ABSTRACT
PURPOSE: Sphingolipid metabolites, such as sphingosine and ceramide, are highly bioactive compounds and are involved in diverse cell processes, including cell-cell interaction, cell proliferation, differentiation, and apoptosis. However, the physiological roles of phytosphingosine are poorly understood. In this study, we report that phytosphingosine can potently induce apoptotic cell death in human cancer cells via caspase activation and caspase-independent cytochrome c release. EXPERIMENTAL DESIGN: Phytosphingosine-induced apoptosis was determined by Hoechst 33258 staining, flow cytometric analysis, and DNA fragmentation assay. Involvement of caspases was determined by immunoblot analysis and cell death detection assays after treatment with synthetic inhibitor z-Val-Ala-Asp-fluoromethyl ketone, z-DEVD-fmk, or z-IETD-fmk. Death receptor (DR) dependency was analyzed by examining expression of DRs (Fas, DR4, DR5, TNFR1, and R2), and interaction of Fas-associated death domain and caspase 8. Involvement of the mitochondria pathway was examined by monitoring of the mitochondria membrane potential, cytochrome c release, and Bax translocation. RESULTS: Phytosphingosine-treated cells displayed several features of apoptosis, including increase of sub-G(1) population, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. We observed that phytosphingosine cause activation of caspase 8 in a DR-independent fashion. Phytosphingosine also induced activation of caspase 9 and 3, loss of mitochondrial membrane potential, and the cytochrome c release from mitochondria. However, we failed to detect Bid cleavage. Moreover, caspase 8 inhibitor z-IETD-fmk did not affect phytosphingosine-induced cytochrome c release and caspase 9 activation, suggesting that phytosphingosine-induced cytochrome c release is caused by caspase 8-independent manner. Phytosphingosine induced mitochondrial translocation of Bax from the cytosol without changes in the protein levels of Bcl-2, Bcl-xL, and Bax. In addition, Bcl-2/Bax interaction was diminished after addition of phytosphingosine. CONCLUSION: These findings indicate that phytosphingosine induces apoptotic cell death in human cancer cells by direct activation of caspase 8, and by mitochondrial translocation of Bax and subsequent release of cytochrome c into cytoplasm, providing a potential mechanism for the anticancer activity of phytosphingosine.
Subject(s)
Apoptosis/drug effects , Caspases/metabolism , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/metabolism , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Carcinoma, Non-Small-Cell Lung , Caspase 8 , Caspase 9 , Caspases/drug effects , Enzyme Activation , Flow Cytometry , Humans , Lung Neoplasms , Lymphoma, T-Cell , Tumor Cells, Cultured , bcl-2-Associated X ProteinABSTRACT
Melatonin is known to have a neuroprotective effect by preventing epileptic seizures, which are normally induced by cyanide. To demonstrate the neuroprotective function of melatonin, we examined cell death and changes in plasma melatonin level in KCN-treated mice. Neuronal cell death is shown in substantial nigra of KCN-treated groups. In melatonin-treated groups, this cell death decreased in substantia nigra. Plasma melatonin level at 12:00 was significantly decreased to 52.6% after KCN injection as compared to the normal group. In contrast, melatonin level was significantly decreased (74.5%) in KCN + melatonin group. Melatonin level at 24:00 was significantly decreased to 57.0% after KCN injection and also significantly decreased to 81.0% in KCN-melatonin group as compared to the normal group. Results from the present study suggest that melatonin prevents neuronal cell death in KCN-induced brain.
