TNF-alpha induces apoptosis mediated by AEBSF-sensitive serine protease(s)that may involve upstream caspase-3/CPP32 protease activation in a human gastric cancer cell line

In the present study, we investigated the role of caspase-3/CPP32 and serin e protease(s) in cell death induced by TNF-alpha in SNU-16 human gastric ad enocarcinoma cells. Apoptosis induced in SNU-16 cells by TNF-alpha was acco mpanied by the activation of caspase-3/CPP32. After treatment with TNF-alph a, PKC delta cleaved to its characteristic 40 kDa fragment in a caspase-3/C PP32 dependent manner. Incubation with z-DEVD-fmk completely abrogated TNF- alpha-induced DNA fragmentation, indicating that activation of caspase-3/CP P32 was crucially involved in TNF-alpha-induced apoptosis. In addition, ser ine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), c learly inhibited all the features of apoptosis including DNA fragmentation and chromatin condensation. Furthermore, in the AEBSF treated SNU-16 cells, only intact PKC delta was detected by immunoblot analysis, suggesting that activation of caspase-3/CPP32 was blocked. Thus, the AEBSF-sensitive step may involve an upstream caspase-3/CPP32 protease activation. Taken together , these results suggest that both caspase-3/CPP32 and serine protease(s) ar e activated and play an important role in TNF-alpha induced apoptosis in SN U-16 cells.