Effects of tocotrienols on cell viability and apoptosis in normal murine liver cells (BNL CL.2) and liver cancer cells (BNL 1ME A.7R.1), in vitro

The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32(mu)g mL-1. Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T3), alpha tocopherol (a-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T3 significantly (P < 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a doseresponsive manner (8-16 (mu)g mL-1), whereas T did not show any significant (P > 0.05) inhibition in cell viability with increasing treatment doses of 0 - 16 (mu)g mL-1. The IC50 for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 (mu)g mL-1 at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T3 treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC50 of 8.98 (mu)g mL-1 tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98(mu)g mL-1) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T3-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T3-treatment (8.98(mu)g mL-1) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T3-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T3-treated and non-treated normal liver cells. These results suggest that tocotrienols were able to reduce the cell viability in the murine liver cancer cells at a dose of 8-32 (mu)g mL-1 and that this decrease in percentage cell viability may be due to apoptosis..