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 γ-tocotrienol (γT3) in DS neurons from hydrogen peroxide (H(2)O(2)) -induced oxidative stress. The pro-apoptosis tendency of γT3 was compared to α-tocopherol (αT) in non-stress condition as well. METHODS: Primary culture of DS and euploid neurons were divided into six groups of treatment: control, H(2)O(2), γT3 pre-treatment with H(2)O(2), γT3 only, αT pre-treatment with H(2)O(2 )and αT 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 γT3 and αT 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 γT3 was able to reduced apoptosis of DS neurons by 10%, however γT3 was cytotoxic at longer incubation period (14 days) and at concentrations ≥ 100 μM. Pre-treatment of αT and γT3 only attenuate apoptosis and increase cell viability in H(2)O(2)-treated DS and euploid neurons by 10% in which the effects were minimal to maintain most of the DS cells' morphology. γT3 act as a free radical scavenger by reducing ROS generated by H(2)O(2). In untreated controls, DS neurons showed lower Bcl-2/Bax ratio and p53 expression compared to normal neurons, while cPKC and PKC-δ expressions were higher in DS neurons. On the other hand, pre-treatment of γT3 in H(2)O(2)-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of γT3 did not promote DS cell survival. Meanwhile γT3 and αT treatments without H(2)O(2 )as well as pre-treatment of γT3 and αT induced changes in cPKC and PKC-δ expression in DS neurons suggesting interaction of γT3 and αT with PKC activity. CONCLUSION: Our study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H(2)O(2)-induced oxidative assault, instead induced the apoptosis process.