PURPOSE: The proteasome inhibitor bortezomib (PS-341) has displayed significant 
efficiency against pancreatic cancer cells. However, the underlying mechanisms 
are not fully understood. Here, we tested if ceramide production was involved in 
the bortezomib's effect.
METHODS: Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the 
primary pancreatic cancer cells were used. Cell death was analyzed by MTT 
viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone 
DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal 
protein changes. The cellular ceramide level after bortezomib treatment was also 
determined.
RESULTS: In cultured pancreatic cancer cells, bortezomib increased cellular 
ceramide production to promote cell apoptosis. The ceramide de novo synthase 
inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production 
and apoptosis, while exogenously added C6-ceramide facilitated 
bortezomib-induced pancreatic cancer cell death. Meanwhile, 
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of 
glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors 
(SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and 
subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun 
N-terminal kinase (JNK) activation was also associated with ceramide production. 
JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II 
and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP 
dramatically enhanced bortezomib-induced cytotoxicity in primary cultured 
pancreatic cancer cells.
CONCLUSIONS: We found that bortezomib-induced apoptosis was associated with 
ceramide production in primary and transformed pancreatic cancer cells.