Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells

Cell Death Differ. 2000 Sep;7(9):761-72. doi: 10.1038/sj.cdd.4400711.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis* / physiology
  • Biological Assay
  • Caspase 3
  • Caspase Inhibitors
  • Caspases / metabolism*
  • Ceramides / biosynthesis*
  • Ceramides / metabolism
  • Ceramides / pharmacology
  • DNA Fragmentation
  • Enzyme Activation
  • Etoposide / pharmacology*
  • Humans
  • Mitochondria / enzymology
  • Mitochondria / metabolism
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • RNA, Messenger / metabolism
  • Rats
  • Signal Transduction
  • Sphingomyelin Phosphodiesterase / metabolism
  • Time Factors
  • Tumor Cells, Cultured
  • bcl-2-Associated X Protein


  • BAX protein, human
  • Bax protein, rat
  • Caspase Inhibitors
  • Ceramides
  • Nucleic Acid Synthesis Inhibitors
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Messenger
  • bcl-2-Associated X Protein
  • Etoposide
  • Sphingomyelin Phosphodiesterase
  • CASP3 protein, human
  • Casp3 protein, rat
  • Caspase 3
  • Caspases