Abstract
BackgroundDown 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 (H2O2) -induced oxidative stress. The pro-apoptosis tendency of γT3 was compared to α-tocopherol (αT) in non-stress condition as well.MethodsPrimary culture of DS and euploid neurons were divided into six groups of treatment: control, H2O2, γT3 pre-treatment with H2O2, γT3 only, αT pre-treatment with H2O2 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.ResultsOne 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 H2O2-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 H2O2. 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 H2O2-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 H2O2 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.ConclusionOur study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.
Highlights
Vitamin E is a generic term for lipid-soluble, chain breaking antioxidants which consists of four tocopherol isomers (a, b, g, δ) and four tocotrienol isomers (a, b, g, δ)
From the propidium iodide (PI) assays, 1 μM and 10 μM of gT3 and aT maintained cell viability but did not improve cell survival when it was added to the Down syndrome (DS) culture for 14 days, gT3 was cytotoxic to DS cortical neurons at concentration ≥ 100 μM compared to aT, with increased apoptosis of 25-35% and 5-8% respectively [Figure 1 (a)]
The previous landmark study has shown that aT was able to attenuate apoptosis and improve cell viability [11], whereas prolonged incubation time of gT3 up to 14 days increased membrane damage and apoptosis to DS neurons, as detected from the PI assay at a dose dependent manner
Summary
Vitamin E is a generic term for lipid-soluble, chain breaking antioxidants which consists of four tocopherol isomers (a, b, g, δ) and four tocotrienol isomers (a, b, g, δ). The tocopherol and tocotrienol isomers differ in the dementia have lower plasma levels of vitamin E than controls without DS [4] These results suggest that intake of essential nutrients such as folate, vitamin B6, vitamin E, selenium, a-lipoic acid might be important in preventing cognitive deterioration in DS and Alzheimer disease (AD) [5]. There was no significant effect of antioxidant supplementation on the superoxide dismutase and glutathione peroxidase activities, on the superoxide dismutase to glutathione peroxidase ratio and on the urinary isoprostane concentrations [6] Another recent review that looked at five different studies on antioxidants and cognitive functions revealed that only three studies examining vitamin E and C supplements gave significantly different results-i. The pro-apoptosis tendency of gT3 was compared to a-tocopherol (aT) in non-stress condition as well
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