Caspase-3 activation and induction of PARP cleavage by cyclic dipeptide cyclo(Phe-Pro) in HT-29 cells.

Background: Cyclo(Phe-Pro) has been shown to inhibit cancer cell growth and induce apoptosis in HT-29 colon cancer cells. Materials and Methods: The molecular mechanisms mediating cyclo(Phe-Pro)-induced apoptosis in HT-29 cells were investigated. Cells were treated with 5 mM or 10 mM cyclo(Phe-Pro) for varying times. Immunoblot analysis was used to detect poly(ADP-ribose)polymerase (PARP) cleavage. A fluorescence-based enzymatic assay was used to measure caspase-3 activity. Results: Cyclo(Phe-Pro) (10 mM) induced time-dependent cleavage of PARP, detected as early as 8 hours post treatment. PARP cleavage was blocked by co-administration with the broad-range caspase inhibitor Z-VAD-FMK. Cyclo(Phe-Pro) also induced a time-dependent increase (p<0.01) in caspase-3 activity. This increase in activity was blocked in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Conclusion: These results provide evidence that cyclo(Phe-Pro)-induced apoptosis in HT-29 cells is mediated by a caspase cascade. These findings warrant further investigation into the potential antitumour activity of cyclo(Phe-Pro) and its related cyclic dipeptide derivatives. An increasing number of cyclic dipeptides (CDPs) and other diketopiperazine derivatives have been shown to modulate important physiological activities in vitro and in vivo (1-6). In particular, the CDP cyclo(Phe-Pro) has been shown to exhibit antifungal activity (4), antibacterial activity (7) and the potential to induce differentiation in HT-29 colon cancer cells (3). We have previously shown that cyclo(Phe- Pro) inhibits cell proliferation and induces apoptosis in HT-29 colon cancer cells (8). However, the molecular mechanisms mediating cyclo(Phe-Pro)-induced apoptosis have not been studied. Apoptosis or programmed cell death is a distinct, intrinsic cell death programme that occurs in various homeostatic or pathological conditions in which the affected cell orchestrates its own demise. Apoptosis has been described as the physiological counterpart of necrosis or "accidental" cell death, which is an uncontrolled, catabolic and degenerative process that often leads to tissue damage (9). The induction of tumour cell death by various approaches such as anticancer drugs, gamma-irradiation, suicide genes or immunotherapy has been shown to be mediated through induction of apoptosis in the target cells (10, 11). A family of cysteine proteases, the caspases, are known to play a central role in various models of cell death (12-14). The activation of effector caspases such as caspase-3 leads to downstream cleavage of various cytoplasmic or nuclear substrates including PARP. These downstream cleavage events mark many of the morphological features of apoptotic cell death (15). Given the important role of caspases as effector molecules in various forms of cell death including drug-induced apoptosis, the ability of anticancer agents to trigger caspase activation appears to be a critical determinant of sensitivity or resistance to cytotoxic therapies (16, 17). The aim of the present study was to investigate the potential of cyclo(Phe-Pro) to induce caspase-3 activation and PARP cleavage in HT-29 colon cancer cells.