Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellu lar protein degradation in eukaryotes. Here, we show that human malignant g lioma cells are susceptible to apoptotic cell death induced by the proteaso me inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluo romethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor . crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and t he formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression o f Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cy tochrome c release and disruption of mitochondrial membrane potential are p rominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c relea se downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive c aspases. The partial inhibition of cytochrome c release by zVAD-fmk indicat es a positive feedback loop that may involve cytochrome c release and zVAD- fmk-sensitive caspases. Finally, death ligand/receptor interactions, includ ing the CD95/CD95 ligand system, do not mediate apoptosis induced by protea some inhibition in human malignant glioma cells.