CV, a unicellular microalgae, has been shown to inhibit cell proliferation and to induce apoptosis in liver cancer-induced rats as well as in hepatoma cell line, HepG2 (Md Saad et al., 2006
; Sulaiman et al., 2006
). The results of our study clearly exhibit that CV extract inhibited the liver tumor growth in CDE-treated rats. Since tumor growth is closely related to increased proliferation, a compound that inhibits proliferation and induces apoptosis is expected to have chemopreventive effect on tumors. In this study, we observed that CV can induce apoptosis via caspase 8 by inhibiting the anti-apoptotic protein Bcl-2. Apoptosis is a way of removing damaged and “bad cells” from the system. Apoptotic pathway consists of several families of proteins such as Bcl-2, Bax and caspases 3, 6, 7, 8 and 9 which could be traced via immunocytochemistry or Western blot techniques. Potential chemopreventive agents including ginger extract, Spirulina, curcumin and other herbs have been linked to a good inducer of apoptosis and inhibitor of tumour cell growth (Surh, 2002
; Lampe, 2003
Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma, and prevention of this proliferative response can reduce the risk of subsequent carcinoma (Akhurst et al., 2001
; Lowes et al., 2003
; Sulaiman et al., 2006
). Oval cell apoptosis has been shown to be mediated via Akt activity (Davies et al., 2006
). We observed that a few oval cells stained positively for Bcl-2 and caspase 8 especially in the periportal areas. This may suggest that CV inhibits tumors by inducing apoptosis in damaged hepatocytes as well as oval cells.
Enhancement of Bcl-2 expressions in CDE group indicated the increased cell proliferation in cancerous cells, while no Bcl-2 expressions were observed in the control and CV groups, which showed normal tissue growth. However, the percentage of Bcl-2 expressions declined to almost zero score at the highest dose of CV (300 mg/kg body weight), showing potent growth inhibitory effect of CV on liver cancer cells. Perhaps CV is involved in the inhibition of initiation and promotion stages in chemical carcinogenesis (Surh, 1999
). This pattern of Bcl-2 expression was seen only in the CDE group at 12 weeks but not at 8 weeks due to numerous oval cells found in the CDE rats at 8 weeks that differentiated into bile-duct like cells at 12 weeks (Tee et al., 1994
). As such we would expect the expression of Bcl-2, an anti-apoptotic protein, to be high, even with CV treatment. Decreased expression of Bcl-2 with increasing concentration of CV administered to the CDE rats at 12 weeks could be associated with increased apoptosis and inhibition of cellular proliferation.
Similar to Bcl-2 expression, there were no caspase 8 expressions in the control and CV groups (for all concentrations) at 8 and 12 weeks. It was evident that caspase 8 expression increased with increasing doses of CV (50, 150, and 300 mg/kg body weight) administered to the CDE group at 8 and 12 weeks (Fig.), indicating higher apoptosis rate when increasing doses of CV were administered. Miyao et al.(2006
) showed high rate of apoptotic cells in human cholesteatoma as indicated by high rate of expressions of caspases 3 and 8 compared to normal skin, since both caspases are pro-apoptotic proteins involved in the death signaling pathway. Loss of caspase 8 expression in neuroblastoma was shown to be related to malignancy and resistance to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis (Hopkins-Donaldson et al., 2000
We have also correlated our findings on the expressions of Bcl-2 and caspase 8 with increased proliferation by BrdU labeling index and apoptosis via TUNEL assay in the CDE rats that were treated with CV extract (Figs.~). The CDE rats at 8 and 12 weeks showed high proliferation rate as indicated with increased BrdU labeling of the nuclei. However, when treated with CV at increasing doses, proliferation of neoplastic cells decreased significantly, which correlated with increased apoptotic rate as evidenced by increased TUNEL staining. Increased apoptosis seen in the CDE rats may reflect a normal physiological event, in which the “bad cells are killed” and they were augmented in the presence of CV.
To the best of our knowledge, this study for the first time defined the chemopreventive role of CV by inhibiting cellular proleferation (decreasing Bcl-2 expresion) and inducing apoptosis (increasing caspase 8 expression) in liver cancer cells. The results of the study could justify the role of CV in the treatment of thousands of liver cancer patients, thereby opening a new door for future research.