key literature to date around the actions, antitumor activity, and mechanisms of action for this broad range of compounds. Cannabinoids are largely defined by an ability to activate the cannabinoid receptors – CB1 or CB2. The action of the cannabinoids is very dependent on the exact ligand tested, the dose, and the duration of exposure. Some cannabinoids, synthetic or plant-derived, show potential as therapeutic agents, and evidence across a range of cancers and evidence in vitro and in vivo is starting to be accumulated. Studies have now been conducted in a wide range of cell lines, including glioma, breast, prostate, endothelial, liver, and lung. This work is complemented by an increasing body of evidence from in vivo models. However, many of these results remain contradictory, an issue that is not currently able to be resolved through current knowledge of mechanisms of action. While there is a developing understanding of potential mechanisms of action, with the extracellular signal-regulated kinase pathway emerging as a critical signaling juncture in combination with an important role for ceramide and lipid signaling, the relative importance of each pathway is yet to be determined. The interplay between the intracellular pathways of autophagy versus apoptosis is a recent development that is discussed. Overall, there is still a great deal of conflicting evidence around the future utility of the cannabinoids, natural or synthetic, as therapeutic agents.

Farber disease: understanding a fatal childhood disorder and dissecting ceramide biology

Farber disease: understanding a fatal childhood disorder and dissecting ceramide biology

Specific Lipidome Signatures in Central Nervous System from Methionine-Restricted Mice

Membrane lipid composition is an important correlate of the rate of aging of animals. Dietary methionine restriction (MetR) increases lifespan in rodents. The underlying mechanisms have not been elucidated but could include changes in tissue lipidomes. In this work, we demonstrate that 80% MetR in mice induces marked changes in the brain, spinal cord, and liver lipidomes. Further, at least 50% of the lipids changed are common in the brain and spinal cord but not in the liver, suggesting a nervous system-specific lipidomic profile of MetR. The differentially expressed lipids includes (a) specific phospholipid species, which could reflect adaptive membrane responses, (b) sphingolipids, which could lead to changes in ceramide signaling pathways, and (c) the physiologically redox-relevant ubiquinone 9, indicating adaptations in phase II antioxidant response metabolism. In addition, specific oxidation products derived from cholesterol, phosphatidylcholine, and phosphatidylethanolamine were significantly decreased in the brain, spinal cord, and liver from MetR mice. These results demonstrate the importance of adaptive responses of membrane lipids leading to increased stress resistance as a major mechanistic contributor to the lowered rate of aging in MetR mice.

Abstract 2960: Flavopiridol-mediated multiple modulatory effects synergistically increase sensitivity to TRAIL-induced cell death in human leukemia cells.

Abstract Previously, we described the antileukemic synergistic interactions between the cyclin-dependent kinase inhibitor flavopiridol (F) and TRAIL in multiple cell lines representing a broad spectrum of human leukemia. With the introduction of new hybrid regimens for flavopiridol-based therapies and advances in the clinical development of TRAIL/TRAIL agonists, a renewed interest in this therapeutic concept has emerged. In an attempt to identify key mechanistic signaling networks, human U937 leukemia cells were treated simultaneously with F (SelleckChem, Houston TX) and TRAIL (Alexis, Enzo, Farmingdale, NY) and analyzed for signaling events associated to cell death induction. We found a dramatic F-mediated shift from cytoprotective NF-kB, AKT and ERK pathways to pro-cell death ceramide and JNK signals. Functional studies using both pharmacological inhibitors and genetically modified cells revealed the functional relevance of these signals. Furthermore, F-mediated transcriptional modifications played key roles in determining and regulating the cross-talk between anti- and pro-cell death pathways. Co-exposure to F resulted in transcriptional down-regulation of genes involved in the regulation of pro-apoptotic JNK including XIAP, Gadd45β and phosphatases DUSP1, -10 and -11. In addition, F induced a significant reduction of multiple anti-apoptotic genes/proteins directly implicated in the pro-apoptotic cascades including (Mcl-1, XIAP, Bcl-2, Bcl-xL, c-Flip, cIAP2/3), and cell cycle modulators (cyclin D1, -E1/E2, CDKs 1,7,9,13,17, p15, p57, p21). However, although in many cases similar changes were observed in F- and TRAIL/F-treated cells, additional factors may have to exist that lead to the potent synergistic interactions observed with the drug combination. In fact, by using clonogenic assays (long term effects), we observed that while TRAIL or F-treated cells were able to recover after treatment, that was not the case in TRAIL/F-exposed cells (% clonogenic survival: T= 82, F=47, TF= 1.4). Time-course analysis by flow cytometry of cell surface localized TRAIL Death Receptors DR4 and DR5 showed that in the presence of F, either alone or in combination with TRAIL, the membrane levels of both receptors were significantly increased during the first 2-6h of treatment. Together, these findings indicate that combined exposure of human acute leukemia cells to TRAIL/FP results in an early mobilization of TRAIL death receptors (DR4, DR5) to the cell surface, effect that is temporally associated with activation of anti-apoptotic ERK, AKT and NF-kB signals; however, activation of DR4/5-mediated apoptotic pathway is later potentiated by a shift toward pro-cell death JNK/ceramide signals that, in association to F-mediated transcriptional effects, results in a dramatic increase in the sensitivity to TRAIL in otherwise TRAIL-resistant human leukemia cells. Citation Format: Swaminathan P. Iyer, Jaime Mejia, Adriana E. Rosato, Steven Grant, Roberto R. Rosato. Flavopiridol-mediated multiple modulatory effects synergistically increase sensitivity to TRAIL-induced cell death in human leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2013-2960