Vol. 289, Issue 3, 1306-1312, June 1999

Betulinic Acid-Induced Apoptosis in Glioma Cells: A Sequential Requirement for New Protein Synthesis, Formation of Reactive Oxygen Species, and Caspase Processing

Wolfgang Wick, Cornelia Grimmel, Bettina Wagenknecht, Johannes Dichgans and Michael Weller

Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, School of Medicine, Tübingen, Germany

Betulinic acid (BA), a pentacyclic triterpene, is an experimental cytotoxic agent for malignant melanoma. Here, we show that BA triggers apoptosis in five human glioma cell lines. BA-induced apoptosis requires new protein, but not RNA, synthesis, is independent of p53, and results in p21 protein accumulation in the absence of a cell cycle arrest. BA-induced apoptosis involves the activation of caspases that cleave poly(ADP ribose)polymerase. Interactions of death ligand/receptor pairs of the CD95/CD95 ligand family do not mediate BA-induced caspase activation. BA enhances the levels of BAX and BCL-2 proteins but does not alter the levels of BCL-x_S or BCL-x_L. Ectopic expression of BCL-2 prevents BA-induced caspase activation, DNA fragmentation, and cell death. Furthermore, BA induces the formation of reactive oxygen species that are essential for BA-triggered cell death. The generation of reactive oxygen species is blocked by BCL-2 and requires new protein synthesis but is unaffected by caspase inhibitors, suggesting that BA toxicity sequentially involves new protein synthesis, formation of reactive oxygen species, and activation of crm-A-insensitive caspases.