Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells.
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.
Park
Moon-Taek
MT
Laboratory of Radiation Effect, Korea Cancer Center Hospital, Seoul 139-706.
Kang
Jung A
JA
Choi
Jung-A
JA
Kang
Chang-Mo
CM
Kim
Tae-Hwan
TH
Bae
Sangwoo
S
Kang
Seongman
S
Kim
Sujong
S
Choi
Weon-Ik
WI
Cho
Chul-Koo
CK
Chung
Hee-Yong
HY
Lee
Yun-Sil
YS
Lee
Su-Jae
SJ
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
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