TPCK is widely used as an inhibitor of chymotrypsin-like proteases but has recently been identified as an inhibitor of the PDK1/Akt pathway. In this study, we show that TPCK inhibits TRAIL-induced caspase activity but potentiates wortmannin-dependent caspase activity in prostatic carcinoma cell lines. The inhibitory activity of TPCK was found to be death ligand-specific since TPCK inhibits TRAIL-mediated caspase activity but does not affect Fas-induced caspase activity. Our data also show that impaired TRAIL-DISC formation in the presence of TPCK is responsible for caspase inhibition. Further, TPCK induces p53 expression and inhibits the PDK1/Akt pathway resulting in BAD dephosphorylation, and the release of cytochrome c and Smac/DIABLO from mitochondria. TPCK also selectively decreases the levels of androgen receptor and caspase-2 whereas it does not change the levels of other proteins (caspases-3, -7, -8, -9; heat shock proteins 27, 70, 90). Finally, TPCK-induced degradation of caspase-2 is protected by Bcl-2 overexpression, apparently by an adapter protein since direct interaction between caspase-2 and Bcl-2 was not detected. Together, these features suggest that TPCK could be used as a therapeutic agent for treatment of those tumor cells that are resistant to ligand-induced treatment because of aberrant signaling pathways downstream of the DISC.