Josefina Casas

Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.

Inhibition of cytosolic epoxide hydrolase by trans-3-phenylglycidols

The inhibition of murine cytosolic epoxide hydrolase has been studied with both racemic and enantiomerically pure trans-3-phenylglycidols. These compounds are the first enantioselective, slow binding inhibitors of cytosolic epoxide hydrolase. The (2S,3S)-3-phenylglycidol enantiomer was always a better inhibitor than the (2R,3R)-enantiomer. When the I50 values of (2S,3S)- and (2R,3R)-3-(4-nitrophenyl)glycidol were compared, the (2S,3S)-enantiomer was at least a 750-fold better inhibitor (I50 = 1.6 microM) than the (2R,3R)-enantiomer (I50 = 1200 microM), and it was the most potent inhibitor tested in the 3-phenylglycidol series. If the hydroxyl group of the glycidol was masked or converted to another functionality, the potency of the inhibitor decreased and the (2S,3S)-enantiomer was not necessarily the better inhibitor. In addition, trans-3-phenylglycidols demonstrated slow binding inhibition of cytosolic epoxide hydrolase. Inhibitors without a hydroxyl group, or with a blocked hydroxyl group, were not slow binding inhibitors. These results suggested that the hydroxyl group was important in both enantioselectivity and time dependence of inhibition of cytosolic epoxide hydrolase by trans-3-phenylglycidols. The hydration pattern of (2S,3S)- and (2R,3R)-2,3-epoxy-3-(4- nitrophenyl)glycidol by cytosolic epoxide hydrolase also differed. When incorporation of [18O] from water catalyzed by cytosolic epoxide hydrolase was measured, the (2S,3S)-enantiomer gave 12% incorporation into the benzylic carbon and the (2R,3R)-enantiometer gave 40% incorporation into the benzylic carbon. Finally, trans-3-phenylglycidols were found to be poor inhibitors of microsomal epoxide hydrolase.

Use of dimethyldioxirane in the preparation of epoxy derivatives related to insect juvenile hormones

Abstract A study on the epoxidation of methyl farnesoate using the title reagent has been carried out. In addition, dimethyldioxirane has shown to be a convenient reagent for the synthesis of putative metabolites of juvenile hormone III, such as diepoxide 4 , triepoxide 5 and epoxy diol 7 . This reagent has also shown to be an excellent tool for performing the preparation of tritium labelled derivatives 4 and 7 . Finally, molecular mechanics and NMR studies for the characterisation of cis and trans tetrahydrofuran derivatives 8a and 8b , compounds resulting from the acid or enzymatic hydrolysis of epoxy diol 7 , are also reported.

Straightforward access to spisulosine and 4,5-dehydrospisulosine stereoisomers: probes for profiling ceramide synthase activities in intact cells.

A stereoselective synthesis of spisulosine (ES285) and 4,5-dehydrospisulosine stereoisomers is described. Hydrozirconation of 1-pentadecyne with Schwartz reagent, followed by diastereocontrolled addition to L- or D-alaninal afforded the required 2-amino-1,3-diol framework. The resulting sphingoid bases revealed as excellent probes for the profiling of ceramide synthase activity in intact cells. Among the sphingoid bases described in this work, spisulosine (ES285), RBM1-77, and RBM1-73 were the most suitable ones because of their highest acylation rates. These molecules should prove useful to study the role of the different ceramide synthases and the resulting N-acyl (dihydro)ceramides in cell fate.

Epoxide hydrolase activities in Drosophila melanogaster

Abstract Epoxide hydrolase (EH) activity in Drosophila melanogaster cell fractions was characterized using juvenile hormone (JH III) and cis- stilbene oxide (CSO) as substrates. A comparison of detergents indicated that 0.3% Lubrol PX was relatively effective for solubilizing EH activity from the 20,000 and 100,000 g pellets. The effect of inhibitors, pH, temperature, salt and organic solvents on EH activity depended on the substrate and cell fraction tested, which suggested there were multiple activities present. For initial purification, polyethylene glycol was useful for precipitating EH activity from the 100,000 g supernatant.

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.

Coordinated Regulation of the Orosomucoid-like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells

Background: Knockdown of orosomucoid-like (ORMDL) proteins releases serine palmitoyltransferase (SPT) activity. Results: Significant changes in SPT activity were detected when all three ORMDLs were overexpressed. Sphingolipids do not modify SPT-ORMDL interaction but rearrange ORMDLs. Macrophages suppress ORMDLs to induce de novo ceramide synthesis. Conclusion: Coordinated ORMDL expression regulation strongly influences SPT activity. Significance: SPT-ORMDL complex presents transcriptional and post-translational regulation. The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.

Inhibition of rat liver microsomal lipid peroxidation elicited by 2,2-dimethylchromenes and chromans containing fluorinated moieties resistant to cytochrome P-450 metabolism.

2,2-Dimethylchromenes and chromans containing cytochrome P-450 resistant 2,2,2-trifluoroethoxy aryl substituents were synthesized and their activity as lipid peroxidation inhibitors evaluated and compared with that exhibited by the corresponding non-fluorinated derivatives. Lipid peroxidation was stimulated in rat liver microsomes by addition of Fe-ascorbate or NADPH, and determined with the TBARS (thiobarbituric acid reactive substances) test. In assays using Fe-ascorbate stimulation, only those derivatives with a OH group at C6 (i.e. 6 and 12) elicited good inhibitory activities (IC50 = 6.0 and 5.3 microM, respectively). In respect to the NADPH dependent incubations, inhibitory activity of compound 11 (IC50 = 6.0 microM) was the highest found within the 6,7-dialkoxy derivatives tested. Results on metabolism assays with this compound showed the generation of phenol 12 (i.e. the putative active antioxidant species); on the other hand, no metabolite resulting from dealkylation at C7 was detected, thus confirming the resistance conferred by the CF3CH2O group to the cytochrome P-450 promoted cleavage. Finally, in assays where incubations in the presence of NADPH were prolonged up to three hours, inhibitory activity of the non-fluorinated 6,7-dialkoxychroman 8 remained constant, thus suggesting that a continued release of the species responsible for the inhibitory activity was produced. However, inhibition elicited by the fluorinated analog 11 showed a small decrease during the third hour of incubation. This decrease could be attributed to the slight inhibition of the cytochrome P-450 metabolism exerted by substrates bearing the CF3CH2O substituent, which would decelerate the generation of the active phenol species.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of epoxide hydrolase from human, monkey, bovine, rabbit and murine liver by trans-3-phenylglycidols

1. trans-3-Phenylglycidols were potent inhibitors of cytosolic epoxide hydrolases in all species tested. 2. The order of inhibitor potency varied from species to species but trans-3-(4-nitrophenyl)glycidols were always the most potent inhibitors tested for cytosolic epoxide hydrolase. 3. The S,S-enantiomer was a more potent cytosolic epoxide hydrolase inhibitor than the R,R-enantiomer when a free hydroxyl group was present. However, (2R,3R)-1-benzoyloxy-2,3-epoxy-3-(4-nitrophenyl)propane was always a better inhibitor than the (2S,3S)-enantiomer. 4. All microsomal epoxide hydrolases were poorly inhibited by the trans-3-phenylglycidols, and related compounds, tested. The best new microsomal epoxide hydrolase inhibitor tested was (1S,2S)-1-phenylpropylene oxide which gave 18-63% inhibition, at 2 mM, depending on the species tested.