Inhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells

Abstract 

Objective

γ-Tocotrienol is a major component of the tocotrienol-rich fraction of palm oil, but there is limited evidence that it has antitumor activity. In particular, the effects of γ-tocotrienol on human colon carcinoma cells have not been reported. To investigate the chemopreventive effects of γ-tocotrienol on colon cancer, we examined its capacity to inhibit proliferation and induce apoptosis in HT-29 cells and explored the mechanism underlying these effects.

Methods

We cultured HT-29 cells in the presence of γ-tocotrienol. The effect of γ-tocotrienol on cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mitotic index, and colony formation. The cell-cycle distribution was investigated by flow cytometry. We measured apoptosis by nuclear staining, transmission electron microscopy, and DNA fragmentation. Apoptosis-related proteins and the nuclear factor-κB p65 protein were determined by western blotting and immunofluorescence.

Results

γ-Tocotrienol inhibited cell growth and arrested HT-29 cells in G₀/G₁ phase. The 50% inhibitory concentration was 31.7 μmol/L (48 h). γ-Tocotrienol–induced apoptosis in HT-29 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of caspase-3. Furthermore, we found that γ-tocotrienol reduced the expression level of total nuclear factor-κB p65 protein and inhibited its nuclear translocation.

Conclusion

The results indicated that γ-tocotrienol inhibits cell proliferation and induces apoptosis in HT-29 cells in a time- and dose-dependent manner, and that this process is accompanied by cell-cycle arrest at G₀/G₁, an increased Bax/Bcl-2 ratio, and activation of caspase-3. Our data also indicated that nuclear factor-κB p65 protein may be involved in these effects.

Keywords: γ-Tocotrienol, Cell proliferation, Cell cycle, Apoptosis, Colon cancer cells