γ-Tocotrienol does not substantially protect DS neurons from hydrogen peroxide-induced oxidative injury

Background: Down syndrome (DS) neurons are more susceptible to oxidative stress and previous studies have shown that vitamin E was able to reduce oxidative stress and improve DS neurons' viability. Therefore, this study was done to investigate the protective role of g-tocotrienol (gamma T3) in DS neurons from hydrogen peroxide (H2O2) induced oxidative stress. The pro-apoptosis tendency of gamma T3 was compared to alpha-tocopherol (alpha T) in non-stress condition as well. Methods: Primary culture of DS and euploid neurons were divided into six groups of treatment: control, H2O2, gamma T3 pre-treatment with H2O2, gamma T3 only, alpha T pre-treatment with H2O2 and alpha T only. The treatments were assessed by MTS assay and apoptosis assay by single-stranded DNA (ssDNA) apoptosis ELISA assay, Hoechst and Neu-N immunofluorescence staining. The cellular uptake of gamma T3 and alpha T was determined by HPLC while protein expressions were determined by Western blot. Comparison between groups was made by the Student's t test, one-way ANOVA and Bonferroni adjustment as well as two-way ANOVA for multiple comparisons. Results: One day incubation of gamma T3 was able to reduced apoptosis of DS neurons by 10%, however gamma T3 was cytotoxic at longer incubation period (14 days) and at concentrations >= 100 mu M. Pre-treatment of alpha T and gamma T3 only attenuate apoptosis and increase cell viability in H2O2-treated DS and euploid neurons by 10% in which the effects were minimal to maintain most of the DS cells' morphology. gamma T3 act as a free radical scavenger by reducing ROS generated by H2O2. In untreated controls, DS neurons showed lower Bcl-2/Bax ratio and p53 expression compared to normal neurons, while cPKC and PKC-delta expressions were higher in DS neurons. On the other hand, pretreatment of gamma T3 in H2O2-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of gamma T3 did not promote DS cell survival. Meanwhile gamma T3 and alpha T treatments without H2O2 as well as pre-treatment of gamma T3 and alpha T induced changes in cPKC and PKC-delta expression in DS neurons suggesting interaction of gamma T3 and alpha T with PKC activity. Conclusion: Our study suggests that gamma T3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.