title: Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Radiation Effect, Korea Cancer Center Hospital, Seoul 139-706.
authors.forename: Moon-Taek
authors.initials: MT
authors.lastname: Park
authors.forename: Jung A
authors.initials: JA
authors.lastname: Kang
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Choi
authors.forename: Chang-Mo
authors.initials: CM
authors.lastname: Kang
authors.forename: Tae-Hwan
authors.initials: TH
authors.lastname: Kim
authors.forename: Sangwoo
authors.initials: S
authors.lastname: Bae
authors.forename: Seongman
authors.initials: S
authors.lastname: Kang
authors.forename: Sujong
authors.initials: S
authors.lastname: Kim
authors.forename: Weon-Ik
authors.initials: WI
authors.lastname: Choi
authors.forename: Chul-Koo
authors.initials: CK
authors.lastname: Cho
authors.forename: Hee-Yong
authors.initials: HY
authors.lastname: Chung
authors.forename: Yun-Sil
authors.initials: YS
authors.lastname: Lee
authors.forename: Su-Jae
authors.initials: SJ
authors.lastname: Lee
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D008175:Lung Neoplasms; D016399:Lymphoma, T-Cell; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C012491:phytosphingosine; D013110:Sphingosine