Binding of tumour necrosis factor-alpha (TNF-alpha) to TNF-RI induces caspase(s)-dependent apoptosis in human cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA), a tumour of the bile duct epithelium, occurs with a higher incidence in South-east Asian countries than in Europe and North America. The prognosis is poor, due to the unavailability of early diagnosis and the tumours being relatively resistant to chemotherapy. In the present study one of the fatal routes of this tumour was studied. This death was stimulated by TNF-alpha. TNF-alpha at a concentration of 760 pg/ml and 100 pg/ml in the presence of 1 microgram/ml actinomycin D induced 50% cell death of the two established human cholangiocarcinoma cell lines HuCCA-1 and HuCCA-INu, respectively. Preincubation of both cell lines with MoAb to TNF-RI or TNF-RII before TNF-alpha treatment showed that only the MoAb specific to TNF-RI inhibited death. The death of these two cell lines was proved to be apoptosis. Western blot analysis of extracts from both cell lines demonstrated a cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 h following TNF-alpha treatment. The degradation of PARP was prevented by a MoAb to TNF-RI indicating that the TNF-RI but not TNF-RII was involved in TNF-induced apoptosis in these two human cholangiocarcinoma cell lines. Moreover, peptide inhibitor for caspase II subfamily, Ac-DEVD-CHO, reduced the cytolysis of TNF-alpha-treated cholangiocarcinoma cells. The inhibitor also prevented degradation of PARP. These results indicate that the interaction between TNF-alpha and TNF-RI alone generated a sufficient signal to activate a caspase II subfamily-dependent apoptosis in human cholangiocarcinoma cell lines.