Inhibition of ceramide accumulation in AdipoR1-/- mice increases photoreceptor survival and improves vision.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.

Mechanistic insights into ceramidase inhibitor LCL521-enhanced tumor cell killing by photodynamic and thermal ablation therapies.

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).

Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

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.

The ATP-Binding Cassette Transporter-2 (ABCA2) Overexpression Modulates Sphingosine Levels and Transcription of the Amyloid Precursor Protein (APP) Gene.

The ATP-binding cassette transporter-2 (ABCA2) is a member of a family of multipass transmembrane proteins that use the energy of ATP hydrolysis to transport substrates across membrane bilayers. ABCA2 has also been genetically linked with Alzheimer's disease but the molecular mechanisms are unknown. In this report, we hypothesized that ABCA2 modulation of sphingolipid metabolism activates a signaling pathway that regulates amyloid precursor protein transcription. We found that ABCA2 overexpression in N2a cells was associated with increased mass of the sphingolipid sphingosine, derived from the catabolism of ceramide. ABCA2 overexpression increased in vitro alkaline and acid ceramidase activity. Sphingosine is a physiological inhibitor of protein kinase C (PKC) activity. Pharmacological inhibition of ceramidase activity or activation PKC activity with 12-myristate 13-acetate (PMA) or diacylglycerol (DAG) decreased endogenous APP mRNA levels in ABCA2 overexpressing cells. Treatment with PMA also decreased the expression of a transfected human APP promoter reporter construct, while treatment with a general PKC inhibitor, GF109203x, increased APP promoter activity. In N2a cells, chromatin immunoprecipitation experiments revealed that a repressive complex forms at the AP-1 site in the human APP promoter, consisting of c-jun, c-jun dimerization protein 2 (JDP2) and HDAC3 and this complex was reduced in ABCA2 overexpressing cells. Activation of the human APP promoter in A2 cells was directed by the upstream stimulatory factors USF-1 and USF-2 that bound to an E-box element in vivo. These findings indicate that ABCA2 overexpression modulates sphingosine levels and regulates transcription of the endogenous APP gene.

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

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

Benzoxazolone carboxamides: potent and systemically active inhibitors of intracellular acid ceramidase.

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

Small molecule inhibitors of ceramidases.

The equilibrium between the pro-apoptotic ceramide and pro-vital sphingosine-1-phosphate is considered to be decisive for cell death or survival. The different ceramidases thus play key roles in cell fate and might offer attractive targets for pharmacological intervention. Although until recently only moderately active inhibitors have been described, first in vivo experiments suggest activity against cancer cell survival and multi-drug resistance. Here, we provide a brief overview on the different ceramidases, and we will review the known inhibitors and current strategies for further inhibitor development.

Endogenous ceramide contributes to the transcytosis of oxLDL across endothelial cells and promotes its subendothelial retention in vascular wall.

Oxidized low density of lipoprotein (oxLDL) is the major lipid found in atherosclerotic lesion and elevated plasma oxLDL is recognized to be a risk factor of atherosclerosis. Whether plasma oxLDL could be transported across endothelial cells and initiate atherosclerotic changes remains unknown. In an established in vitro cellular transcytosis model, the present study found that oxLDL could traffic across vascular endothelial cells and further that the regulation of endogenous ceramide production by ceramide metabolizing enzyme inhibitors significantly altered the transcytosis of oxLDL across endothelial cells. It was found that acid sphingomyelinase inhibitor, desipramine (DES), and de novo ceramide synthesis inhibitor, myriocin (MYR), both decreasing the endogenous ceramide production, significantly inhibited the transcytosis of oxLDL. Ceramidase inhibitor, N-oleoylethanolamine (NOE), and sphingomyelin synthase inhibitor, O-Tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609), both increasing the endogenous ceramide production, significantly upregulated the transcytosis of oxLDL. In vivo, injection of fluorescence labeled oxLDL into mice body also predisposed to the subendothelial retention of these oxidized lipids. The observations provided in the present study demonstrate that endogenous ceramide contributes to the transcytosis of oxLDL across endothelial cells and promotes the initiating step of atherosclerosis-the subendothelial retention of lipids in vascular wall.

A review of ceramide analogs as potential anticancer agents.

Ceramide serves as a central mediator in sphingolipid metabolism and signaling pathways, regulating many fundamental cellular responses. It is referred to as a 'tumor suppressor lipid', since it powerfully potentiates signaling events that drive apoptosis, cell cycle arrest, and autophagic responses. In the typical cancer cell, ceramide levels and signaling are usually suppressed by overexpression of ceramide-metabolizing enzymes or downregulation of ceramide-generating enzymes. However, chemotherapeutic drugs as well as radiotherapy increase intracellular ceramide levels, while exogenously treating cancer cells with short-chain ceramides leads to anticancer effects. All evidence currently points to the fact that the upregulation of ceramide levels is a promising anticancer strategy. In this review, we exhibit many anticancer ceramide analogs as downstream receptor agonists and ceramide-metabolizing enzyme inhibitors.

Novel chemotherapeutic drugs in sphingolipid cancer research.

Sphingolipid-metabolizing enzymes are becoming targets for chemotherapeutic development with an increasing interest in the recent years. In this chapter we introduce the sphingolipid family of lipids, and the role of individual species in cell homeostasis. We also discuss their roles in several rare diseases and overall, in cancer transformation. We follow the biosynthesis pathway of the sphingolipid tree, focusing on the enzymes in order to understand how using small molecule inhibitors makes it possible to modulate cancer progression. Finally, we describe the most used and historically significant inhibitors employed in cancer research, their relationships to sphingolipid metabolism, and some promising results found in this field.

Sphingomyelinase activity in mother's milk is essential for juvenile development: a case from lactating tsetse flies.

Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mother's milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4-5-day extension in pregnancy, 10%-15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.

Sphingolipid modulation: a strategy for cancer therapy.

Sphingolipids are membrane lipids that play important roles in the regulation of cell functions and homeostasis. Alterations in their metabolism have been associated with several pathologies. For this reason, therapeutic strategies based on the design of small molecules to restore sphingolipid levels to their physiological condition have rapidly emerged. In addition, some of these new chemical entities, even if they fail to succeed along the pipeline, can become valuable pharmacological tools for the study of sphingolipid function. Implications of altered sphingolipid metabolism in cancer progression have allowed the identification of new targets for the development of potential anticancer agents. Based on these premises, this review is focused on the most recent achievements in the field, with special attention to the development of small molecules, mainly enzyme inhibitors, able to disrupt some of the key sphingolipid metabolic pathways implicated in cancer progression. On the other hand, metabolic dysregulation can also be modulated by the use of sphingolipid analogs, which can alter the sphingolipid balance driving cells to death or survival and thus becoming useful candidates for subsequent drug development.

Discovery and evaluation of inhibitors of human ceramidase.

The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analogue (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analogue, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell-cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development and provide the first nonlipid inhibitors of human ceramidase activity.

Ceramidases in hematological malignancies: senseless or neglected target?

Ceramidases are ubiquitous amidohydrolases that catalyze the cleavage of ceramides into sphingosine and fatty acids. This reaction exerts a cytoprotective role in physiological conditions, while altered ceramidase activities favour a number of human diseases. Among these diseases, several reports point to important roles of ceramidases, mainly the acid ceramidase, in the initiation and progression of cancer, and the response of tumors to radio- or chemotherapy. Multiple reports confirm the interest of acid ceramidase inhibitors as anticancer drugs, either alone or in combination with other therapies. Sphingolipid metabolism plays a role in hematological malignancies and appears as an interesting target for therapeutic intervention. Although the use of ceramidase inhibitors in chemotherapy of hematologic cancers has not been widely investigated, a number of indirect evidence suggest that inhibition of specific ceramidases could potentiate the effect of drugs in clinical use to treat hematologic malignancies and may afford strategies to combat relapses. The arsenal of ceramidase inhibitors so far available is wide and hopefully, upcoming research will assess the feasibility of this approach.

Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases.

Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease.

Inhibition of acid ceramidase by a 2-substituted aminoethanol amide synergistically sensitizes prostate cancer cells to N-(4-hydroxyphenyl) retinamide.

BACKGROUND: The purpose of this study was to determine whether the therapeutic efficacy of fenretinide (4-HPR), a ceramide-generating anticancer agent, could be enhanced in prostate cancer cells by inclusion of a novel synthetic acid ceramidase (AC) inhibitor, DM102, a pivaloylamide of a 2-substituted aminoethanol. In prostate cancer, AC plays a role in progression and resistance to chemotherapy. METHODS: PC-3 and DU 145 hormone-refractory human prostate cancer cell lines were used. Cells were exposed to 4-HPR, DM102, and combinations; viability, apoptosis, cell migration, ceramide metabolism, and levels of reactive oxygen species (ROS) were assessed. RESULTS: Single agent 4-HPR and DM102 (2.5-10 µM) were weakly cytotoxic; however, combinations synergistically decreased cell viably to as low as 1.5% of control. N-oleoylethanolamine (NOE), a frequently employed AC inhibitor, was not effective in producing synergy. The 4-HPR/DM102 regimen enhanced caspase activity and increased [(3) H](dihydro)ceramide and ROS levels 6- and 30-fold over control, respectively. The antioxidant vitamin E, but not the de novo ceramide synthesis inhibitor myriocin, partially rescued cells from 4-HPR/DM102 cytotoxicity. The 4-HPR/DM102 combination also elicited synergistic cytotoxicity in DU 145 cells, another human hormone-refractory prostate cancer cell line. CONCLUSION: This study shows that 4-HPR cytotoxicity is enhanced in a synergistic fashion by inclusion of the AC inhibitor DM102, by a mechanism that enlists generation of ROS, and thus provides a system to raise 4-HPR therapeutic potential. The role of ceramide however in the cytotoxic response is not clear, as blocking ceramide generation failed to rescue PC-3 cells from 4-HPR/DM102 cytotoxicity.

Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease.

Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.

Improved synthesis of a fluorogenic ceramidase substrate.

Substantial interest has focused on the roles of sphingolipid metabolizing enzymes in a variety of hyperproliferative and inflammatory diseases. A key family of enzymes involved in these pathologies is the ceramidases. Ceramidases cleave the pro-apoptotic lipid ceramide into a long-chain fatty acid and sphingosine, which can then be further metabolized to the mitogenic and inflammatory lipid sphingosine 1-phosphate. Consequently, development of ceramidase inhibitors would provide useful pharmacologic probes for further studies of sphingolipid metabolism, as well as lead compounds for drug development. This effort has been hampered by the lack of in vitro and cellular ceramidase assays that are amenable to high-throughput screening. Recently, a fluorogenic ceramide analog has been described as a substrate for use in ceramidase assays. The synthesis of this compound has now been substantially improved in terms of both the required effort and the overall yield of the process. Key improvements include: reduction in number of required steps, use of a hydroboration reaction; incorporation of a Mitsunobu reaction; improved acylation by the addition of triethylamine; together providing a fourfold increase in the overall yield. In addition, it has been demonstrated that the ceramide analog can be used in high-throughput assays to identify ceramidase inhibitors. Overall, the improved efficiency in the preparation of this ceramidase substrate should accelerate discovery efforts relating to sphingolipid metabolism.