RESEARCH Open Access

Then et al. Nutrition & Metabolism 2012, 9:1 http://www.nutritionandmetabolism.com/content/9/1/1 RESEARCH Open Access g-Tocotrienol does not substantially protect DS neurons from hydrogen peroxide-induced oxidative injury Sue-Mian Then1*, Coral Sanfeliu2, Gapor M Top4, Wan Zurinah Wan Ngah1,3 and Musalmah Mazlan1,3 Abstract 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 (gT3) in DS neurons from hydrogen peroxide (H2O2) -induced oxidative stress. The pro-apoptosis tendency of gT3 was compared to a-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 Neu-N immunofluorescence staining. The cellular uptake of gT3 and aT 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 gT3 was able to reduced apoptosis of DS neurons by 10%, however gT3 was cytotoxic at longer incubation period (14 days) and at concentrations ≥ 100 μM. 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-δ 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-δ 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. Keywords: Apoptosis, Down syndrome, human neurons, oxidative stress, γ-tocotrienol, vitamin E Introduction number and position of methyl substitutions on the Vitamin E is a generic term for lipid-soluble, chain chromanol head. Although tocopherols and tocotrienols breaking antioxidants which consists of four tocopherol are closely related chemically, they differ in their biolo- isomers (a, b, g, δ) and four tocotrienol isomers (a, b, g, gical effectiveness [1]. Studies have shown that vitamin δ). The tocopherol and tocotrienol isomers differ in the E deficiency impairs cognitive performance in mice sub- jected to oxidative stress [2]. Meanwhile, one study found that Down syndrome (DS) children have signifi- * Correspondence: [email protected] 1 UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan cantly less vitamin E levels than normal children [3]; Malaysia (UKM), Kuala Lumpur, Malaysia while another study showed that DS patients with Full list of author information is available at the end of the article © 2012 Then et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Then et al. Nutrition & Metabolism 2012, 9:1 Page 2 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 dementia have lower plasma levels of vitamin E than apoptosis in murine C6 glioma cell line and Tet21N controls without DS [4]. These results suggest that neuroblastoma cell line [16,17] but not tocotrienols. intake of essential nutrients such as folate, vitamin B6, g-Tocotrienol (gT3) was reported to activate the apop- vitamin E, selenium, a-lipoic acid might be important in tosis pathway via the mitochondrial death pathway of preventing cognitive deterioration in DS and Alzheimer the Bcl-2 family proteins in pancreatic stellate cells [18], disease (AD) [5]. while tocotrienols was shown to be anti-proliferative in However, intervention studies of antioxidant supple- mammary epithelial cells by reducing PKCa (Protein mentation in DS and AD have not been conclusive. A Kinase C) activation [19]. Our previous studies in pri- recent randomized controlled trial on antioxidant sup- mary rat’s astrocytes and cerebellar neuron cultures plementation, including vitamin E for DS children did revealed that high dosage of gT3 was cytotoxic and have not show any significant difference in developmental a high tendency to induce the expressions of proteins outcome after a two-year research period. There was that were involved in the apoptosis pathway such as also no significant effect of antioxidant supplementation Bax, p53 and p38 MAPK [20,21]. Another study also on the superoxide dismutase and glutathione peroxidase showed that high doses of vitamin E and vitamin C activities, on the superoxide dismutase to glutathione enhanced the toxic effect of H 2O 2 to cells [22]. Since peroxidase ratio and on the urinary isoprostane concen- most trials of vitamin E supplementations utilized high trations [6]. Another recent review that looked at five dosage of vitamin E for maximum effects, the concern different studies on antioxidants and cognitive functions for the safety of vitamin E supplementation at the mole- revealed that only three studies examining vitamin E cular and cellular level has yet to be fully addressed. DS and C supplements gave significantly different results-i. cells are known to be highly susceptible to oxidative e. one study found a positive association with specific damage compared to normal cells [23]. Genomic and cognitive test, while the other two studies showed a link functional profiling of DS neural progenitor cell line with global cognitive functions [7]. Other double-blind exposed to S100B suggested that dysregulation of chro- studies reported that vitamin E has no benefit in mosome 21 genes led to increased ROS and thereby patients with mild cognitive impairment and Alzhei- altered transcriptional regulation of cytoprotective genes mer’s disease [8]. In all these trials, subjects partake in response to oxidative stress [24]. Vitamin E treatment high doses of vitamin E (2000 IU or 1500 mg) daily, induced neuroinflammatory processes by increasing which is more than the upper tolerable intake level for microglial activation in animals overexpressing S100B, vitamin E (1500 IU or 1000 mg per day) [9]. which is involved in the neuropathology of DS and AD Vitamin E mainly function as free radical scavenger, [25]. Therefore, the purpose of this study is to further but recent studies showed that tocopherols and tocotrie- investigate the effects of aT and gT3 in the apoptosis nols have other non-antioxidant roles: a-tocopherol signaling pathway of human DS neurons as a model of (aT) was shown to modulate signal transduction and oxidative stress susceptible system, while normal human gene expression in various cell lines, while tocotrienols neurons were used as control. possess powerful neuroprotective, anti-inflammatory anti-angiogenic, anti-artherogenic, anti-cancer and cho- Materials and methods lesterol lowering properties (for a comprehensive review, Materials refer [10]). Vitamin E has been shown to be neuropro- The Malaysia Palm Oil Board (MPOB) supplied the palm tective in various studies: firstly in a landmark study of gT3 and aT isomers of 87% and 80% purity respectively, neurodegeneration of in vitro culture of DS neurons which was isolated as described previously [26]. Culture [11]; followed by a study that reported that aT was able dishes were from Nunc while antibodies (p53, Bax, Bcl-2, to attenuate oxidative stress-induced apoptosis in striatal cPKC, PKC-δ, b-actin) were from Santa Cruz Technolo- neuron cultures via its free radical scavenger function gies. Reagents for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- [12]; while other studies showed that a-tocotrienol pro- nyltetrazolium bromide (MTT) assays were from tects neurons from glutamate-induced cell death by the Promega while single-stranded DNA (ssDNA) Apoptosis c-src activation molecular pathway [13]. However, not ELISA kits were from Chemicon. All other chemicals and many studies have address the possible pro-apoptotic reagents were from Sigma unless indicated. tendency of vitamin E in neurons, especially tocotrie- nols, which has shown to have greater apoptotic activity Primary cortical neuron cultures from normal and towards various cancer cell lines such as mammary DS fetal brain tumor cells and prostate tumor cells compared to toco- The cultures were established using human cortical pherols [14,15]. Current studies has shown that toco- brain tissues obtained from normal euploid and DS leg- pherol and vitamin E analogues were able to induced ally aborted fetuses at 14-21 weeks of gestation. The Then et al. Nutrition & Metabolism 2012, 9:1 Page 3 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 permission to use human fetal tissues was obtained from fixed with 4% paraformaldehyde before being permeabi- the ethics committee of the Spanish National Research lized with 0.25% Triton in PBS for 30 mins. The cells Council (CSIC) (Approval date: August 6th, 2009; ref. were then washed with PBS, followed by incubation no: SAF2009-13093-C02-02). Enriched neuron cultures with goat serum at room temperature to block unspeci- were prepared as described elsewhere [27]. fic binding site, and incubation with mouse Neu-N antibody (Chemicon, USA) in 1:200 dilution overnight Cell culture treatments at 4°C. Subsequently, cultures were washed with PBS Neuron cultures were incubated with varying concentra- and incubated with anti-mouse Alexa Fluor 488 (Mole- tions of gT3 and aT, with aT as a positive control cular Probes, The Netherlands) in 1:2000 dilution for 1 based on previous studies showing aT having non-toxic hr at room temperature. After washing with PBS, and neuroprotective effects on neurons [12,21]. Stock nuclei were counterstained with Hoescht before visuali- solutions of 0.5 M gT3 and aT (in 100% ethanol) were zation under fluorescence microscope (Nikon, Japan) at first resolved overnight in fetal calf serum at 37°C and 40× magnification. The intracellular production of ROS diluted to 100 times the final concentration with culture was determined using DCFH-DA assay. Non-fluores- media containing 50% ethanol. Final dilution of aT and cent DCFH-DA was permeable to cell membrane and gT3 in the cell culture contained 0.5% ethanol, which oxidation of hydroperoxides produced fluorescent 2’,7’- did not significantly affect cell survival (data not shown). dichlorofluorescein (DCF), which was detected by All experiments utilized freshly prepared dilutions of fluorescence plate reader at 485 nm excitation/530 nm H2O2, aT and gT3. emission [30]. Cytotoxicity of gT3 Determination of Vitamin E Uptake by HPLC The human DS neuron cultures were incubated with The uptake of gT3 and aT was analyzed using reverse- gT3 (1-200 μM) for 24 hours at 37°C. DS neurons were phase high performance liquid chromatography (HPLC) also given gT3 treatment of 7 days and 14 days to deter- Fluorescent EM 330 nm, EX 294 nm detector (Shi- mine possible cytotoxicity or protective effects of gT3. madzu, Japan) as described previously [20]. Concentra- Cytotoxicity of gT3 and aT was assessed by propidium tion peaks of the samples were compared with iodide (PI) assay. Briefly, for PI assay, the cultures in 96 tocotrienol rich fraction (TRF) standard and the concen- wells were stained with PI (7 μM) for one hour prior to trations of aT and gT3 uptake in cells were calculated the end of the incubation period (24 h. 7 days and 14 as μM/106 cells. days). At the end of the incubation period, the fluores- cence intensity was determined and expressed relative to SDS-PAGE and Western Blot cultures treated with 0.2% Triton X-100 (to permeabilize Western blot of DS and euploid (normal) cortical neu- all cells). The fluorescence signal was measured by a rons in various treatment groups were used to elucidate fluorescence plate reader (Molecular Devices, USA) at the expression of proteins involved in the apoptosis sig- 530-nm excitation ⁄ 645-nm emission to quantify cell naling pathway including p53, Bax, Bcl-2, cPKC (for membrane damage as described elsewhere [28]. detection of common isoforms PKC-a, PKC-b and PKC-g) and PKC-δ; while b-actin were used as house- Detection of Cell Survival keeping protein and loading control. A maximum pro- DS and euploid cortical neurons were pre-treated with tective dosage of 10 μM gT3 and aT was used to test if varying concentrations of aT and gT3 (1-100 μM) for this concentration could induce apoptosis in DS and one hour at 37°C, followed by addition of H 2 O 2 (100 euploid neurons. The western blots were performed as μM) to the cells and a further incubation for 24 hours previously described. at 37°C before cell viability and apoptosis were assessed. Cell viability was assessed using MTT assay. Briefly, the Statistical Analysis cell culture media was loaded with 0.5 mg/mL MTT to Each experiment of cultures in microplates was carried detect any decrease in the cell metabolic activity using out in triplicate wells with at least three independent MTT reduction assay following standard procedures cultures. The data were reported as mean ± SD of at [29]. Meanwhile, the rate of apoptosis was measured least three experiments. Comparison between groups using the ssDNA ELISA kit as described previously [20]. was made by the Student’s t test, one-way ANOVA and In addition, cell viability was also assessed utilizing the Bonferroni adjustment as well as two-way ANOVA for PI assay as described above. For cell imaging, DS neuron multiple comparisons. p < 0.05 was considered as statis- cultures were stained with of Neu-N (neuron- specific tically significant for Student t-test whereas p < 0.0001 nuclear protein) antibody to confirm the results shown was considered as statistically significant for multi-factor by the MTT and ssDNA ELISA assay. Briefly, cells were comparisons. Then et al. Nutrition & Metabolism 2012, 9:1 Page 4 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 Results and discussion previous landmark study has shown that aT was able to From the PI assays, 1 μM and 10 μM of gT3 and aT attenuate apoptosis and improve cell viability [11], maintained cell viability but did not improve cell survi- whereas prolonged incubation time of gT3 up to 14 val when it was added to the DS culture for 14 days, days increased membrane damage and apoptosis to DS gT3 was cytotoxic to DS cortical neurons at concentra- neurons, as detected from the PI assay at a dose depen- tion ≥ 100 μM compared to aT, with increased apopto- dent manner. Similar to our previous studies, long term sis of 25-35% and 5-8% respectively [Figure 1 (a)]. For incubation of gT3 was shown to be cytotoxic to neurons neurons incubated with gT3, more apoptotic cells were at high dose (≥ 100 μM) while aT showed minor toxic observed at 50 μM, and at 100 μM almost all of the effects to human neurons as illustrated in Figure 1 (a) cells undergo apoptosis. From Figure 1 (b), a short 1 and Figure 1 (b)[20,21]. Fluorescence detection of DCF day incubation of gT3 at 10 μM and 100 μM in DS neu- showed that gT3 act as a free radical scavenger by redu- rons was only able to reduced apoptosis by 10%. The cing ROS generated by H 2 O 2 in a dose dependent v (a) (b) # * * # $ $ # # # * * * * * * # * * (c) (d) * # * * * * * * * * * * * * * * * * # # # # #   Figure 1 (a) The effects of a-tocopherol (aT) and g-tocotrienol (gT3) towards DS neuronal cell death for 14 days in vitro as detected by Propidium Iodide (PI) assay. *Denotes P < 0.05 compared to control while # denotes P < 0.05 when gT3 was compared to aT at their corresponding concentration. The data are presented as mean ± SD, n = 6. (b) The effects of gT3 at various incubation periods (1 day, 4 days and 14 days). *Denotes P < 0.05 compared to the control, while # denotes P < 0.05 compared to day 1 and $ denotes P < 0.05 compared to day 4. The data are presented as mean ± SD, n = 3. (c) The effects of gT3 on ROS generated by H2O2 in DS neurons with intracellular accumulation of hydroperoxides after one hour exposure to the indicated concentration of H2O2 measured by DCFH oxidation to DCF. *Denotes P < 0.05 compared to control while # denotes P < 0.05 compared to 10 μM gT3. The data are presented as mean ± SD, n = 3. (d) Uptake of a- tocopherol (aT) and g-tocotrienol (gT3) in human cortical neurons measured by HPLC. The cellular uptake of gT3 and aT was significantly higher than the untreated control, with the uptake of gT3 significantly higher than that of aT. *Denotes P < 0.05 compared to the control, while # denotes P < 0.05 when gT3 uptake of gT3 incubated cultures were compared with aT uptake of aT incubated cultures. The data are presented as mean ± SD, n = 3. Then et al. Nutrition & Metabolism 2012, 9:1 Page 5 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 manner at concentration ≤ 1000 μM of gT3 after one dose of vitamin E compounds the toxic effect of H2O2, hour H2O2 exposure [Figure 1 (c)]. The HPLC analysis here we further investigate whether non-lethal doses of revealed that the uptake of gT3 was higher compared to high concentration aT and gT3 will further exacerbate the uptake of aT in neurons [Figure 1 (d)]. The absorp- the detrimental effects of H2O2 via the mitochondrial tion of aT had been associated to ATP-binding cassette Bcl-2 family pathway or the PKC signalling pathway as (ABC) transporter (MDR1) which acts as a cellular both cPKC and PKC-δ are redox sensitive and were exporter of tocopherols to ApoA-I and HDL by reported to be involved in the initiation of apoptosis sig- ABCA1-dependent and ABCA1-independent processes nalling [35,36]. [31,32]. Hence, this may explain why high doses of aT From our investigation, euploid neurons showed a did not exhibit toxicity to neurons, as aT was effluxed completely different protein expression profile compared from the cells by ABC transporter 1 (MDR1) to elimi- to DS neurons. In the control group, DS neurons were nate excess aT. On the other hand, gT3 was not selec- shown to have lower Bcl-2/Bax ratio and p53 expression tively transported by TTP. Thus, excess gT3 was not compared to euploid neurons, while cPKC and PKC-δ being effluxed from the cells, leading to the accumula- expressions were higher in DS neurons. Figure 5 (a) (i), tion of gT3 that might exacerbate cell death and contri- Figure 5 (a) (ii) and Figure 6a revealed that for DS neu- bute to the possible toxicity of gT3 at high dose. rons, pre-treatment of gT3 followed by H2O2 has signifi- In both DS and euploid neurons, the pre-treatment of cantly lower Bcl-2/Bax ratio than the controls, whereas gT3 and aT at concentration ≤ 50 μM was able to other treatment showed changes which were not statisti- reduce cell death induced by H 2 O 2 [Figure 2 (a) and cally significant. This result suggests that gT3 does not Figure 3 (a)] and increase cell viability [Figure 2 (b) and contribute to the survival of DS neurons under H 2 O2 Figure 3 (b)]. Figure 2 (c) and Figure 3 (c) shows that assault via the Bcl-2/Bax heterodimer complex forma- both pre-treatments of up to 10 μM gT3 and 50 μM aT tion. However, the analysis of Western blot for the were able to attenuate apoptosis in H2 O2-induced DS euploid neurons in Figure 5 (b) (i), Figure 5 (b) (ii) and and euploid neurons respectively. However, the effec- Figure 6 (a) showed a different picture: Bcl-2/Bax ratio tiveness of gT3 and aT pre-treatment was more pro- increased significantly in euploid neurons when neurons nounced in euploid neurons compared to DS neurons, were pre-treated with either gT3 or aT followed by as two-way ANOVA analysis showed that vitamin E iso- H2O2 which suggested that gT3 pre-treatment attenu- mer type and concentration contributed significantly to ated apoptosis and improved cell survival of normal cell viability and apoptosis rate in euploid neurons but euploid neurons. However, p53 expression was not sig- not in DS neurons. This indicated that the protective nificantly different across various treatments in both DS effect of the vitamin E in euploid neurons is dose and and euploid neurons, as depicted in Figure 5 (a) (iii), isomer type dependent, while protective effects of vita- Figure 5 (b) (iii) and Figure 6 (b). Nevertheless, the min E in DS neurons is not dependent on dose and iso- comparison of p53 expression between DS and euploid mer type. Figure 4 shows the morphology of H 2 O 2 - neurons showed lower p53 expression in DS neurons treated DS neurons as stained by Neu-N, a marker for for these treatment groups: control, H2O2, gT3 followed differentiated neurons [33] which had undergone apop- by H2O2 and gT3 treatments only. Taking it all together, tosis; pre-treatment with 10 μM of aT or gT3 retained these results show that lower Bcl-2/Bax ratio DS neu- some of the neurons’ viability but cell morphologies rons in the untreated groups, was in agreement with the were not fully maintained. Reduced Neu-N expression results from previous study which reported that fetal DS in differentiated neurons indicated perturbed cell mor- neurons had increased Bax and p53 expressions phology induced by H2O2 assault, which pre-treatment mediated by the transcription factor est-2 when treated of either aT or gT3 were not substantial enough to pro- with H 2 O 2 [37]. On the other hand, another study tect cells from oxidative assault. However, comparatively reported that APO-1, caspase-3 and Bcl-2 protein aT seems to be more protective to the DS neurons than expression levels were unaltered in the fetal DS neurons gT3 as more cells were stained with Neu-N, although [38]. A previous study also stated that incubation of aT the staining was dim. This is not surprising since the induced the up-regulation of Bcl-2 as preventive effects previous study has shown that in vitro DS cortical neu- from neuronal cell death [39]. Thus, treatment of only ron culture had higher sensitivity to H2O2-induced oxi- aT and gT3 without the presence of H2 O 2 in human dative damage compared to euploid neurons [27]. H2O2 neuron did not show pro-apoptosis tendency (from the directly induces cellular damage and has been reported Bcl-2/Bax ratio and p53 expression) compared to rat to induce parallel apoptosis and autophagy [34], making cerebellar culture as reported previously [21]. it more difficult for aT and gT3 to protect the cells Aberrant expression of PKC signalling has been especially if the pre-treatment incubation period was reported in fetal DS post-mortem tissues [40], while DS short. Since our previous studies have shown that high patients’ fibroblast was reported to be hyposensitive to Then et al. Nutrition & Metabolism 2012, 9:1 Page 6 of 10 http://www.nutritionandmetabolism.com/content/9/1/1  (a) (b) † † † † † † † * * * * * * * * * * * * †  (c) * * * * * * * * * † † † † †  Figure 2 The effects of a-tocopherol (aT) and g-tocotrienol (gT3) against H2O2-induced cell death in human DS neuron cultures, whereby (a) the cell death was determined using propidium iodide (PI) assay, (b) the cell viability was determined using MTT assay, (c) the apoptosis assay was determined using ELISA kits for ssDNA. The neurons were pre-treated with varying concentrations of aT and gT3 for one hour before the exposure to 100 μM H2O2 for 24 hours at 37°C. *Denotes P < 0.05 compared to control, † denotes P < 0.0001 compared to H2O2. The data are presented as mean ± SD, from 3 independent experiments of triplicate wells (n = 9). One-way ANOVA showed that there are significant differences between groups, F13, 83 = 14.47, P < 0.001. Meanwhile, two-way ANOVA showed both types of vitamin E isomer and vitamin E concentration are not significant factors contributing to the cell survival and no significant interaction between types of vitamin E isomer and vitamin E concentration (F5, 71 = 0.45). PKC [41]. However, both studies did not specify the fold increase of PKC-δ expression in H2O2-treated neu- location of PKC isoforms involvement. From previous rons, suggesting an accumulation of PKC-δ in the cyto- study, cPKC was shown to be activated when exposed sol, which signified pro-apoptotic activities in neurons to oxidative stress in neuronal death induced by ische- [44] were suppressed by the pre-treatment of gT3, while mia, hypoxia and exitotoxicity [42], whereas other stu- the pre-treatment of aT did not alter PKC-δ expression. dies revealed that an increase in PKC-δ expression was In normal euploid neurons, H 2 O 2 induced increased needed for glutamate-induced neuronal death [43], Par- cPKC expression [Figure 5 (b) (iv) and Figure 6 (c)] but kinson’s disease model [44] and AGE-induced neuronal suppressed PKC-δ [Figure 5 (b) (v) and Figure 6 (d)]. death [45] as well as H2O2-induced oxidative stress [46]. Pre-treatment of gT3 suppressed the cPKC expression Across the various treatment groups, the expression of but elevated the PKC-δ expression; while the pre-treat- cPKC was higher in DS neurons compared to euploid ment of aT was found to increase the cPKC expression neurons. However, the cPKC expression of DS neurons but down-regulated PKC-δ expression [Figure 6 (c) and was down-regulated in all other treatment groups (gT3 Figure 6 (d)]. aT has been known to inhibit PKC-a followed by H2O2, gT3, aT followed by H2O2, and aT), activities [47,48] which was not shown in euploid neu- as shown in Figure 5 (a) (iv) and Figure 6 (c). From Fig- rons pre-treated with aT in Figure 5 (b) (iv) and Figure ure 5 (a) (v) and Figure 6 (d), DS neurons showed a 2- 6 (c). Meanwhile a high concentration of aT (500 μM) Then et al. Nutrition & Metabolism 2012, 9:1 Page 7 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 Figure 3 The effects of a-tocopherol (aT) and g-tocotrienol (gT3) against H2O2-induced cell loss in human euploid neuron cultures, whereby (a) the cell death was determined using propidium iodide (PI) assay, (b) the cell viability was determined using MTT assay, and (c) the apoptosis assay was determined using ELISA kits for ssDNA. The neurons were pre-treated with varying concentrations of aT and gT3 for one hour before the exposure to 100 μM H2O2 for 24 hours at 37°C. *Denotes P < 0.05 compared to control, † denotes P < 0.0001 compared to H2O2. The data are presented as mean ± SD, from 3 independent experiments of triplicate wells (n = 9). One-way ANOVA showed that there are significant differences between groups, F13, 83 = 9.81, P < 0.001. Two-way ANOVA showed both types of vitamin E isomer and vitamin E concentration are significant factors contributing to the cell survival and no significant interaction between types of vitamin E isomer and vitamin E concentration (F5, 71 = 27.15, P < 0.0001). Control H2O2 ĮT + ȖT3 + H2O2 H2O2 Hoechst stain Apoptotic bodies      Control H2O2 ĮT + ȖT3 + H2O2 H2O2 NeuN     Figure 4 Fluorescence image of DS neurons stained with anti Neu-N antibody in various treatments: control, 100 μM of H2O2 only, 10 μM of aT or gT3 pre-treatment followed by H2O2 exposure Then et al. Nutrition & Metabolism 2012, 9:1 Page 8 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 has been shown to inhibit PKC-δ activation in AGE- induced neuronal death [45] in which lower dose of aT pretreatment in euploid neurons showed similar result [Figure 5 (b) (v) and Figure 6 (d)]. However, the incuba- tion of only gT3 also showed a decrease in cPKC, similar to a previous study which showed that gT3 sup- pressed PKC-a expression [19]. This suggests that besides functioning as an antioxidant, gT3 might also play a role in modulating PKC-δ expression as PKC-δ is a redox sensitive molecule. Conclusion This study revealed that in DS neurons, even though gT3 pre-treatment provided initial slight improvement in neuron viability, the protection from both aT and gT3 pre-treatment was not substantial to protect DS Figure 6 Densitometric analysis of Bcl-2 and Bax as Bcl-2/Bax neurons from H2O2 assault. Furthermore, pre-treatment ratio in (a), p53 in (b), cPKC in (c) and PKC-δ in (iv). * Denotes of gT3 would reduce the Bcl-2/Bax ratio that indicates to P < 0.05 compared to control, † denotes P < 0.05 compared to H2O2-treated neurons, ‡ denotes P < 0.05 compared to cells pre- cell survival while aT pre-treatment did not suppress incubated with gT3 or aT followed by H2O2 treatment while pro-apoptotic PKC-δ expression in the cells. However, # denotes P < 0.05 compares to euploid neurons. in non-oxidative stress condition, aT and gT3 did not exert strong pro-apoptosis tendency in human DS and euploid neurons compared to our previous studies in rat neuronal cell model subjected to oxidative stress. Our neurons [21]. DS neurons has been shown to have present results further underlie the importance of more chronic overexpression of S100B, in which oxidation of study to be done on the safety of vitamin E supplemen- S100B preferentially induced the neurotrophic processes tation in neurodegenerative diseases such as DS and (which is beneficial for cell survival and differentiation) AD. Although gT3 act as a free radical scavenger which over neuroinflammation processes. However treatment could quench ROS generated from H2 O 2 , it may also with antioxidants such as vitamin E interrupts this feed- synergistically induce apoptosis and autophagy through back and leads to increase glial activation and cell death the mitochondrial death pathway, including the Bcl-2 [25]. This may explain the reason why in DS neurons, family proteins [49]. Meanwhile, our study also showed aT and gT3 at the concentrations used in this study that aT has a different mechanism of action compared may aggravate cellular damages in a highly susceptible to gT3, which remains to be further elucidated. (a) H2O2 GTT - - + - - - + - - + + +  (b) H2O2 GTT - - + - - - + - - + + + Acknowledgements  ATF - - + + - -  ATF - - + + - - We are grateful to Dr Santiago Barambio, Yolanda Trejo and Raquel Lopez i. Bcl-2  i. Bcl-2    from Tutor Medica Clinics, Barcelona, and Dr Nuria Toran from Hospital de la  ii. Bax  ii. Bax Vall d’Hebron, Barcelona, for their assistance in providing the tissue samples   for this work. This study was funded by the Ministry of Science, Technology iii. p53 iii. p53  and Innovation Malaysia under the Intensified Research Prioritized Area  iv. PKC iv. PKC (IRPA) grant 06-02-02-002/PR0008/09-07, SAF2009-13093-C02-02 from   MICINN and RD06/0013/1004 from ISCIII, Spain. v. PKC-G v. PKC-G   vi. E-Actin vi. E-Actin Author details   1  UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Figure 5 Bcl-2, Bax, p53, cPKC and PKC-δ were differentially Malaysia (UKM), Kuala Lumpur, Malaysia. 2Institut d’Investigacions expressed in euploid and DS neurons which were given Biomèdiques de Barcelona (IIBB), CSIC-IDIBAPS, Barcelona, Spain. 3 various treatments in the following fashion: untreated control, Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia. 4Malaysian Palm Oil Board, Bangi, incubation of neurons with H2O2 for 24 hours (H2O2), Selangor, Malaysia. incubation of neurons with gT3 (10 μM) for 24 hours (gT3), one hour of gT3 (10 μM) pre-treatment in neurons followed by Authors’ contributions H2O2 incubation for 24 hours (gT3 + H2O2), incubation of SMT carried out the most of the experiments, performed the statistical neurons with aT (10 μM) for 24 hours (aT) and one hour of aT analysis and drafted the manuscript. WZWN and MM played a major role in (10 μM) pre-treatment in neurons, followed by H2O2 the experimental procedures of this study and revised the manuscript. CS incubation for 24 hours (aT + H2O2). (a) Western blot of Bcl-2, carried out the cell imaging work and help revised the manuscript. Bax, p53, cPKC and PKC in DS neurons; (b) Western blot of the Meanwhile, GMT extracted the vitamin E isomers and tested the purity of same proteins in euploid neurons. the vitamin E isomers. All authors have read and approved the final manuscript. Then et al. Nutrition & Metabolism 2012, 9:1 Page 9 of 10 http://www.nutritionandmetabolism.com/content/9/1/1 Competing interests 20. Mazlan M, Sue Mian T, Mat Top G, Zurinah Wan NW: Comparative effects The authors declare that they have no competing interests. of alpha-tocopherol and gamma-tocotrienol against hydrogen peroxide induced apoptosis on primary-cultured astrocytes. 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