Proteasome inhibition blocks caspase-8 degradation and sensitizes prostate cancer cells to death receptor-mediated apoptosis.
BACKGROUND
Proteasome inhibition through the administration of Velcade is a viable chemotherapeutic strategy that is approved to treat multiple myeloma and is being evaluated for efficacy against prostate cancer. Currently, the apoptotic pathways that contribute to this anticancer response are poorly understood. Our goal is to test the extent to which proteasome inhibition modulates apoptosis through death receptor pathways.


METHODS
Several prostate cancer cell lines and primary prostate epithelial cells (PrECs) were used as models. The death receptor pathway was activated by the expression of Fas ligand (FasL) or addition of TNF-related apoptosis-inducing ligand (TRAIL) in the presence or absence of proteasome inhibitors. The apoptotic response was quantified by annexin V, TUNEL and nuclear condensation assays. Western blot analysis was conducted to quantify protein levels and enzyme assays were used to measure caspase activity.


RESULTS
Proteasome inhibition markedly sensitized prostate cancer cells to apoptosis initiated by Fas ligand (FasL) or TRAIL. In the presence of either death ligand, procaspase-8 processing occurred, but led to minimal amounts of active caspase-8. The addition of Velcade, however, led to robust active caspase-8 protein abundance and activity. In the presence of Velcade the caspase-8 p18 subunit half-life increased from 22 min to over 2 hr.


CONCLUSIONS
These findings demonstrate that proteasome inhibition can sensitize cells to apoptosis elicited by tumor necrosis factor ligands and retarding caspase-8 degradation provides one explanation for this activity. This study suggests that the clinical efficacy of Velcade may result, at least in part, from the activity of death ligands.
Thorpe
Jeffery A
JA
Department of Molecular and Cellular Biochemistry and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, USA.
Christian
Perry A
PA
Schwarze
Steven R
SR
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D053181:Caspase 8 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D053222:Fas Ligand Protein / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008297:Male; D011467:Prostate / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011480:Protease Inhibitors / Q000494:pharmacology; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology; D053218:Receptors, Death Domain / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology
D001897:Boronic Acids; D053222:Fas Ligand Protein; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011719:Pyrazines; D053218:Receptors, Death Domain; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D000069286:Bortezomib; D053181:Caspase 8