Enzymes Of Sphingolipid Metabolism Research Articles

Vaticanol C, a phytoalexin, induces apoptosis of leukemia and cancer cells by modulating expression of multiple sphingolipid metabolic enzymes.

ABSTRACTResveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.

Fingolimod Affects Transcription of Genes Encoding Enzymes of Ceramide Metabolism in Animal Model of Alzheimer\u2019s Disease

The imbalance in sphingolipid signaling may be critically linked to the upstream events in the neurodegenerative cascade of Alzheimer’s disease (AD). We analyzed the influence of mutant (V717I) amyloid β precursor protein (AβPP) transgene on sphingolipid metabolism enzymes in mouse hippocampus. At 3 months of age AβPP/Aβ presence upregulated enzymes of ceramide turnover on the salvage pathway: ceramide synthases (CERS2, CERS4, CERS6) and also ceramidase ACER3. At 6 months, only CERS6 was elevated, and no ceramide synthase was increased at 12 months. However, sphingomyelin synthases, which utilize ceramide on the sphingomyelinase pathway, were reduced (SGMS1 at 12 and SGMS2 at 6 months). mRNAs for sphingomyelin synthases SGMS1 and SGMS2 were also significantly downregulated in human AD hippocampus and neocortex when compared with age-matched controls. Our findings suggest early-phase deregulation of sphingolipid homeostasis in favor of ceramide signaling. Fingolimod (FTY720), a modulator of sphingosine-1-phosphate receptors countered the AβPP-dependent upregulation of hippocampal ceramide synthase CERS2 at 3 months. Moreover, at 12 months, FTY720 increased enzymes of ceramide-sphingosine turnover: CERS4, ASAH1, and ACER3. We also observed influence of fingolimod on the expression of the sphingomyelinase pathway enzymes. FTY720 counteracted the AβPP-linked reduction of sphingomyelin synthases SGMS1/2 (at 12 and 6 months, respectively) and led to elevation of sphingomyelinase SMPD2 (at 6 and 12 months). Therefore, our results demonstrate potentially beneficial, age-specific effects of fingolimod on transcription of sphingolipid metabolism enzymes in an animal model of AD.

The Role of Ceramide Synthases Response to the Heavy Metal Cadmium Chloride

Cadmium is an extremely toxic heavy metal that is hazardous to human health. In the United States, the primary source of cadmium exposure is in the food supply. Ingestion results in a buildup of cadmium in the body, specifically in the kidneys, which can lead to organ damage and cancer. This toxin has been known to induce stress in organisms by causing an accumulation of reactive oxidative species (ROS). To counter stress, cells respond with a variety of signaling pathways. We are interested in studying the sphingolipid pathway, which is known to mediate stress responses, but its role in defending against cadmium toxicity is unclear. We hypothesized that ceramide synthase, a sphingolipid metabolism enzyme, is required for survival to cadmium stress and decreased accumulation of ROS in animals. To test this, we utilized the model organism Caenorhabditis elegans. Ceramide synthase converts sphingosine to ceramide in the cells. We examined animals with mutations in ceramide synthase (hyl‐2 mutants) by treating worms to acute and chronic cadmium exposure and examining changes in survival, reproduction, and locomotion. We present data showing that cadmium does not affect animal reproduction, but it does affect survival upon exposure. Cadmium exposure resulted in decreased survival, however, and this result was less prominent in organisms possessing hyl‐2/ceramide synthase. We also found that the hyl‐2 gene is vital for oxidative stress response mechanisms. In addition, we present data on the effects of cadmium exposure on gene expression of sphingolipid metabolism enzymes using qPCR. These results are important because stress plays a role in aging, and if we better understand how stress is regulated via sphingolipids, it could become a potential pharmaceutical target to slow the degradative aging process.Support or Funding InformationNIH‐ 2R15AG052933

Targeting Mitochondrial Sphingolipids to Improve Brain Function in Gulf War Illness

Gulf War Illness (GWI) affects Veterans who were deployed in Southwest Asia during the Gulf War. GWI is multi‐symptom disease, which includes cognitive difficulties, depression, and anxiety. Meta‐analyses of epidemiological studies have causally linked these symptomatic profiles to prolonged chemical exposure to different pesticides and pyridostigmine bromide, a medication used as prophylaxis against chemical warfare attacks. Compelling evidence suggests that mitochondrial malfunction is a centerpiece of multiple converging processes implicated in GWI, including oxidative stress, altered synaptic transmission and inflammatory reactions. The focus of these studies is on the role of toxic sphingolipids and mitochondria in the pathogenesis of GWI. Sphingolipids are important structural components of cell membranes and pleiotropic messengers in intracellular signaling pathways. Mitochondria support energy production for biosynthetic reactions, maintenance of ion/metabolite gradients and muscular contractions. Our central hypothesis is that chemical exposure disrupts the lipid metabolism network leading to activation of acid sphingomyelinase (ASMase), a crucial enzyme of sphingolipid metabolism, and generation of toxic sphingolipids in mitochondria.To induce GWI, animals were administered a combination of pyridostigmine bromide and permethrin, i.e. GW agents (GWA), in a single dose daily for 10 days. Quantitative assessment of mitochondrial sphingolipidome was conducted using a state‐of‐the‐art metabolomics technique, tandem mass spectrometry. The cognitive and memory deficits were assessed by video‐recording using an EthovisionXT system (Noldus) and data analysis with EthoVision 13 software.Consistent with our hypothesis, the results of the studies demonstrate a long‐lasting post‐transcriptional activation of ASMase during the six months following animal exposure to GWA. The sphingolipidome analysis revealed significant disturbances in both structural and toxic sphingolipids in response to GWA. Importantly, we have identified several ceramide species, well‐known endogenous inhibitors of mitochondrial function, upregulated in cerebral mitochondria post‐GWA exposure. In mitochondria, ceramide could inhibit mitochondrial respiratory chain Complex III, which would result in an increased ROS production and the loss of mitochondrial integrity. The data indicate that the source of mitochondrial ceramide accumulation is the ASMase‐mediated hydrolysis of sphingomyelin which occurs in lysosomes. The data highlight the importance of the mitochondrial compartment in GWI and accentuate the novel role of ASMase in brain impairment. An inhibitor of ASMase activity, Amitriptyline, ameliorated GWA‐induced changes in mitochondrial sphingolipidome and behavioral abnormalities. The data provide experimental evidence to support the concept that blocking ASMase activity confers mitochondrial protection and improves the cognitive function in an animal model of GWI that provide a foundation for further investigation of novel treatments aimed to improve brain function in GWI patients.Support or Funding InformationThe studies were supported by the DOD grantW81XWH‐18‐1‐0230 (SAN) and VA grant I01BX002991 (TIG)

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels.We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Interaction between nitric oxide and storage temperature on sphingolipid metabolism of postharvest peach fruit

Both nitric oxide (NO) and cold storage have positive effects on the maintenance of fruit quality during storage. However, the roles of NO and storage temperatures in regulating the responses of sphingolipids metabolism to chilling injury of peach fruit during storage remain unknown. Peaches were treated by immersion in distilled water and 15 μmol L−1 NO solution, then stored at 25 °C and 0 °C, respectively. The effects of NO-treatment and storage temperature on the activities of enzymes in sphingolipid metabolism and the contents of sphingolipids in peach fruits were studied. NO maintained higher activities of acid phosphatase (AP) and alkaline phosphatase (ALP) in peach fruits at 25 °C, but promoted the decrease in the activities of AP and ALP at 0 °C, suggesting the regulation by NO on AP and ALP could be modulated by temperature. Compared with the storage at 25 °C, cold storage at 0 °C decreased the activities of phospholipase A (PLA), alkaline phosphatase (ALP), 3-ketodihydrosphingosine reductase (KDSR), sphingosine kinase (SPHK), ceramide synthase (CERS), ceramide kinase (CERK), and the contents of sphingosine (SPH), ceramide (CER), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), sphingomyelin (SM), and increased the activities of phospholipase C (PLC), phospholipase D (PLD), sphingomyelin synthase (SMS). NO significantly increased the contents of sphingolipid metabolites, and the activities of PLA, KDSR, SPHK, CERS, CERK, but decreased the activities of PLC, PLD, SMS of peaches. The results suggested that NO could maintain sphingolipid metabolism to relieve the response of the postharvest fruit to low temperature.

Sphingolipids as mediators of inflammation and novel therapeutic target in inflammatory bowel disease.

Morbidity of inflammatory gastrointestinal (GI) diseases continues to grow resulting in worsen quality of life and increased burden on public medical systems. Complex and heterogenous illnesses, inflammatory bowel diseases (IBDs) encompass several inflammation -associated pathologies including Crohn's disease and ulcerative colitis. IBD is often initiated by a complex interplay between host genetic and environmental factors, lifestyle and diet, and intestinal bacterial components. IBD inflammatory signature was linked to the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) signaling pathway that is currently targeted by IBD therapies. Sphingolipid signaling was identified as one of the key mediators and regulators of pro-inflammatory conditions, and, specifically, TNF-α related signaling. All GI tissues and circulating immune/blood cells contain activated sphingolipid-metabolizing enzymes, including sphingosine kinases (SphK1 and SphK2) that generate sphingosine-1-phosphate (S1P), a bioactive lipid and ligand for five G-protein coupled membrane S1P receptors (S1PRs). Numerous normal and pathogenic inflammatory responses are mediated by SphK/S1P/S1PRs signaling axis including lymphocyte trafficking and activation of cytokine signaling machinery. SphK1/S1P/S1PRs axis has recently been defined as a target for the treatment of GI diseases including IBD/colitis. Several SphK1 inhibitors and S1PRs antagonists have been developed as novel anti-inflammatory agents. In this review, we discuss the mechanisms of SphK/S1P signaling in inflammation-linked GI disorders. The potential role of SphK/S1PRs inhibitors in the prevention and treatment of IBD/colitis is critically evaluated.

Impaired \u03b2-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations

Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only β-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature β-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated β-glucocerebrosidase. Immunostaining revealed loss of neuronal β-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on β-glucocerebrosidase outside of the context of neurodegeneration, we investigated β-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn−/− mice had lower β-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs β-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired β-glucocerebrosidase processing.

Evaluation of ellagic acid as an inhibitor of sphingosine kinase 1: A targeted approach towards anticancer therapy

Sphingosine kinase 1 (SphK1) is one of the central enzymes of sphingolipid metabolism whose high expression level is presumed to be correlated with cancer and other inflammatory diseases. Using a virtual screening approach and in vitro studies, we have identified the ellagic acid (EA), a dietary polyphenol, as a potent inhibitor of SphK1. Molecular docking study has suggested a strong binding affinity of EA to the SphK1. Fluorescence binding and isothermal titration calorimetry (ITC) measurements has also indicated an appreciable binding affinity. Kinase inhibition assay revealed an excellent inhibitory action of EA towards SphK1 (IC50 = 0.74 ± 0.06 μM). Cell viability studies point towards the antiproliferative effects of EA on lung cancer cell line (A549) without affecting human embryonic kidney cells (HEK293). Binding and inhibition mechanism of EA was unveiled by docking analysis of SphK1-EA complex. EA binds to the SphK1 and forms several interactions with catalytically important residues of ATP-binding pocket. Structural stability and dynamics analysis of SphK1-EA complex during 100 ns molecular dynamic simulation studies suggested that EA forms a stable complex with SphK1 without inducing any significant conformational shift. Taken together, our study suggests that EA can be utilized as a chemical prototype to develop potent therapeutics targeting SphK1-associated pathologies.

RED LIGHT IRRADIATION REGULATES ROS SCAVENGING AND ANTI-INflAMMATION THROUGH SPHK1/NF-ΚB PATHWAY IN HACAT CELLS

Objectives Oxidative stress is a well-accepted pathogenesis of several human diseases, which is an increased amount of the oxidants exceeding the capacity of antioxidant defense system. Light-emitting diode irradiation (LEDI) represents an efficient strategy to counteract this condition. The purpose of the present study was to evaluate the ROS scavenging and anti-inflammatory mechanism of LEDI. Findings cDNA microarray, semi quantitative PCR (semi-qPCR), western blotting and small-interfering RNA (siRNA) transfection were processed on PMA induced oxidative stress and inflammation in HaCaT cells. In this study, 625 nm LEDI showed the effect of ROS scavenging and anti-inflammation. One of the most important genes which identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key enzyme in sphingolipid metabolism. SPHK1 knockdown drastically reduced the viability of ROS scavenging in the presence of PMA-stimulated HaCaT cells. Furthermore, results with cyclooxygenease-2 (COX-2) and prostaglandin E2(PGE2) further indicated the importance of the SPHK1 in anti-inflammatory process in HaCaT cells. Conclusions The results obtained in this work highlight the possible role of SPHK1 in ROS scavenging and anti-inflammation in PMA-stimulated HaCaT cells, investigating for the first time the possibility its involved molecular mechanisms. And SPHK1 can be used as a therapeutic target in LEDI treatments for treating skin disorders through ROS scavenging and/or anti-inflammation.

Influence of Resveratrol on Sphingolipid Metabolism in Hepatocellular Carcinoma Cells in Lipid Overload State.

Obesity is characterized by increased long chain fatty acids (LCFA) uptake and impaired lipid metabolism in hepatocytes. Consequently, an enhanced intracellular lipid content, including sphingolipids, may lead to lipotoxicity. It is believed that resveratrol (RSV), one of the most extensively studied plant-derived polyphenols, and its interaction with sphingolipid metabolism may constitute one of the major therapeutic targets for cancer and metabolic diseases treatment. The aim of this study was to ascertain, whether resveratrol may affect sphingolipid metabolic pathways, enzymes and transporters in a lipid overload state. The experiments were conducted on hepatocellular carcinoma cells (HepG2) incubated with RSV and/or Palmitic Acid (PA) at the concentration of 0.5 mM and 50 µM, respectively for 16h. Intra- and extracellular sphingolipid concentrations were assessed by high-performance liquid chromatography and gas liquid chromatography. Moreover, the expression of caspase 3, selected fatty acid transporters and sphingolipid metabolism pathway proteins were estimated by Western Blot. RSV alone and together with PA significantly increased the intracellular concentration of ceramide, sphinganine and sphingosine as well as the expression of enzymes related to de novo ceramide synthesis pathway. Moreover, in our study, we observed augmented ceramide and sphingomyelin efflux into the incubation media in these groups. In addition, RSV substantially reduced intracellular triacylglycerols accumulation in lipid overload conditions. The above-mentioned findings suggest that RSV, at least partially, demonstrates a potential protective effect on HepG2 cells in a lipid overload state.

Potential therapeutic targets for atherosclerosis in sphingolipid metabolism.

Sphingolipids, such as sphingomyelins, ceramides, glycosphingolipids, and sphingosine-1-phosphates (S1P) are a large group of structurally and functionally diverse molecules. Some specific species are found associated with atherogenesis and provide novel therapeutic targets. Herein, we briefly review how sphingolipids are implicated in the progression of atherosclerosis and related diseases, and then we discuss the potential therapy options by targetting several key enzymes in sphingolipid metabolism.

The role of acid sphingomyelinase and modulation of sphingolipid metabolism in bacterial infection.

Acid sphingomyelinase (ASM) is a key enzyme in sphingolipid metabolism that converts sphingomyelin to ceramide, thereby modulating membrane structures and signal transduction. Bacterial pathogens can manipulate ASM activity and function, and use host sphingolipids during multiple steps of their infection process. An increase in ceramides upon infection results in the formation of ceramide-enriched membrane platforms that serve to cluster receptor molecules and organize intracellular signaling molecules, thus facilitating bacterial uptake. In this review, we focus on how extracellular bacterial pathogens target ASM and modulate membrane properties and signaling pathways to gain entry into eukaryotic cells or induce cell death. We describe how intracellular pathogens interfere with the intralysosomal functions of ASM to favor replication and survival. In addition, bacteria utilize their own sphingomyelinases as virulence factors to modulate sphingolipid metabolism. The potential of ASM as a target for treating bacterial infections is also discussed.

NSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

Epidermal permeability barrier function and sphingolipid content in the skin of sphingomyelin synthase 2 deficient mice.

Sphingomyelin synthase (SMS) is an enzyme that generates sphingomyelin (SM) from ceramide (CER) and phosphatidylcholine. SM in the epidermis is a precursor of CER, an important lipid for epidermal permeability barrier function. However, the physiological role of SMS in skin is unclear. To uncover the function of SMS in skin, we investigated sphingolipid metabolism enzyme activity in skin, SM content in the epidermis, CER content in the stratum corneum (SC) and transepidermal water loss (TEWL) as an indicator of barrier function in SMS2-knockout (KO) mice. The activities of sphingolipid metabolism enzymes in skin homogenates were measured using a fluorescently labelled substrate. Enzymatic reaction products were detected by high-performance liquid chromatography (HPLC). Lipids in the epidermis or SC were extracted and quantified by high-performance thin layer chromatography (HPTLC). TEWL was measured using a Tewameter TM300. In SMS2-KO mice, SMS activity in skin homogenates, epidermal SM content and SC CER content were significantly decreased relative to wild-type (WT) mice. The TEWL of SMS2-KO mice was significantly increased compared to WT mice. Our data indicate that SMS2 generates SM in the epidermis and contributes to epidermal permeability barrier function and will support understanding of SM-related metabolic disorders.

The sphingosine kinase 2 inhibitor ABC294640 displays anti-non-small cell lung cancer activities in vitro and in vivo.

Non-small cell lung cancer (NSCLC) accounts for about 85-90% of lung cancer cases, and is the number one killer among cancers in the United States. The majorities of lung cancer patients do not respond well to conventional chemo- and/or radio-therapeutic regimens, and have a dismal 5-year survival rate of ∼15%. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but even with these agents, not all patients respond, and responses are rarely complete. Thus, there is still an urgent need to identify new therapeutic targets in NSCLC and develop novel anti-cancer agents. Sphingosine kinase 2 (SphK2) is one of the key enzymes in sphingolipid metabolism. SphK2 expression predicts poor survival in NSCLC patients, and is associated with Gefitinib-resistance. In this study, the anti-NSCLC activities of ABC294640, the only first-in-class orally available inhibitor of SphK2, were explored. The results obtained indicate that ABC294640 treatment causes significant NSCLC cell apoptosis, cell cycle arrest and suppression of tumor growth in vitro and in vivo. Moreover, lipidomics analyses revealed the complete signature of ceramide and dihydro(dh)-ceramide species in the NSCLC cell-lines with or without ABC294640 treatment. These findings indicate that sphingolipid metabolism targeted therapy may be developed as a promising strategy against NSCLC.

Sphingolipid biosynthesis in man and microbes.

A new review covering up to 2018 Sphingolipids are essential molecules that, despite their long history, are still stimulating interest today. The reasons for this are that, as well as playing structural roles within cell membranes, they have also been shown to perform a myriad of cell signalling functions vital to the correct function of eukaryotic and prokaryotic organisms. Indeed, sphingolipid disregulation that alters the tightly-controlled balance of these key lipids has been closely linked to a number of diseases such as diabetes, asthma and various neuropathologies. Sphingolipid biogenesis, metabolism and regulation is mediated by a large number of enzymes, proteins and second messengers. There appears to be a core pathway common to all sphingolipid-producing organisms but recent studies have begun to dissect out important, species-specific differences. Many of these have only recently been discovered and in most cases the molecular and biochemical details are only beginning to emerge. Where there is a direct link from classic biochemistry to clinical symptoms, a number a drug companies have undertaken a medicinal chemistry campaign to try to deliver a therapeutic intervention to alleviate a number of diseases. Where appropriate, we highlight targets where natural products have been exploited as useful tools. Taking all these aspects into account this review covers the structural, mechanistic and regulatory features of sphingolipid biosynthetic and metabolic enzymes.

De novo Synthesis of Sphingolipids Is Defective in Experimental Models of Huntington's Disease.

Alterations of lipid metabolism have been frequently associated with Huntington's disease (HD) over the past years. HD is the most common neurodegenerative disorder, with a complex pathogenic profile, typically characterized by progressive striatal and cortical degeneration and associated motor, cognitive and behavioral disturbances. Previous findings from our group support the idea that disturbed sphingolipid metabolism could represent an additional hallmark of the disease. Although such a defect represents a common biological denominator among multiple disease models ranging from cells to humans through mouse models, more efforts are needed to clearly define its clinical significance and the role it may play in the progression of the disease. In this study, we provided the first evidence of a defective de novo biosynthetic pathway of sphingolipids in multiple HD pre-clinical models. qPCR analysis revealed perturbed gene expression of sphingolipid-metabolizing enzymes in both early and late stage of the disease. In particular, reduction in the levels of sptlc1 and cerS1 mRNA in the brain tissues from manifest HD mice resulted in a significant decrease in the content of dihydroSphingosine, dihydroSphingosine-1-phospahte and dihydroCeramide [C18:0] as assessed by mass spectrometry. Moreover, in vitro studies highlighted the relevant role that aberrant sphingolipid metabolism may have in the HD cellular homeostasis. With this study, we consolidate the evidence of disturbed sphingolipid metabolism in HD and demonstrate for the first time that the de novo biosynthesis pathway is also significantly affected in the disease. This finding further supports the hypothesis that perturbed sphingolipid metabolism may represent a crucial factor accounting for the high susceptibility to disease in HD.

S1P: the elixir of life for naive T cells.

During their life, T cells are constantly circulating throughout the body. Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, triggers T cell egress from the thymus and secondary lymphoid organs (SLO) into the lymph and the blood. This phenomenon is dependent on the S1P gradient between lymphoid organs (including thymus and SLO) and the lymphatic and blood vessels, which exhibit low, intermediate and high S1P levels, respectively. T cells follow the S1P gradient via the engagement of the G-protein-coupled receptor S1P receptor 1 (S1P1) expressed on T cells.1 S1P production is regulated by various sphingolipid-metabolizing enzymes. The sphingosine kinases 1 and 2 (SKs), encoded by Sphk1 and Sphk2, phosphorylate the sphingosine (Sph) into S1P. Conversely, S1P can be dephosphorylated to Sph by S1P phosphatases 1 and 2, encoded by Sgpp1 and Sgpp2. Alternatively, S1P can be irreversibly degraded by the S1P Lyase (SPL), encoded by Sgpl1, into phosphoethanolamine and hexadecenal. The secretion of S1P was described to involve two transporters, the ATP-binding cassette (ABC) transporter, in a non-specific manner, and the recently identified specific transporter Spinster 2 (SPNS2), encoded by the Spns2 gene.2 As a result, Spns2-deficient mice exhibit decreased concentrations of S1P into the lymph but not blood.3 In a recent study, Mendoza et al. reported that S1P, which is secreted by lymphatic endothelial cells via SPNS2, favors naive T-cell survival by maintaining their mitochondrial content in an S1P1-dependent manner.4 Herein, we discuss the main findings and pathophysiological implications of the latter study as regard to the state of the art.

Lipid environment induces ER stress, TXNIP expression and inflammation in immune cells of individuals with type 2 diabetes.

Obesity and type 2 diabetes are concomitant with low-grade inflammation affecting insulin sensitivity and insulin secretion. Recently, the thioredoxin interacting protein (TXNIP) has been implicated in the activation process of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we aim to determine whether the expression of TXNIP is altered in the circulating immune cells of individuals with type 2 vs type 1 diabetes and whether this can be related to specific causes and consequences of inflammation. The expression of TXNIP, inflammatory markers, markers of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress and enzymes involved in sphingolipid metabolism was quantified by quantitative reverse transcription real-time PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) of 13 non-diabetic individuals, 23 individuals with type 1 diabetes and 81 with type 2 diabetes. A lipidomic analysis on the plasma of 13 non-diabetic individuals, 35 individuals with type 1 diabetes and 94 with type 2 diabetes was performed. The effects of ER stress or of specific lipids on TXNIP and inflammatory marker expression were analysed in human monocyte-derived macrophages (HMDMs) and THP-1 cells. The expression of TXNIP and inflammatory and UPR markers was increased in the PBMCs of individuals with type 2 diabetes when compared with non-diabetic individuals or individuals with type 1 diabetes. TXNIP expression was significantly correlated with plasma fasting glucose, plasma triacylglycerol concentrations and specific UPR markers. Induction of ER stress in THP-1 cells or cultured HMDMs led to increased expression of UPR markers, TXNIP, NLRP3 and IL-1β. Conversely, a chemical chaperone reduced the expression of UPR markers and TXNIP in PBMCs of individuals with type 2 diabetes. The lipidomic plasma analysis revealed an increased concentration of saturated dihydroceramide and sphingomyelin in individuals with type 2 diabetes when compared with non-diabetic individuals and individuals with type 1 diabetes. In addition, the expression of specific enzymes of sphingolipid metabolism, dihydroceramide desaturase 1 and sphingomyelin synthase 1, was increased in the PBMCs of individuals with type 2 diabetes. Palmitate or C2 ceramide induced ER stress in macrophages as well as increased expression of TXNIP, NLRP3 and IL-1β. In individuals with type 2 diabetes, circulating immune cells display an inflammatory phenotype that can be linked to ER stress and TXNIP expression. Immune cell ER stress can in turn be linked to the specific exogenous and endogenous lipid environment found in type 2 diabetes.