Research
Gamma-tocotrienol does not substantially protect DS neurons from hydrogen peroxide-induced oxidative injury
Nutrition & Metabolism 2012, 9:1 doi:10.1186/1743-7075-9-1
Published: 5 January 2012Abstract (provisional)
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 gamma-tocotrienol (gT3) in DS neurons from hydrogen peroxide (H2O2) -induced oxidative stress. The pro-apoptosis tendency of gT3 was compared to alpha-tocopherol (aT) in non-stress condition as well.
Methods
Primary culture of DS and euploid neurons were divided into six groups of treatment: control, H2O2, gT3 pre-treatment with H2O2, gT3 only, aT pre-treatment with H2O2 and aT only. The treatments were assessed by MTS assay and apoptosis assay by single-stranded DNA (ssDNA) apoptosis ELISA assay, Hoechst and NeuN immunofluorescence staining. The cellular uptake of gT3 and aT were 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 gT3 was able to reduced apoptosis of DS neurons by 10%, however gT3 was cytotoxic at longer incubation period (14 days) and at concentrations [greater than or equal to]100uM. Pre-treatment of aT and gT3 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. gT3 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, pre-treatment of gT3 in H2O2-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of gT3 did not promote DS cell survival. Meanwhile gT3 and aT treatments without H2O2 as well as pre-treatment of gT3 and aT induced changes in cPKC and PKC-delta expression in DS neurons suggesting interaction of gT3 and aT with PKC activity.
Conclusion
Our study suggests that gT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.