Proteasome inhibition-induces endoplasmic reticulum dysfunction and cell death of human cholangiocarcinoma cells.
OBJECTIVE
To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms.


METHODS
Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 cholangiocarcinoma, and normal rat cell lines. MG132, a peptide aldehyde, which inhibits the chymotrypsin-like activity of the proteasome was employed for this study. Apoptosis was assessed morphologically by 4'-6-Diamidino-2-phenylindole (DAPI) nuclear staining and fluorescence microscopy. Mitochondrial membrane potential was examined using a fluorescent unquenching assay. Ultrastructural changes during cell death were examined using transmission electron microscopy (TEM). Caspase 3/7 activity was assessed using an enzymatic-based fluorescent assay. Cytosolic-free calcium concentrations were measured using Fura-2 and digitized fluorescent microscopy.


RESULTS
MG132, a proteasome inhibitor, induced apoptosis in all the cholangiocarcinoma cell lines examined. In contrast, minimal cytotoxicity was observed in normal rat cholangiocytes. Apoptosis was time- and -concentration-dependent. There was no change in the mitochondrial membrane potential between treated and untreated cells. Ultrastructural examination by transmission electron microscopy displayed the classic features of apoptosis, but in addition, there was also dramatic vacuolization of the endoplasmic reticulum (ER). Unexpectedly, no increase in caspase 3/7 activity was observed in MG132 treated cells, nor did the pancaspase inhibitor, Q-VD-OPh prevent cell death. The protein synthesis inhibitor, cycloheximide, blocked apoptosis induced by proteasome inhibitor indicating that ER dysfunction was dependent upon the formation of new proteins.


CONCLUSIONS
Proteasome inhibition induces ER dysfunction and caspase-independent cell death selectively in human cholangiocarcinoma cells. Proteasome inhibitors warrant evaluation as anticancer agents for the treatment of human cholangiocarcinoma.
Ustundag
Yucel
Y
Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota 55905, USA.
Bronk
Steven F
SF
Gores
Gregory J
GJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001650:Bile Duct Neoplasms / Q000473:pathology* / Q000503:physiopathology; D001653:Bile Ducts, Intrahepatic / Q000473:pathology* / Q000503:physiopathology; D020169:Caspases / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000473:pathology* / Q000503:physiopathology; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D016328:NF-kappa B / Q000502:physiology; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde