Intracellular Ceramide Research Articles

Deciphering the Link Between Hyperhomocysteinemia and Ceramide Metabolism in Alzheimer-Type Neurodegeneration.

Aging is one of the strongest risk factor for Alzheimer's disease (AD). However, several data suggest that dyslipidemia can either contribute or serve as co-factors in AD appearance. AD could be examined as a metabolic disorder mediated by peripheral insulin resistance. Insulin resistance is associated with dyslipidemia, which results in increased hepatic ceramide generation. Hepatic steatosis induces pro-inflammatory cytokine activation which is mediated by the increased ceramides production. Ceramides levels increased in cells due to perturbation in sphingolipid metabolism and upregulated expression of enzymes involved in ceramide synthesis. Cytotoxic ceramides and related molecules generated in liver promote insulin resistance, traffic through the circulation due to injury or cell death caused by local liver inflammation, and because of their hydrophobic nature, they can cross the blood-brain barrier and thereby exert neurotoxic responses as reducing insulin signaling and increasing pro-inflammatory cytokines. These abnormalities propagate a cascade of neurodegeneration associated with oxidative stress and ceramide generation, which potentiate brain insulin resistance, apoptosis, myelin degeneration, and neuro-inflammation. Therefore, excess of toxic lipids generated in liver can cause neurodegeneration. Elevated homocysteine level is also a risk factor for AD pathology and is narrowly associated with metabolic diseases and non-alcoholic fatty liver disease. The existence of a homocysteine/ceramides signaling pathway suggests that homocysteine toxicity could be partly mediated by intracellular ceramide accumulation due to stimulation of ceramide synthase. In this article, we briefly examined the role of homocysteine and ceramide metabolism linking metabolic diseases and non-alcoholic fatty liver disease to AD. We therefore analyzed the expression of mainly enzymes implicated in ceramide and sphingolipid metabolism and demonstrated deregulation of de novo ceramide biosynthesis and S1P metabolism in liver and brain of hyperhomocysteinemic mice.

Defining the roles of host lipids in flavivirus infection

The flavivirus family includes Zika virus (ZIKV), Dengue virus, West Nile virus, Japanese Encephalitis and Yellow Fever virus, each of which causes a major global health threat. During their long‐lasting co‐existence with their host, these viruses have developed sophisticated strategies to co‐opt thecellular machinery including, host lipid metabolism, to effectively establish infection. They enter the host cells through receptor‐mediated endocytosis. In endosomes, the acidic environment induces fusion between the virus and the host membrane resulting in viral genome release. The viral RNA is translated into a polyprotein, which is post‐translationally cleaved by cellular and viral proteases into three structural proteins including capsid, membrane, and envelope, and seven nonstructural proteins. During viral infection cell membranes are co‐opted for virtually every step of the viral life cycle: entry, replication, assembly and release. The role of host factors, especially that of lipids, in pathogenesis of flaviviruses is largely unknown, and there exists a gap in understanding the basic biology of these viruses. This is especially true for ZIKV, which until recently was one of the most understudied flaviviruses. Because lipids are an important interface in host‐virus interactions, studying these biomolecules may provide strategies to combat diseases caused by these pathogens. Zika virus (ZIKV) is a positive‐sense RNA virus that remodels host membrane to establish sites of replication and assembly. To determine how ZIKV modulates the host lipid repertoire in an unbiased manner, we performed comprehensive lipid profiling of human cells infected with ZIKV. We found that ZIKV replication triggers significant changes in the cellular sphingolipidomes especially that of ceramide and sphingomyelin highly enriched during infection. Disruption of sphingolipid biosynthesis with CRISPR/Cas9 gene‐editing or small molecule inhibitors effectively blocks ZIKV replication. Both sphingomyelin and ceramide redistribute to ZIKV replication sites, and the sphingomyelinase‐catalyzed degradation of sphingomyelin to ceramide is the critical pathway to establish infection effectively. Inhibition of sphingomyelinase attenuated ZIKV replication, while the accumulation of intracellular ceramide through knockout of sphingomyelin synthesis enhanced ZIKV infection. Thus, we identify a sphingolipid salvage pathway with a critical role in ZIKV replication and show that ceramide is a key mediator of ZIKV pathogenesis.Support or Funding InformationThis work was supported by the Collins Medical Trust and NIH grant R21AI133631. W.B.M. and J.B.W. were supported by the Medical Research Foundation of Oregon, the Oregon Clinical and Translational Research Institute, and the Sunlin and Priscilla Chou Foundation. D.K. was supported by the Summer Equity Internship Program of the OHSU Center for Diversity and Inclusion. E.B. was supported by NIH grants R01GM060170 and R01GM101983. E.A.D. and A.R.N. were supported by NIH grants R01DK105961, U19AI106754, and U54GM069338.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oleate increase neutral lipid accumulation, cellular respiration and rescues palmitate-exposed GLP-1 secreting cells by reducing ceramide-induced ROS

BackgroundFatty acids (FAs), and especially monounsaturated FAs (MUFAs) stimulate GLP-1 release. However, lipotoxicity is indicated in GLP-1 secreting cells following long-term exposure to elevated levels of saturated FAs (SFAs) in vivo and in vitro, where in vitro studies indicate that cosupplementation with MUFAs confers lipoprotection. SFAs and MUFAs differentially affect the fate of cells in ways that depend on the cell type, concentration and ratio of the FAs. The present study was designed to further elucidate the mechanisms underlying the effects of SFAs/MUFAs on GLP-1-producing cells in terms of lipotoxicity/lipoprotection and GLP-1 secretion. MethodsCultured GLP-1 secreting cells were exposed to hyperlipidemia simulated by SFA-albumin complexes where the molar ratio was 2:1. The cellular response to simulated hyperlipidemia was assessed in the presence/absence of MUFA cosupplementation by determining intracellular ceramide, ROS, neutral lipid accumulation, and cellular respiration. The role for cellular respiration in GLP-1 secretion in response to SFAs/MUFAs was assessed. ResultsGeneration of intracellular ceramide mediate a detrimental increased in ROS production following long term exposure to SFAs in GLP-1-secreting cells. Cosupplementation with MUFAs increases cellular respiration, triglyceride synthesis, and the expression of ceramide kinase, while reducing ceramide synthesis and attenuating ROS production, caspase-3 activity and DNA fragmentation. Further, acute secretory effects of unsaturated FAs are independent of FAO, but mediated by a FFAR1 induced increase in cellular respiration. ConclusionThis study demonstrates novel data supporting effects of MUFAs on the ceramide biosynthetic pathway, triglyceride storage respiration and secretion in GLP-1 secreting cells. These findings may be of value for nutritional interventions, as well as for identification of novel targets, to help preserve L-cell mass and potentiate GLP-1 secretion in diabesity.

Ceramide kinase regulates the migration of bone marrow-derived mesenchymal stem cells

Endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) are mobilized into peripheral blood and injured tissues by various growth factors and cytokines that are expressed in the injured tissues, such as substance P (SP), stromal cell derived factor-1 (SDF-1), and transforming growth factor-beta (TGF-β). Extracellular bioactive lipid metabolites such as ceramide-1-phosphate and sphingosine-1-phosphate also modulate BM-MSC migration as SP, SDF-1, and TGF-β. However, the roles of intrinsic lipid kinases of BM-MSCs in the stem cell migration are unclear. Here, we demonstrated that ceramide kinase mediates the chemotactic migration of BM-MSCs in response to SP, SDF-1, or TGF-β. Furthermore, a specific inhibitor of ceramide kinase inhibited TGF-β-induced migration of BM-MSCs and N-cadherin that is necessary for BM-MSCs migration in response to TGF-β. Therefore, these results suggest that the intracellular ceramide kinase is required for the BM-MSCs migration and the roles of the intrinsic ceramide kinase in the migration are associated with N-cadherin regulation.

The effect of enterolactone on sphingolipid pathway and hepatic insulin resistance development in HepG2 cells

AimsObesity and type 2 diabetes mellitus, correlate with increased tissue concentration of sphingolipids, which directly interfere with insulin signaling pathway. Phytoestrogens are a group of plant-derived compounds that have been studied in the case of metabolic disorders treatment. Therefore, the aim of this study was to ascertain whether enterolactone (ENL), a commonly known phytoestrogen, may affect sphingolipid metabolism and decrease hepatic insulin resistance development in a lipid overload state. Main methodsThe study was conducted on HepG2 cells incubated with ENL and/or palmitic acid (PA) for 16 h. Intra- and extracellular sphingolipid concentrations were assessed by high performance liquid chromatography. The expression of sphingolipid pathway enzymes, apoptosis and insulin signaling pathway proteins and glucose metabolism regulators were evaluated by Western Blot. Key findingsIn HepG2 cells, a considerable augmentation of intracellular ceramide and sphingosine concentration in ENL with PA group were indicated with simultaneous increase in extracellular ceramide concentration. The ENL treatment increased expression of selected enzymes from de novo ceramide synthesis pathway with lower expression of ceramide transfer protein. We also observed a decreased expression of insulin-stimulated phosphorylation of AKT and AMPK after exposure to ENL with PA. Our research demonstrated that ENL with PA resulted in an increased expression of caspase-3. SignificanceEnterolactone, in a higher fatty acids availability, led to the development of hepatic IR in HepG2 cells. This phenomenon may be the result of elevated intracellular ceramide accumulation caused by increased de novo synthesis pathway what led to enhanced apoptosis of HepG2 cells.

StAR-related lipid transfer domain 11 (STARD11)–mediated ceramide transport mediates extracellular vesicle biogenesis

Extracellular vesicles are important carriers of cellular materials and have critical roles in cell-to-cell communication in both health and disease. Ceramides are implicated in extracellular vesicle biogenesis, yet the cellular machinery that mediates the formation of ceramide-enriched extracellular vesicles remains unknown. We demonstrate here that the ceramide transport protein StAR-related lipid transfer domain 11 (STARD11) mediates the release of palmitate-stimulated extracellular vesicles having features consistent with exosomes. Using palmitate as a model of lipotoxic diseases and as a substrate for ceramide biosynthesis in human and murine liver cell lines and primary mouse hepatocytes, we found that STARD11-deficient cells release fewer extracellular vesicles. Moreover, STARD11 reciprocally regulated exosome ceramide enrichment and cellular ceramide depletion. We further observed that in STARD11 knockout cells intracellular ceramide accumulates and that this apparent inability to transfer cellular ceramide into extracellular vesicles reduces cellular viability. Using endogenous markers, we uncovered structural and functional colocalization of the endoplasmic reticulum (ER), STARD11, and multivesicular bodies. This colocalization increased following palmitate treatment, suggesting a functional association that may mediate ceramide trafficking from the ER to the multivesicular body. However, the size and number of multivesicular bodies were comparable in WT and STARD11-knockout cells. In conclusion, we propose a model of how STARD11 mediates ceramide trafficking in palmitate-treated cells and stimulates exosome biogenesis.

FTY720 enhances the anti-tumor activity of carboplatin and tamoxifen in a patient-derived xenograft model of ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related deaths among women in the United States. Although most patients respond to frontline therapy, virtually all patients relapse with chemoresistant disease. This study addresses the hypothesis that carboplatin or tamoxifen + FTY720, a sphingosine analogue, will minimize or circumvent drug-resistance in ovarian cancer cells and tumor models.In vitro data demonstrate that FTY720 sensitized two drug-resistant (A2780. cp20, HeyA8. MDR) and two high-grade serous ovarian cancer cell lines (COV362, CAOV3) to carboplatin, a standard of care for patients with ovarian cancer, and to the selective estrogen receptor modulator tamoxifen. FTY720 + tamoxifen was synergistic in vitro, and combinations of FTY720 + carboplatin or + tamoxifen were more effective than each single agent in a patient-derived xenograft model of ovarian carcinoma. FTY720 + tamoxifen arrested tumor growth. FTY720 + carboplatin induced tumor regressions, with tumor volumes reduced by ∼86% compared to initial tumor volumes. Anti-tumor efficacy was concomitant with increases in intracellular proapoptotic lipid ceramide. The data suggest that FTY720 + tamoxifen or carboplatin may be effective in treating ovarian tumors.

Inhibition of pannexin-1 channel activity by adiponectin in podocytes: Role of acid ceramidase activation

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs+in/Na+out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P.

Palmitic acid triggers inflammatory responses in N42 cultured hypothalamic cells partially via ceramide synthesis but not via TLR4

A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα. L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα. However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.

Adiponectin Regulation and Function.

Adipose tissue is now recognized as an important endocrine organ, capable of secreting a large number of endocrine factors which regulate a wide variety of physiological functions. Adiponectin is one such factor, secreted in large quantities primarily from adipose tissue. Adiponectin is posttranslationally modified from a 30-kDa monomeric protein into different multimers (low molecular weight or trimer, middle molecular weight or hexamer, and high molecular weight) and secreted into the circulation. Upon binding to its receptors, AdipoR1 and R2, adiponectin initiates a series of tissue-dependent signal transduction events, including phosphorylation of adenosine monophosphate (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK), and increased peroxisome proliferator-activated receptor alpha (PPARα) ligand activity. These signal transduction events are regulated by adaptor protein containing a pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL1), which binds directly to the intracellular regions of AdipoR1 and R2. AdipoR1 and R2 also possesses inherent ceramidase activity, resulting in a decrease in intracellular ceramide, a sphingolipid that has been implicated in insulin resistance, cell death, inflammation, and atherosclerosis. Adiponectin stimulates fatty acid oxidation in skeletal muscle and inhibits glucose production in the liver, resulting in an improvement in whole-body energy homeostasis. Adiponectin is also a classic anti-inflammatory agent, reducing inflammation in various cell types through AdipoR1 and R2 signaling mechanisms. Adiponectin's anti-inflammatory and anti-apoptotic properties results in protection of the vasculature, heart, lung, and colon. In this review, we provide a comprehensive overview of the discovery, protein structure, receptors, expression, regulation, and physiological functions of adiponectin. © 2017 American Physiological Society. Compr Physiol 8:1031-1063, 2018.

Emerging concepts of ganglioside metabolism.

Gangliosides (GGs) are sialic acid-containing glycosphingolipids (GSLs) and major membrane components enriched on cellular surfaces. Biosynthesis of mammalian GGs starts at the cytosolic leaflet of endoplasmic reticulum (ER) membranes with the formation of their hydrophobic ceramide anchors. After intracellular ceramide transfer to Golgi and trans-Golgi network (TGN) membranes, anabolism of GGs, as well as of other GSLs, is catalyzed by membrane-spanning glycosyltransferases (GTs) along the secretory pathway. Combined activity of only a few promiscuous GTs allows for the formation of cell-type-specific glycolipid patterns. Following an exocytotic vesicle flow to the cellular plasma membranes, GGs can be modified by metabolic reactions at or near the cellular surface. For degradation, GGs are endocytosed to reach late endosomes and lysosomes. Whereas membrane-spanning enzymes of the secretory pathway catalyze GSL and GG formation, a cooperation of soluble glycosidases, lipases and lipid-binding cofactors, namely the sphingolipid activator proteins (SAPs), act as the main players of GG and GSL catabolism at intralysosomal luminal vesicles (ILVs).

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.

Indomethacin induced glioma apoptosis involving ceramide signals

Nonsteroidal anti-inflammatory drugs (NSAIDs) are increasingly implicated in the prevention and treatment of cancers apart from their known inhibitory effects on eicosanoid production. One of the NSAIDs, indomethacin, in particular shows promising antineoplastic outcome against glioma. To extend such finding, we here studied in human H4 and U87 glioma cells the possible involvement of the ceramide/protein phosphatase 2 A (PP2A)/Akt axis in the indomethacin-induced apoptosis. We found that the induced apoptosis was accompanied by a series of biochemical events, including intracellular ceramide generation, PP2A activation, Akt dephosphorylation, Mcl-1 and FLICE inhibiting protein (FLIP) transcriptional downregulation, Bax mitochondrial distribution, and caspase 3 activation. Such events were also duplicated with a cell-permeable C2-ceramide and Akt inhibitor LY294002. Pharmacological inhibition of ceramide synthase by fumonisin B1 and PP2A by okadaic acid moderately attenuated indomethacin-induced Akt dephosphorylation along with the apoptosis. Results suggested that the ceramide/PP2A/Akt axis is involved in the apoptosis and a possible cyclooxygenase-independent target for indomethacin. Furthermore, apoptosis regulatory proteins such as Mcl-1 and FLIP are potential downstream effectors of this axis and their downregulation could turn on the apoptotic program.

Abstract B019: Developing new ceramide analogs against AIDS-related lymphoma

Abstract The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including primary effusion lymphoma (PEL), a subtype of highly aggressive non-Hodgkin lymphoma (NHL). PEL preferentially arises in immunocompromised patients, e.g., AIDS patients, but lacks of effective therapeutic options. Ceramide serves as a central mediator in sphingolipid metabolism and is referred to as a “tumor suppressor lipid”; however, its functional role in PEL survival and therapeutic value remain largely unknown. Our previous work demonstrated that targeting sphingolipid metabolism by either sphingosine kinase 2 (SphK2) inhibitor or exogenous ceramides induced significant PEL apoptosis and suppressed tumor progression. Recently, we have synthesized a series of new ceramide analogs with better stability and solubility. Our first screening indicated that at least 2 new compounds effectively inhibited PEL cell growth, through the complex mechanisms including induction of tumor cell apoptosis, cell cycle arrest, intracellular ceramide production, and viral lytic gene expression. By using an immunodeficiency mice xenograft model, we found that these selective ceramide analogs significantly repressed PEL progression in vivo. Ongoing work includes the identification of global gene profile affected by these new compounds within PEL cells using the NGS technology. We hope these new ceramide analogs can be developed as promising therapeutic strategies for prolonging the survival time of PEL patients and/or improving their life quality. Citation Format: Lu Dai, Maryam Foroozesh, Zhiqiang Qin. Developing new ceramide analogs against AIDS-related lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B019.

ABC294640, A Novel Sphingosine Kinase 2 Inhibitor, Induces Oncogenic Virus-Infected Cell Autophagic Death and Represses Tumor Growth.

Kaposi sarcoma-associated herpes virus (KSHV) is the etiologic agent of several malignancies, including Kaposi sarcoma and primary effusion lymphoma (PEL), which preferentially arise in HIV+ patients and lack effective treatment. Sphingosine kinase 2 (SphK2) is a key factor within sphingolipid metabolism, responsible for the conversion of proapoptotic ceramides to antiapoptotic sphingosine-1-phosphate (S1P). We have previously demonstrated that targeting SphK2 using a novel selective inhibitor, ABC294640, leads to the accumulation of intracellular ceramides and induces apoptosis in KSHV-infected primary endothelial cells and PEL tumor cells but not in uninfected cells. In this study, we found that ABC294640 induces autophagic death instead of apoptosis in a KSHV long-term-infected immortalized endothelial cell-line, TIVE-LTC, but not in uninfected TIVE cells, through the upregulation of LC3B protein. Transcriptomic analysis indicates that many genes related to cellular stress responses, cell cycle/proliferation, and cellular metabolic process are altered in TIVE-LTC exposed to ABC294640. One of the candidates, Egr-1, was found to directly regulate LC3B expression and was required for the ABC294640-induced autophagic death. By using a Kaposi sarcoma-like nude mice model with TIVE-LTC, we found that ABC294640 treatment significantly suppressed KSHV-induced tumor growth in vivo, which indicates that targeting sphingolipid metabolism, especially SphK2, may represent a promising therapeutic strategy against KSHV-related malignancies. Mol Cancer Ther; 16(12); 2724-34. ©2017 AACR.

Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma.

Omics-based identification of the combined effects of idiosyncratic drugs and inflammatory cytokines on the development of drug-induced liver injury

The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process.To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I− and N−) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis.We found that all I+ treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria-independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions.

Abstract 2017: The role of ceramide in the dual cytotoxic and metabolic stimulative effects of sigma-2 receptors

Abstract Sigma-2 receptors are highly overexpressed in cancer cells compared to normal cells, as well as in proliferative versus quiescent cells. Sigma-2 receptors have been of interest as an antineoplastic target, as sigma-2 agonists cause cell death via a variety of pathways. Recently, a new class of sigma-2 agonists has been discovered, with ligands such as CM764, that do not induce cell death but that affect cellular metabolism, as shown by increased MTT reduction, increased ATP level, and HIF-1α stabilization. Previously, our lab has shown that treatment of breast tumor cells with sigma-2 receptor agonists CB-184 and BD737 caused dose dependent increases in ceramide and simultaneous decreases in sphingomyelin, suggesting that sigma-2 receptors modulate the sphingolipid pathway. The ceramide pathway has been shown to play both prosurvival and pro-apoptotic roles in cancer cells. Here, we examine the role of ceramide in sigma-2 receptor mediated pathways in human SK-N-SH neuroblastoma cells. A 24 h treatment of SK-N-SH neuroblastoma cells with 20 μM exogenous C6-ceramide caused less than 20% cell death. Interestingly, this compared to over 50% cell death in MCF-7 breast adenocarcinoma cells and PANC-1 pancreatic cancer cells, indicating a differential role of ceramide in cell death pathways across cell lines. SK-N-SH cells were treated with 10 μM of either CM572, a classic sigma-2 agonist that causes cell death, or CM764, a sigma-2 agonist that stimulates metabolism. Fumonisin B1 is a potent inhibitor of ceramide synthase. Treatment of cells with fumonisin B1 (10 μM) did not significantly alter CM572-induced cytotoxicity. Conversely, treatment with fumonisin B1 did significantly inhibit the MTT reduction caused by CM764. Taken together, these results suggest that ceramide formation may play more of a role in the metabolic stimulative effect of sigma-2 receptors, as opposed to the cytotoxic effect. Decreased levels of ceramide have been shown to increase cellular resistance to apoptosis. Therefore, it has been of interest to increase intracellular ceramide levels in drug resistant cancer cells in order to sensitize the resistant cells to chemotherapeutics. Multidrug resistance (MDR) in cancer cells is often attributed to the overexpression of particular ABC transporters that effectively pump drugs out of the cells. One such protein is the multidrug resistance-associated protein 1 (MRP1). It has been shown that increased expression of drug efflux pumps also decreases cell sensitivity to ceramide. Treatment with CM572 caused a dose-dependent decrease in MRP1 protein levels, while treatment with CM764 had little effect on MRP1. We will examine how this modulation of MRP1 affects sensitivity to C6-ceramide. We will also examine whether combination of C6-ceramide with sigma-2 receptor ligands augments the cytotoxic and/or metabolic stimulative effects of sigma-2 receptors. Citation Format: Cheri Z. Liu, Ellen Sukharevsky, Wayne D. Bowen. The role of ceramide in the dual cytotoxic and metabolic stimulative effects of sigma-2 receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2017. doi:10.1158/1538-7445.AM2017-2017

Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death

Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant decrease in cytoplasmic area occupied by autophagic vacuoles. Together, these findings highlight the critical role of GBA1 in mediating enhanced self-consumption of intracellular components and endomembranes, leading to autophagic cell death.

Regulation of heat shock gene expression in response to stress

Heat shock (HS) genes, or stress genes, code for a number of proteins that collectively form the most ancient and universal stress defense system. The system determines the cell capability of adaptation to various adverse factors and performs a variety of auxiliary functions in normal physiological conditions. Common stress factors, such as higher temperatures, hypoxia, heavy metals, and others, suppress transcription and translation for the majority of genes, while HS genes are upregulated. Transcription of HS genes is controlled by transcription factors of the HS factor (HSF) family. Certain HSFs are activated on exposure to higher temperatures or other adverse factors to ensure stress-induced HS gene expression, while other HSFs are specifically activated at particular developmental stages. The regulation of the main mammalian stress-inducible factor HSF1 and Drosophila melanogaster HSF includes many components, such as a variety of early warning signals indicative of abnormal cell activity (e.g., increases in intracellular ceramide, cytosolic calcium ions, or partly denatured proteins); protein kinases, which phosphorylate HSFs at various Ser residues; acetyltransferases; and regulatory proteins, such as SUMO and HSBP1. Transcription factors other than HSFs are also involved in activating HS gene transcription; the set includes D. melanogaster GAF, mammalian Sp1 and NF-Y, and other factors. Transcription of several stress genes coding for molecular chaperones of the glucose-regulated protein (GRP) family is predominantly regulated by another stress-detecting system, which is known as the unfolded protein response (UPR) system and is activated in response to massive protein misfolding in the endoplasmic reticulum and mitochondrial matrix. A translational fine tuning of HS protein expression occurs via changing the phosphorylation status of several proteins involved in translation initiation. In addition, specific signal sequences in the 5'-UTRs of some HS protein mRNAs ensure their preferential translation in stress.