n-oleoylethanolamine and sphingosine-1-phosphate

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

Topics: Animals; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Daunorubicin; Drug Therapy; Endocannabinoids; Ethanolamines; Galactosylgalactosylglucosylceramidase; Humans; Lysophospholipids; Mice; Microscopy, Electron, Transmission; Mitochondria; Oleic Acids; Protease Inhibitors; RNA, Messenger; RNA, Small Interfering; Sphingosine; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases