benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Cholangiocarcinoma

To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms.. 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.. 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.. 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.

Topics: Animals; Apoptosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Caspases; Cell Line; Cell Line, Tumor; Cholangiocarcinoma; Cycloheximide; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Humans; Leupeptins; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Synthesis Inhibitors; Rats

To clarify the innate immunity of the intrahepatic biliary tree, we examined expression of Toll-like receptors and intracellular signalings in biliary epithelial cells in response to bacterial components by using cultured biliary epithelial cells (murine biliary cells and human cholangiocarcinoma cell lines). The expression of Toll-like receptors in cultured cells was examined by reverse transcription and PCR and immunohistochemistry. Intracellular signalings after Toll-like receptors activation by lipopolysaccharide was examined by analysis of nuclear factor (NF)-kappaB activation and inhibition studies using inhibitors for NF-kappaB and mitogen-activated protein kinase and blocking antibody. The mRNAs of Toll-like receptors 2, 3, 4, and 5, and related molecules (MD-2, MyD88, and CD14) were detected, and their proteins were expressed in cultured cells. Lipopolysaccharide was shown to bind to the cell surface of cultured cells. Lipopolysaccharide treatment induced the production of TNF-alpha, and nuclear translocation of NF-kappaB and increased NF-kappaB-DNA binding in cultured cells. This induction of TNF-alpha was partially inhibited by anti-Toll-like receptor 4 antibody. The nuclear translocation and increased binding of NF-kappaB by lipopolysaccharide were blocked by addition of MG132, an inhibitor of NF-kappaB. In conclusion, lipopolysaccharide appears to form a receptor complex of CD14, Toll-like receptor 4, MD-2, and MyD88 in cultured biliary epithelial cells and seems to regulate activation of NF-kappaB and synthesis of TNF-alpha. The recognition of pathogen-associated molecular patterns using Toll-like receptors and related molecules in biliary epithelial cells, which is demonstrated in this in vitro study, may participate in an immunopathology of the intrahepatic biliary tree in vivo.

Topics: Adult; Aged; Animals; Bile; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Nucleus; Cholangiocarcinoma; DNA; DNA Primers; Endotoxins; Epithelium; Escherichia coli; Female; Humans; Leupeptins; Lipopolysaccharides; Male; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Middle Aged; NF-kappa B; Receptors, Cell Surface; RNA, Messenger; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha