Ceramide Analog Research Articles

The sensitivity of chronic myeloid leukemia CD34 cells to Bcr-Abl tyrosine kinase inhibitors is modulated by ceramide levels

Despite BCR-ABL tyrosine kinase inhibitors (TKIs) improved outcome of patients with chronic myeloid leukemia (CML), resistance still develops when progresses to blast phase (BP). The mechanisms underlying resistance to TKIs are not well understood. In this study, we analyzed ceramide levels in CD34 cells derived from BP-CML patients and healthy donor bone marrow (BM) using liquid chromatography mass spectrometry. We found that ceramide level was significantly lower in BP-CML CD34 compared with normal BM counterparts. BP-CML CD34 ceramidelow were more resistant to BCR-ABL TKIs compared to BP-CML CD34 ceramidenormal. Both mRNA and proteins levels of sphingomyelin synthase 1 and 2 are lower in BP-CML CD34 ceramidelow compared to normal BM CD34 cells, suggesting that these two ceramide synthesis enzymes maybe the mechanism of how ceramide level is suppressed. Importantly, up-regulation of cellular ceramide level induces apoptosis of multiple CML cell lines and BP-CML CD34 progenitors. Combination of BCR-ABL TKIs with ceramide analog is synergistic in targeting BP-CML 34 progenitors. Collectively, our work provides evidence that down-regulation of ceramide level is involved in the resistance of BP-CML CD34 progenitors to TKIs treatment. Targeting ceramide metabolism together with BCR-ABL inhibition makes it an attractive addition to the armamentarium in BP-CML treatment.

Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

Abstract Cytotoxic T Lymphocytes (CTLs) are the major effectors of the host cancer immune surveillance. FasL-induced cytotoxicity is one of the two effector mechanisms that CTLs use to kill tumor cells. Fas is the physiological ligand of FasL and its expression and function is often deregulated in cancer cells. Ceramide is a sphingolipid metabolite that mediates Fas function. We aimed at testing the hypothesis that ceramide analogs are effective in modulating Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis by tumor-specific CTLs. We show that Fas is expressed in human colon carcinoma cells. However, human colon carcinoma cells are not sensitive to FasL-induced apoptosis. Based on structures of existing ceramide analogs and ceramidase inhibitors, we used rationale design and synthesized twenty ceramide analogs as Fas function modulators. Six of these twenty ceramide analogs, IG4, IG7, IG8, IG14, IG17 and IG19, exhibit potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. At the molecular level, we observed that these six ceramide analogs dramatically increased FasL-induced activation of caspase 8, an essential initiator caspase of the Fas receptor-death-inducing signaling complex. Finally, a sublethal dose of ceramide analog significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed six novel ceramide analogs that act at sublethal doses to enhance the efficacy of tumor-specific CTLs and hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.

Seborrheic Keratoses: The Rodney Dangerfield of Skin lesions, and Why They Should Get Our Respect

Neel etal. have demonstrated that seborrheic keratosis, the most common of all skin tumors, is dependent on acutely transforming retrovirus AKT8 in rodent T-cell lymphoma signaling. The authors found that these lesions are hypersensitive to Akt inhibitors which bind to the ATP binding site of Akt. Cutaneous squamous cell carcinoma is resistant to Akt inhibitors. The implications of this study are not limited to seborrheic keratosis. The presence of wild type p53 (seborrheic keratosis) or mutant p53 (cutaneous squamous cell carcinoma) appears to dictate whether a lesion is sensitive to Akt inhibition or not.

Investigation of the Efficacy of Transdermal Penetration Enhancers Through the Use of Human Skin and a Skin Mimic Artificial Membrane

The aim of this study was to investigate the behavior of promising penetration enhancers through the use of 2 different skin test systems. Hydrogel-based transdermal formulations were developed with ibuprofen as a nonsteroidal anti-inflammatory drug. Transcutol and sucrose esters were used as biocompatible penetration enhancers. The permeability measurements were performed with ex vivo Franz diffusion cell methods and a newly developed Skin Parallel Artificial Membrane Permeability Assays (PAMPA) model. Franz diffusion measurement is commonly used as a research tool in studies of diffusion through synthetic membranes in vitro or penetration through ex vivo human skin, whereas Skin PAMPA involves recently published artificial membrane-based technology for the fast prediction of skin penetration. It is a 96-well plate-based model with optimized artificial membrane structure containing free fatty acid, cholesterol, and synthetic ceramide analog compounds to mimic the stratum corneum barrier function. Transdermal preparations containing 2.64% of different sucrose esters and/or Transcutol and a constant (5%) of ibuprofen were investigated to determine the effects of these penetration enhancers. The study demonstrated the good correlation of the permeability data obtained through use of human skin membrane and the in vitro Skin PAMPA system. The Skin PAMPA artificial membrane serves as quick and relatively deep tool in the early stages of transdermal delivery systems, through which the enhancing efficacy of excipients can be screened so as to facilitate the choice of effective penetration components.

Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway.

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

Systemic Codelivery of a Homoserine Derived Ceramide Analogue and Curcumin to Tumor Vasculature Inhibits Mouse Tumor Growth.

Prior studies reported significant anticancer activities of ceramides. However, anticancer activities of homoserine based ceramides have not been tested. With a view to compare the anticancer activity of ceramides and homoceramides, in the present study, we have synthesized four serine based and four homoserine based C8-ceramide analogues. Since many cancer cells have shown resistance to ceramides, curcumin is now being used in combination with ceramides because of its ability to reverse multidrug resistance. Aimed at targeting curcumin-ceramide combination to tumor endothelial cells, herein we have used a tumor vasculature targeting liposomes of a newly synthesized pegylated RGDGWK-lipopeptide. Importantly, the liposomal formulations of the homoserine based C8-ceramide analogue containing oleyl chain showed more promising antineoplastic activities under both in vitro and systemic settings than the liposomal formulations of commercially available C8-ceramide. Findings in the mouse tumor growth inhibition study revealed synergistic therapeutic benefit from simultaneous delivery of curcumin and a homoserine based ceramide containing oleyl chain to tumor vasculature. Results in RT-PCR and Western blot experiments suggest that inhibition of solid tumor growth is mediated via inhibition of PI3K-Akt signaling pathway. The present structure-activity study is the first report to demonstrate therapeutic promise of curcumin-homoserine based ceramide combination in antiangiogenic cancer therapy.

Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis.

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C2-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.

Inhibition of Tumors in Mice Models by a Synthetic Ceramide-analog AD2725

A synthetic analog of ceramide (AD2725) elevated ceramide levels in MDA-MB-435 breast cancer cells inducing apoptosis and resulted in cell death. In animal studies, using a model of xenograft breast cancer cancer cells in nude mice, treatment with AD2725 resulted in a significant reduction of the tumor volume and weight, and a significantly prolonged survival of the treated mice. The analog also significantly elevated INF gamma and IL-12 cytokine levels and reduced the level of IL-10 in the plasma. These data indicate that use of suitable synthetic analogs that most probably elevate the ceramide levels may have beneficial anti-cancer therapeutic effects.

Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells.

The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.

Long-Term Hematologic Response to Eliglustat in Patients with Gaucher Disease Type 1: Results from a Phase 2 and Two Phase 3 Trials

Introduction: Gaucher disease is caused by a deficiency in lysosomal acid β-glucosidase, which leads to accumulation of glucosylceramide primarily in macrophages. These pathogenic macrophages (Gaucher cells) deposit in the spleen, liver, bone marrow and lungs and lead to hepatosplenomegaly, pancytopenia and skeletal disease. As thrombocytopenia, anemia and splenomegaly are common presenting symptoms of Gaucher patients, hematologists are often the healthcare provider to identify, evaluate and manage the disease. The current standard of care is biweekly intravenous enzyme replacement therapy (ERT) that supplements lysosomal acid β-glucosidase in mononuclear phagocytes. Eliglustat, a ceramide analogue, is a novel oral substrate reduction therapy (SRT) that was recently approved as a first-line treatment of adults with Gaucher disease type 1 (GD1) who have a compatible CYP2D6 metabolizer phenotype (>90% of patients). Rather than supplement lysosomal acid β-glucosidase in mononuclear phagocytes, eliglustat partially inhibits glucosylceramide synthase and reduces glucosylceramide production to offset the attenuated rate of degradation. Methods: We report on improvements in hemoglobin level and platelet counts in adults with GD1 from 3 ongoing clinical trials of eliglustat sponsored by Genzyme, a Sanofi company. Two trials are in treatment-naïve patients: an open-label Phase 2 trial (N=26, NCT00358150) for which 4-year data are available and a randomized, placebo-controlled Phase 3 trial (ENGAGE, N=40, NCT00891202) for which 18-month data are available. The third trial (ENCORE, Phase 3 imiglucerase-controlled, N=159, NCT00943111) involved patients previously stabilized on ≥3 years of ERT and 24-month data are available. Results: In the Phase 2 study, the primary endpoint was met after 1 year, with 77% of patients improving in at least 2 of 3 main efficacy parameters (spleen volume, hemoglobin level and platelet count) (Lukina et al. Blood 2010;116:816). After 4 years, mean hemoglobin level had increased by 2.3±1.5 g/dL from the baseline mean of 11.3±1.5 g/dL, and mean platelet count had increased by 95% from the baseline mean of 69±21x109/L. In the ENGAGE study, all primary and secondary endpoints were met after 9 months, with statistically significant improvements in spleen volume, hemoglobin level, liver volume and platelet counts seen in eliglustat-treated but not placebo patients (Mistry et al. JAMA 2015;313;695). Improvements continued in patients who received eliglustat for 18 months in the trial extension; mean hemoglobin level increased by 1.02±0.84 g/dL from a baseline of 12.1±1.8 g/dL, mean platelet count increased by 58%±41 from a baseline of 75±14x109/L, and mean spleen volume decreased by 44.6%±10 from a baseline of 13.9±6.0 multiples of normal (MN). For the 20 patients who switched from placebo to eliglustat, mean hemoglobin increased by 0.79±0.82 g/dL from a baseline of 12.2±2.0 g/dL, mean platelet count increased by 40%±37 from a baseline of 71.5±25.2 x109/L and mean spleen volume decreased by 31.1%±10 from a baseline of 12.5±6.0 MN, after 9 months of eliglustat. In the ENCORE study, eliglustat was found to be non-inferior to imiglucerase in maintaining stability with respect to hemoglobin concentration, platelet count, spleen volume and liver volume after 12 months (Cox et al. Lancet 2015;385:2355). Stable hemoglobin was defined as not decreasing by more than 1.5 g/dL from baseline and stable platelet count as not decreasing by more than 25% from baseline. For patients who received eliglustat for 24 months in the ENCORE extension, hemoglobin levels remained stable in 97% of patients (mean change: -0.10±0.77 g/dL) and platelet count remained stable in 94% of patients (mean change: 2.27±17.6x109/L). In former imiglucerase patients who received eliglustat for 12 months in the ENCORE extension, hemoglobin levels remained stable in 100% of patients at 12 months and platelet counts remained stable in 90%. In all 3 trials, eliglustat was generally well tolerated with 90% of patients overall electing to continue on eliglustat. Most AEs were non-serious, considered unrelated to eliglustat, and either mild or moderate in severity. Conclusions: Long-term use of eliglustat in adults with GD1 is associated with continued improvements over time in hematologic parameters in previously untreated patients and hematologic stability in patients previously stabilized on ERT. Disclosures Weinreb: Genzyme, a Sanofi company: Consultancy, Honoraria, Other: Grant Support, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Cox:Genzyme, a Sanofi company: Consultancy, Honoraria, Other: Grant Support, Speakers Bureau; Shire: Consultancy, Honoraria, Other: Grant support, Speakers Bureau; Actelion: Consultancy, Honoraria, Speakers Bureau. Lukina:Shire: Honoraria, Other: Travel Reimbursement; Genzyme, a Sanofi company: Honoraria, Other: Travel Reimbursement. Mistry:Genzyme, a Sanofi company: Consultancy, Honoraria, Other: Grant Support, Speakers Bureau; Shire: Consultancy, Honoraria, Other: Grant Support, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Synageva: Honoraria, Speakers Bureau; NIH NIMS: Other: Grant Support. Angell:Genzyme, a Sanofi company: Employment. Gaemers:Genzyme, a Sanofi company: Employment. Peterschmitt:Genzyme, a Sanofi company: Employment.

Abstract B17: Ceramide-antiestrogen regimen targets bioenergetic elements in acute myelogenous leukemia

Abstract Acute myelogenous leukemia (AML), the most common type of leukemia in adults, is heterogeneous in nature and aggressive and only about 25% of patients that experience remission with cytotoxic chemotherapy remain disease-free. The present work demonstrates the therapeutic efficacy of a novel antiestrogen-ceramide regimen in AML. Herein we show that the antiestrogen tamoxifen (tam) magnifies the cytotoxic impact of ceramide, a tumor-suppressor sphingolipid, inducing apoptosis through mitochondrial-bioenergetic targeting, an expedient, practicable avenue for disrupting the energy-producing systems of cancer cells and a potentially fruitful therapeutic direction. C6-ceramide (C6-cer), a short-chain, hydrophilic analog of ceramide was used in these studies. Nanoliposomal formulations of C6-cer and tam were also utilized and found equally effective. In human AML cell lines and in patient-derived AML cells, the C6-cer-tam combination was far more active than single agents in depressing viability and in inducing apoptosis (Annexin V binding; DNA fragmentation). Interestingly, the major tam metabolite in humans, and the one with the longest serum half-life, N-desmethyltamoxifen (DMT), was equally effective in combination with C6-cer. As an example, in KG-1 cells, whereas C6-cer (5 μM) and DMT (5 μM) reduced viability to only 95 and 90% of control, respectively, the combination resulted in 10% viability after 72 hr exposure. In-depth investigations revealed that cytotoxic responses to the C6-cer-antiestrogen regimen were accompanied by i) time- and dose-dependent loss of mitochondrial membrane potential, ii) inhibition of mitochondrial Complex I respiration, iii) mitochondrial cytochrome c release, a key initiative step in the apoptotic process, iv) decreases in cellular ATP levels as marked by robust increases the ratio of ADP to ATP, and v) a >50% reduction in cellular glycolytic capacity, a demonstration of commitment to energy severance. As loss of mitochondrial membrane potential constitutes a critical event in cell death and depletion of ATP in conjunction with targeting the “Warburg effect” represent potent metabolic “hits”, we conclude that the C6-cer-tamoxifen regimen could be a potentially effective targeted therapy for AML, independent of the multidrug resistant phenotype, as shown by efficacy in vincristine-resistant AML cells. Although a two-drug combination, this regimen could be classified as a mitocan, an agent that targets mitochondria and exhibits anti-cancer activity. Support: NCI CA171983 Citation Format: Samy A.F. Morad, Terence E. Ryan, Brian M. Barth, David F. Claxton, Mark Kester, Thomas P. Loughran, Jr., Myles C. Cabot. Ceramide-antiestrogen regimen targets bioenergetic elements in acute myelogenous leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B17.

Ceramides with a pentadecasphingosine chain and short acyls have strong permeabilization effects on skin and model lipid membranes

The composition and organization of stratum corneum lipids play an essential role in skin barrier function. Ceramides represent essential components of this lipid matrix; however, the importance of the individual structural features in ceramides is not fully understood. To probe the structure–permeability relationships in ceramides, we prepared analogs of N-lignoceroylsphingosine with shortened sphingosine (15 and 12 carbons) and acyl chains (2, 4 and 6 carbons) and studied their behavior in skin and in model lipid membranes. Ceramide analogs with pentadecasphingosine (15C) chains were more barrier-perturbing than 12C- and 18C-sphingosine ceramides; the greatest effects were found with 4 to 6C acyls (up to 15 times higher skin permeability compared to an untreated control and up to 79 times higher permeability of model stratum corneum lipid membranes compared to native very long-chain ceramides). Infrared spectroscopy using deuterated lipids and X-ray powder diffraction showed surprisingly similar behavior of the short ceramide membranes in terms of lipid chain order and packing, phase transitions and domain formation. The high- and low-permeability membranes differed in their amide I band shape and lamellar organization. These skin and membrane permeabilization properties of some short ceramides may be explored, for example, for the rational design of permeation enhancers for transdermal drug delivery.

Abstract 19536: Lipid Oversupply to Cardiomyocytes Induces Sphingolipid-dependent Oxidative Stress and Induction of Mitophagy Through Ceramide Synthase 2

Introduction: Diabetic cardiomyopathy (DbCM) contributes to the high risk of heart failure (HF) in diabetics, but mechanisms underlying DbCM remain unclear. We previously showed that highly saturated fat feeding in mice altered cardiomyocyte sphingolipid profiles leading to DbCM and that some of these maladaptations were dependent on autophagy and ceramide synthase 5 (CerS5). Hypothesis: In the present study, we sought to further understand the cellular processes in which lipid overload leads to DbCM in in the context of sphingolipids. Methods: Mice fed an obesogenic diet and in vitro studies using H9c2 cardiomyocytes were utilized in this study. Results: Increased oxidative stress and enhanced apoptosis were identified in the hearts of animals subjected to lipid overload, while animals treated with the sphingolipid synthesis inhibitor myriocin were protected. Similarly, cardiomyocytes treated with palmitate showed a sphingolipid-dependent increase of reactive oxygen species (ROS) and mitophagy. Treatment with mitochondria-targeted ceramide analogs induced cardiomyocyte cell death in an increasing chain length manner, which was exacerbated by inhibiting mitophagy. This suggested that lipotoxicity to cardiomyocytes occurs in part through oxidative stress in a sphingolipid-dependent manner, and mitophagy may occur to prevent further damage. Our observation that only longer chain ceramides led to these outcomes suggested involvement of CerS2 which specifically produces very long chain ceramides (C20 and higher). Overexpression of CerS2 showed increased mitophagy in cardiomyocytes and knockdown of CerS2 by CRISPR-CAS9 technology decreased mitophagy. Conclusions: Taken together, our data suggest that lipid overload induces mitophagy potentially as a protective measure in defense from CerS2-induced mitochondrial damage, oxidative stress and cell death in DbCM.

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors.

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200 nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.

Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3.

Cilia are important organelles formed by cell membrane protrusions; however, little is known about their regulation by membrane lipids. We characterize a novel activation mechanism for glycogen synthase kinase-3 (GSK3) by the sphingolipids phytoceramide and ceramide that is critical for ciliogenesis in Chlamydomonas and murine ependymal cells, respectively. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in (phyto)ceramide biosynthesis. Inhibition of SPT in Chlamydomonas by myriocin led to loss of flagella and reduced tubulin acetylation, which was prevented by supplementation with the precursor dihydrosphingosine. Immunocytochemistry showed that (phyto)ceramide was colocalized with phospho-Tyr-216-GSK3 (pYGSK3) at the base and tip of Chlamydomonas flagella and motile cilia in ependymal cells. The (phyto)ceramide distribution was consistent with that of a bifunctional ceramide analogue UV cross-linked and visualized by click-chemistry-mediated fluorescent labeling. Ceramide depletion, by myriocin or neutral sphingomyelinase deficiency (fro/fro mouse), led to GSK3 dephosphorylation and defective flagella and cilia. Motile cilia were rescued and pYGSK3 localization restored by incubation of fro/fro ependymal cells with exogenous C24:1 ceramide, which directly bound to pYGSK3. Our findings suggest that (phyto)ceramide-mediated translocation of pYGSK into flagella and cilia is an evolutionarily conserved mechanism fundamental to the regulation of ciliogenesis.

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.

A Series of Ceramide Analogs Modified at the 1-Position with Potent Activity Against the Intracellular Growth of Chlamydia Trachomatis

Chlamydia trachomatis is an intracellular pathogen that requires different nutrients, including sphingolipids, for survival. Components for the transport and biosynthesis of sphingolipids thus may have a potential as antichlamydial targets. In this study, we synthesized a collection of 24 ceramide derivatives. Three derivatives show pronounced activity with submicromolar IC50. The potency of these compounds was one order of magnitude higher than that of the antibiotic chloramphenicol. We show a detailed structure-activity relationship study for this novel compound class exhibiting a significant effect on the growth of C. trachomatis L2 without penetrating the bacteria itself. The structure-activity relationship presented here defines an important step toward the molecular target of this compound class, which is still elusive.

Synthesis and antiproliferative properties of a new ceramide analog of varacin

A benzopentasulfane was synthesized in 8 steps with a ceramide attached through an amide bond to the 7-position of the heterocycle structure. The anticancer activity of this synthetic ceramide–benzopolysulfane drug conjugate was analyzed against five human cancer cell lines MDA-MB-231 (breast), DU145 (prostate), MIA PaCa-2 (pancreas), HeLa (cervix), and U251 (glioblastoma). The ceramide–benzopolysulfane conjugate had IC50 values ranging from 10 to >20μM with complete cell killing at 12.5μM for MDA-MB-231 and 20μM for DU145 and HeLa cells. The ceramide–benzopolysulfane conjugate had IC50 values 1.8 and 4.0 times lower than a PEG benzopolysulfane, N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzo[f][1,2,3,4,5]-pentathiepine-7-carboxamide, for MDA-MB-231 and DU145 cells, respectively. The parent “unsubstituted” benzopolysulfane, o-C6H4S5, had IC50 values 4.2 times lower and 2.7 times higher than the ceramide benzopolysulfane for MDA-MB-231 and DU145 cells, respectively. The results indicate that the polysulfur linkage is needed for activity since benzenedithiol, o-C6H4(SH)2, had IC50 values greater than 30μM with little effect on MDA-MB-231 and DU145 cells. Thus, to account for the bioactivity, a bimolecular reaction of cellular thiol with the ceramide benzopolysulfane is a proposed followed by thiozone (S3) extrusion.

Stereospecific induction of apoptosis in tumor cells via endogenous C16-ceramide and distinct transcripts.

Concentration and distribution of individual endogenous ceramide species is crucial for apoptosis induction in response to various stimuli. Exogenous ceramide analogs induce apoptosis and can in turn modify the composition/concentrations of endogenous ceramide species and associated signaling. In this study, we show here that the elevation of endogenous C16-ceramide levels is a common feature of several known apoptosis-inducing triggers like mmLDL, TNF-alpha, H2O2 and exogenous C6-ceramide. Vice versa apoptosis requires elevation of endogenous C16-ceramide levels in cells. Enantiomers of a synthetic ceramide analog HPL-1RS36N have been developed as probes and vary in their capacity to inducing apoptosis in macrophages and HT-29 cells. Apoptosis induction by the two synthetic ceramide analogs HPL-39N and HPL-1R36N correlates with generation of cellular C16-ceramide concentration. In contrast to the S-enantiomer HPL-1S36N, the R-enantiomer HPL-1R36N shows significant effects on the expression of distinct genes known to be involved in cell cycle, cell growth and cell death (CXCL10, CCL5 and TNF-alpha), similarly on apoptosis induction. Enantioselective effects on transcription induced by metabolically stable synthetic probes provide clues on molecular mechanisms of ceramide-induced signaling, as well as leads for future anti-cancer agents.

Inhibitors of Ceramidases

The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. Acid ceramidase inhibition sensitizes certain types of cancer to chemo- and radio-therapy and this is suggestive of a role of acid ceramidase inhibitors as chemo-sensitizers which can act synergistically with chemo-therapeutic drugs. In this review, we summarize the development of ceramide analogues as first-generation ceramidase inhibitors together with data on their activity in cells and disease models. Furthermore, we describe the recent developments that have led to highly potent second-generation ceramidase inhibitors that act at nanomolar concentrations. In the third part, various assays of ceramidases are described and their relevance for accurately measuring ceramidase activities and for the development of novel inhibitors is highlighted. Besides potential clinical implications, the recent improvements in ceramidase inhibition and assaying may help to better understand the mechanisms of ceramide biology.