C2 Ceramide Research Articles

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics. Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study, we evaluated the chronic effects of TNFα, IL-6, and IL-1β on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics, including increased proton leak, decreased ΔΨm, increased basal respiration, and decreased ATP turnover. In contrast, although IL-6 and IL-1β decreased maximal respiratory capacity, they had no effect on ΔΨm and varied effects on ATP turnover, proton leak, or basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis, including PGC-1α and eNOS as well as deceased expression of COX IV and Cyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation and decreased the total level of OPA1 while increasing OPA1 cleavage, without expression of levels of mitofusin 2, DRP-1, or mitofilin being affected. In summary, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism, but each leads to decreased mitochondrial function and a reprogramming of fat cell biology.

The Combined Effect of Encapsulating Curcumin and C6 Ceramide in Liposomal Nanoparticles against Osteosarcoma

This study examines the antitumor potential of curcumin and C6 ceramide (C6) against osteosarcoma (OS) cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Three liposomal formulations were prepared: curcumin liposomes, C6 liposomes and C6-curcumin liposomes. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with curcumin liposomes alone. Importantly, C6-curcumin liposomes were found to be less toxic on untransformed primary human cells (human mesenchymal stem cells) in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. The efficiency of the preparations was tested in vivo using a human osteosarcoma xenograft assay. Using pegylated liposomes to increase the plasma half-life and tagging with folate (FA) for targeted delivery in vivo, a significant reduction in tumor size was observed with C6-curcumin-FA liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy.

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression

BackgroundCeramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.MethodsThe activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.ResultsExposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.ConclusionWe have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

Mutation for Nonsyndromic Mental Retardation in the trans-2-Enoyl-CoA Reductase TER Gene Involved in Fatty Acid Elongation Impairs the Enzyme Activity and Stability, Leading to Change in Sphingolipid Profile

Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TER(P182L/P182L) B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TER(P182L/P182L) B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.

A Metabolic Shift Favoring Sphingosine 1-Phosphate at the Expense of Ceramide Controls Glioblastoma Angiogenesis

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

Abstract B104: Liposomal C6 Ceramide appears to potentiate chemotoxicity of paclitaxel, gemcitabine and cetuximab against aggressive pancreatic cancer.

Abstract Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We have previously demonstrated reversal of these resistant pathways by C6 Ceramide and now are pursuing optimum intracellular delivery using selected liposomal preparations ofC6Ceramide. Methods: C6 Ceramide housed in 4 different liposomal vesicle formulations were tested against pancreatic cell lines L3.6, PANC1 and MIA, using combination therapy with chemotherapy agents Gemcitabine, Paclitaxel and Cetuximab with determination of the MTT anti tumor response. Liposomal preparations included : (#1)18:1 PC(1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp and is fluid at bodyT*(# 3). 18:0 PC- has high transition Temperature(T*) is rigid at body T*;#5 DPGG- avoids RE System (is fluid at bodyT*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system) Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro. Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 & 3, but confirmed high activity in preparations #5 & 7. Conclusion: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B104. Citation Format: Harold J. Wanebo, Cong Cao, Shaun Lu, David Shrayer, Yinsheng Wan, Wayne Bowen, Wayne Bowen. Liposomal C6 Ceramide appears to potentiate chemotoxicity of paclitaxel, gemcitabine and cetuximab against aggressive pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B104.

Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice

Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

Plasma Ceramide and Glucosylceramide Metabolism Is Altered in Sporadic Parkinson's Disease and Associated with Cognitive Impairment: A Pilot Study

BackgroundMutations in the gene coding for glucocerebrosidase (GBA), which metabolizes glucosylceramide (a monohexosylceramide) into glucose and ceramide, is the most common genetic risk factor for sporadic Parkinson's disease (PD). GBA mutation carriers are more likely to have an earlier age of onset and to develop cognitive impairment and dementia. We hypothesized that plasma levels of lipids involved in ceramide metabolism would also be altered in PD non-GBA mutation carriers and associated with worse cognition.MethodsPlasma ceramide, monohexosylceramide, and lactosylceramide levels in 26 cognitively normal PD patients, 26 PD patients with cognitive impairment or dementia, and 5 cognitively normal non-PD controls were determined by LC/ESI/MS/MS.ResultsLevels of all lipid species were higher in PD patients versus controls. Among PD patients, levels of ceramide C16:0, C18:0, C20:0, C22:0, and C24:1 and monohexosylceramide C16:0, C20:0 and C24:0 species were higher (all P<0.05) in those with versus without cognitive impairment.ConclusionThese results suggest that plasma ceramide and monohexosylceramide metabolism is altered in PD non-GBA mutation carriers and that higher levels are associated with worse cognition. Additional studies with larger sample sizes, including cognitively normal controls, are needed to confirm these findings.

Acid sphingomyelinase–ceramide system mediates effects of antidepressant drugs

Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

Ultraviolet (UV) and Hydrogen Peroxide Activate Ceramide-ER Stress-AMPK Signaling Axis to Promote Retinal Pigment Epithelium (RPE) Cell Apoptosis

Ultraviolet (UV) radiation and reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelium (RPE) cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD). The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER) stress-AMP activated protein kinase (AMPK) signaling axis in UV and hydrogen peroxide (H2O2)-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal), ceramide production (fumonisin B1) and AMPK activation (compound C) suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) mimicked UV and H2O2’s effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

Abstract 325: Palmitic Acid Enhances the Stimulatory Effect of Lipopolysaccharide on Interleukin 6 Expression in Cardiac Microvascular Endothelial Cells by Increasing Ceramide Production via both De Novo Synthesis and Sphingomyelin Hydrolysis

Host inflammatory response mediated by toll-like receptor (TLR)4, a pattern recognition receptor for lipopolysaccharide (LPS), plays an important role in cardiovascular complication of diabetes. Although it is known that patients with type 2 diabetes or/and obesity have increased plasma free fatty acids (FAs), and saturated FAs such as palmitic acid (PA) increase host inflammatory response, the interaction between saturated FAs and LPS-elicited TLR4 signaling has not been well elucidated. Since it is known that neovascularization in advanced atherosclerotic plaques contributes to plaque destabilization, we investigated the mechanism whereby PA interacts with LPS for pro-inflammatory gene expression in cardiac microvascular (MIC) endothelial cells (ECs). We found that while LPS at 5 ng/ml stimulated a robust interleukin (IL)-6 secretion in cardiac MIC ECs, 100 μM of PA further augmented the stimulation by more than 2-fold (Control, 4.4; LPS, 41.3, PA, 6.0, LPS+PA, 89.0 ng/ml). Besides IL-6, PA also enhanced the stimulatory effect of LPS on tumor necrosis factor (TNF)α (Control, 2.6; LPS, 38.5, PA, 13.8, LPS+PA, 58.0 pg/ml). Time course study showed that IL-6 expression induced by LPS peaked at 4 h and dropped sharply after the peak. In contrast, addition of PA to LPS delayed the peak to 8 h and stimulated a second peak at 24 h after a transient drop at 12 h. Consistently, quantification of IL-6 in medium showed that IL-6 level in culture medium of cells treated by LPS alone plateaued at 8 h, but addition of PA to LPS led to a continuous liner increase in IL-6 in culture medium up to 48 h. Our further investigation showed that PA and LPS had a synergistic effect on the production of total ceramides and, in particular, C16 ceramide. The involvement of the synergistic effect of LPS and PA on ceramide production in IL-6 upregulation was indicated by the findings that myriocin and fumonisin B1, the inhibitors of ceramide de novo synthesis, and GW4869, an inhibitor of sphingomyelin hydrolysis, reduced IL-6 secretion by cells treated with LPS and PA significantly. Taken together, this study has demonstrated that PA enhances the stimulatory effect of LPS on IL-6 expression in cardiac MIC ECs by increasing ceramide production via both de novo synthesis and sphingomyelin hydrolysis.

Ceramide analogue displays stimulatory activity on dendritic cells to promote T cell responses upon virus infection (P6115)

Abstract The ceramide family of lipids has been shown to play various important roles in both cell structure and signaling in a diverse array of cell types, including immune cells. However, very little is known regarding how ceramide affects the activation of dendritic cells (DCs) in response to viral infection. In this study, we demonstrate that a synthetic ceramide analogue (C8) stimulates DCs to increase the expansion of virus-specific CD8 T cells upon virus infection. Exogenously supplied C8 ceramide elevated the expression of DC maturation markers such as MHC class I and the co-stimulatory molecule B7-2 following infection with the immune suppressive Clone 13 strain of lymphocytic choriomeningitis virus (LCMV) in vitro. In addition, the ceramide analogue enhanced the production of IL-12 from DCs infected by the virus. Importantly, ceramide-conditioned, LCMV-infected DCs displayed a heightened activity to promote proliferation of virus-specific, CD8 T cells when compared to virus-infected DCs. Furthermore, locally instilled, but not systemically administered, ceramide analogue via intranasal inoculation significantly increased virus-reactive CD8 T cell responses in vivo. Collectively, these findings provide new insights into ceramide-mediated regulation of DC responses against virus infection and help us establish a foundation for novel immune-stimulatory therapeutics.

Paclitaxel disrupts polarized entry of membrane-permeable C6 ceramide into ovarian cancer cells resulting in synchronous induction of cell death

Exogenous cell-permeable C6 ceramide has been demonstrated to act synergistically with chemotherapeutic drugs, including paclitaxel, cisplatin, doxorubicin and the histone deacetylase inhibitor, trichostatin A, to induce cell death in a variety of cancer cells. We previously demonstrated that C6 ceramide and paclitaxel function synergistically to induce ovarian cancer cell death via modulation of the PI3/AKT cell survival pathway. In the present study, the entry pattern of C6 ceramide into ovarian cancer cells was investigated using fluorescent short chain C6-NBD sphingomyelin (C6-NBD). Confocal microscopy revealed that C6-NBD enters the cells in a polarized pattern, characterized by marked signals at one cellular end, representing a likely mitosis initiation site. Pretreatment of the cells with filipin, an inhibitor of the lipid raft/caveolae endocytosis pathway, decreases C6-NBD entry into the cells. A pretreatment with the water channel inhibitor, CuSO4, was also found to reduce the entry of C6-NBD. Notably, the pretreatment with paclitaxel was shown to disrupt the polarized entry of C6-NBD into the cells, resulting in an even distribution of C6-NBD in the cytoplasm. In addition, the pretreatment of the cells with paclitaxel destabilized the cytoskeletal proteins, releasing an increased number of short tubulin fragments. The results of the present study indicate that C6 ceramide preferentially enters the cells via a predetermined initiation site of mitosis. In addition to diffusion, short chain C6 ceramide may also enter cells via water channels and caveolae-mediated endocytosis. Paclitaxel disrupts the cell cytoskeleton and induces an even distribution of C6 ceramide in the cytoplasm resulting in synergistic ovarian cancer cell death.

Abstract 600: Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer.

Abstract Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We previously demonstrated reversal of these resistant pathways by C6 Ceramide and now pursue optimum intracellular delivery using selected liposomal preparations of C6 Ceramide. Methods: C6 Ceramide(C6Cer) in 4 different liposomal vesicle formulations were tested by MTT assay against pancreatic cell lines L3.6, PANC1 and MIA, combined with chemotherapy: Gemcitabine, Paclitaxel and Cetuximab.Liposomal preparations included : (#1)18:1 PC (1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp.(T*) and is fluid at body T*(# 3). 18:0 PC-is rigid with high transition (T*) . #5 DPGG- avoids RE System (is fluid at body T*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system).Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro. Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 &amp; 3, but confirmed high activity in preparations #5 &amp; 7. MTT ResponseFractionControlTaxolGemcitabineCetuximab100%87%70%90%#5(C6)90%42%40%33%# 6 PPS control108%76%36%77%# 7 C6101%31%45%59%# 8 PBS control110%60%58%62%*Control-cell survival 72 hours ∼ 90%-100%(Drug Dose - C6 1 ug/ml, Pac. 1ug/ml,Gem 5ug/ml, Cetux 25ug/ml) Conclude: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process. Citation Format: Harold J. Wanebo, Cao Cong, Li Shu, David Shrayer, Yeushung Wan, Wayne Bowen. Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2013-600

Abstract 3396: Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism.

Abstract Novel approaches to enhancing the delivery, uptake, and therapeutic effect of sphingolipids are of interest for the treatment of neoplasms. We attempt to promote cellular ceramide accumulation in murine and human models of acute myelogenous leukemia (AML) by modifying ceramide metabolism with either the sphingosine kinase1 inhibitor safingol (Lip-Saf), or the acid ceramidase inhibitor LCL204, preventing ceramide glycosylation by inhibition of p-glycoprotein (P-GP) activity with tamoxifen (Lip-Tam), and supplementing cells with exogenous C6 ceramide (Lip-C6) via liposomal delivery. Ceramide is widely recognized as an apoptotic signal and blocking its catabolism to sphingosisne-1-phosphate has been shown to induce autophagy. Safingol and LCL204 both modify ceramide catabolism. Supplementation of tamoxifen has been shown to promote apoptosis via ceramide accumulation by inhibiting the activity of P-GP at the Golgi membrane, which can participate in ceramide glycosylation. C1498, HL-60, HL-60/VCR, and KG-1 leukemia cell line viability was quantified following treatment with Lip-C6, Lip-Saf, or a 2:1 combination to measure synergy. Likewise, cell line viability was evaluated using Lip-Tam and Lip-C6, independently and in a 1:1 combination to study synergy. C1498 viability was also quantified with increasing concentrations of LCL204. CalcuSyn software was used to analyze dose-response and synergistic therapeutic efficacy. Colony-forming capacity was assessed using a poor-prognosis AML patient sample treated with Lip-C6, Lip-Saf, or the combination of both. Autophagy and apoptosis were further evaluated using flow cytometry with primary AML samples and leukemia cell lines. A reduction in IC50 of the drugs used in combination was noted in several cell lines. Dramatic IC50 shifts were observed in KG-1, HL-60, and the vincristine-resistant HL-60/VCR cell lines after exposure to a combination of Lip-C6 and Lip-Saf in a 2:1 ratio. Flow cytometry confirmed Lip-Saf enhances autophagy, whereas Lip-C6 exerts an effect primarily through apoptosis in C1498 and primary AML samples. Therefore, the observed cell death synergism is likely due to induction of both autophagy and apoptosis. Additionally, it was shown that the anti-AML efficacy of Lip-Tam was dramatically enhanced in combination with Lip-C6, demonstrating that inhibition of ceramide glycosylation is also a valid combinatorial therapeutic approach. Lastly, inhibition of acid ceramidase via LCL204 demonstrated efficacy as a cytotoxic agent both in vitro and in vivo using C1498 cells alone or engrafted into C57BL/6J mice. These studies verify inhibition of ceramide metabolism as an effective strategy to enhance the therapeutic efficacy of ceramide in the treatment of AML. Citation Format: Timothy J. Brown, Aileen M. Garcia, Su-Fern Tan, Lindsey N. Kissinger, David J. Feith, Brian M. Barth, David F. Claxton. Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2013-3396

AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells

Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide's effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramide-induced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.

Engineering of an ω-3 polyunsaturated fatty acid-containing nanoemulsion system for combination C6-ceramide and 17β-estradiol delivery and bioactivity in human vascular endothelial and smooth muscle cells

Delayed endothelial cell (EC) regeneration and the medial vascular smooth muscle cells (VSMCs) proliferation contribute to arterial restenosis. Although ω-3-polyunsaturated fatty acids (PUFAs), 17β-estradiol (17-βE) and C6-ceramide (CER) have shown therapeutic promise in addressing restenosis, extensive protein binding and lipophilicity complicate their (co-)delivery to cellular targets. We report engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and CER cargo to cultured vascular cells. The cargo-free, ω-3-PUFA-rich nanoemulsion itself typically reduced growth factor-stimulated cellular proliferation, as did nanoemulsion-delivered CER alone, through enhanced pro-apoptotic caspase 3/7 activity. 17-βE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-βE and CER loaded nanoemulsions exerted an anti-proliferative effect more pronounced on VSMCs than ECs. These therapeutically beneficial responses to ω-3-PUFA, CER, and/or 17-βE in our nanoemulsion formulation invite evaluation of this novel approach in animal models of restenosis and other occlusive vasculopathies. From the Clinical EditorThis team of investigators report the engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and C6-ceramide cargo to cultured vascular cells in an effort to address vascular restenosis. Further preclinical studies will be needed in animal models before this approach could be considered for clinical trials.

Inhibition of sphingolipid synthesis improves dyslipidemia in the diet-induced hamster model of insulin resistance: Evidence for the role of sphingosine and sphinganine in hepatic VLDL-apoB100 overproduction

Sphingolipids have emerged as important bioactive lipid species involved in the pathogenesis of type 2 diabetes and cardiovascular disease. However, little is known of the regulatory role of sphingolipids in dyslipidemia of insulin-resistant states. We employed hamster models of dyslipidemia and insulin resistance to investigate the role of sphingolipids in hepatic VLDL overproduction, induction of insulin resistance, and inflammation. Hamsters were fed either a control chow diet, a high fructose diet, or a diet high in fat, fructose and cholesterol (FFC diet). They were then treated for 2 weeks with vehicle or 0.3 mg/kg myriocin, a potent inhibitor of de novo sphingolipid synthesis. Both fructose and FFC feeding induced significant increases in hepatic sphinganine, which was normalized to chow-fed levels with myriocin (P < 0.05); myriocin also lowered hepatic ceramide content (P < 0.05). Plasma TG and cholesterol as well as VLDL-TG and -apoB100 were similarly reduced with myriocin treatment in all hamsters, regardless of diet. Myriocin treatment also led to improved insulin sensitivity and reduced hepatic SREBP-1c mRNA, though it did not appear to ameliorate the activation of hepatic inflammatory pathways. Importantly, direct treatment of primary hamster hepatocytes ex vivo with C2 ceramide or sphingosine led to an increased secretion of newly synthesized apoB100. Taken together, these data suggest that a) hepatic VLDL-apoB100 overproduction may be stimulated by ceramides and sphingosine and b) inhibition of sphingolipid synthesis can reduce circulating VLDL in hamsters and improve circulating lipids—an effect that is possibly due to improved insulin signaling and reduced lipogenesis but is independent of changes in inflammation.

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.