Inhibition of caspase activity does not prevent the signaling phase of apoptosis in prostate cancer cells

BACKGROUND. Caspases are a family of cysteine proteases capable of characte ristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical in itiators in the signaling phase, while others (e.g., caspases 3, 6,and 7) h ave been implicated in the effector or execution phase of apoptosis. Thapsi gargin (TG) is capable of inducing cell proliferation independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspas e inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apopt osis in prostate cancer cells. METHODS. Caspase activity was evaluated by Western blot analysis of the cle avage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specifi c fluorescent peptide substrates was assayed in lysates from TG-treated cel ls. Clonogenic survival assays were performed following treatment of rat AT 3 and human TSU-Prl prostate cancer cell lines with TG and 5-FrdU in the pr esence and absence of peptide caspase inhibitors. AT3.1 cells transfected w ith the crmA gene, encoding a viral protein with caspase-inhibitory activit y, were also tested for clonogenic survival following TG and 5-FrdU exposur e. RESULTS. During treatment with TG, Rb is first dephosphorylated and then pr oteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hyd rolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspa se inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect T SU and AT3 cells from apoptosis induced by TG or 5-FrdU. CONCLUSIONS. Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest t hat caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A-strategy for targetin g TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development. (C) 1999 Wiley-Liss, Inc.