During the last decade, a considerable amount of research has focused on cancer cell apoptosis. Apoptosis, or programmed cell death, is the major control mechanism by which cells die if DNA damage is not repaired [19
]. Apoptosis is also a critical protective mechanism against carcinogenesis, eliminating damaged cells or cells proliferating abnormally in response to carcinogens [20
]. Therefore, induction of apoptotic cell death is a promising emerging strategy for the prevention and treatment of cancer [21
]. The results of the present study clearly demonstrate that EDCL suppressed HepG2 cell viability by inducing apoptosis. After exposure to EDCL, chromatin condensation and apoptotic bodies were clearly observed. These results suggest that HepG2 cells exposed to EDCL undergo typical apoptosis. Furthermore, flow cytometric analysis after propidium iodide staining confirmed EDCL-induced apoptosis in HepG2 cells.
Members of the Bcl-2 family of proteins, such as bcl-2, bcl-xl, mcl-1, and bax, are the most prominent actors in controlling the release of cytochrome c and in mitochondria-mediated apoptosis [22
]. Thus, it has been suggested that the ratio between the level of proapoptotic bax protein and the level of antiapoptotic bcl-2 protein determines whether a cell responds to an apoptotic signal [23
]. In this study, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl.
The execution of cellular demolition in apoptosis is also carried out by caspases [24
]. The caspase family of proteins is one of the main executors of the apoptotic process. Caspases belong to a group of enzymes known as cysteine proteases and exist within the cell as inactive proforms or zymogens. These zymogens can be cleaved to form active enzymes following the induction of apoptosis. The IAP family of proteins blocks apoptosis by directly inhibiting at least two members of the caspase family of cell death proteases, caspase-3, and caspase-7. XIAP, cIAP-1, and cIAP-2 can prevent the proteolytic processing of procaspase-3, -6, and -7 by blocking the cytochrome c-induced activation of procaspase-9 [24
]. Studies have shown that exposure of HepG2 cells to EDCL caused proteolytic activation of caspases and down-regulation of XIAP, cIAP-1 and cIAP-2. The enzyme poly(ADP-ribose) polymerase, or PARP, was one of the first proteins identified as a substrate for caspases. PARP is involved in repair of DNA damage and functions by catalyzing the synthesis of poly (ADP-ribose) and by binding to DNA strand breaks and modifying nuclear proteins. PARP helps cells maintain viability, and the cleavage of PARP facilitates cellular disassembly and serves as a marker for cells undergoing apoptosis [26
]. In the present study, we examined whether the PARP protein, a substrate of caspase-3 [28
], was cleaved in cells exposed to EDCL. As expected, PARP was clearly degraded in a concentration- and time-dependent manner that correlated with caspase activation. Under the same experimental conditions, z-vad-fmk prevented EDCL-induced apoptosis by blocking caspase activation. These data indicate that caspases are the key molecules mediating EDCL-induced apoptosis in HepG2 cells.
In conclusion, this study clearly demonstrates that EDCL strongly inhibits cell proliferation and induces apoptosis in HepG2 cells. EDCL induced apoptosis through the mitochondrial pathway, involving the activation of caspase-3/7, -8, and -9, the down-regulation of antiapoptotic proteins, and the degradation of PARP protein. Because induction of apoptosis is thought to be a suitable anticancer therapeutic mechanism, these results confirm the potential of EDCL as a chemotherapeutic agent in human hepatocellular carcinoma cells. In vivo studies are needed to fully establish the potential of EDCL as a chemopreventive and therapeutic agent in cancer.