Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells.
Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD.FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation.
Sawada
M
M
Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan. sawasawa@cc.gifu-u.ac.jp
Nakashima
S
S
Banno
Y
Y
Yamakawa
H
H
Hayashi
K
K
Takenaka
K
K
Nishimura
Y
Y
Sakai
N
N
Nozawa
Y
Y
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001681:Biological Assay; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002518:Ceramides / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494828:Bax protein, rat; D061945:Caspase Inhibitors; D002518:Ceramides; D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D005047:Etoposide; D013108:Sphingomyelin Phosphodiesterase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases