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

Then SM, Sanfeliu C, Top GM, Wan Ngah WZ, Mazlan M - Nutr Metab (Lond) (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia. suemian@ppukm.ukm.my.

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 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.

Methods: Primary 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.

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 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.

Conclusion: Our study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.

No MeSH data available.


Related in: MedlinePlus

The effects of α-tocopherol (αT) and γ-tocotrienol (γT3) 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 αT and γT3 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).
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Figure 3: The effects of α-tocopherol (αT) and γ-tocotrienol (γT3) 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 αT and γT3 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).

Mentions: In both DS and euploid neurons, the pre-treatment of γT3 and αT at concentration ≤ 50 μM was able to reduce cell death induced by H2O2 [Figure 2 (a) and Figure 3 (a)] and increase cell viability [Figure 2 (b) and Figure 3 (b)]. Figure 2 (c) and Figure 3 (c) shows that both pre-treatments of up to 10 μM γT3 and 50 μM αT were able to attenuate apoptosis in H2O2-induced DS and euploid neurons respectively. However, the effectiveness of γT3 and αT pre-treatment was more pronounced in euploid neurons compared to DS neurons, as two-way ANOVA analysis showed that vitamin E isomer type and concentration contributed significantly to cell viability and apoptosis rate in euploid neurons but not in DS neurons. This indicated that the protective effect of the vitamin E in euploid neurons is dose and isomer type dependent, while protective effects of vitamin E in DS neurons is not dependent on dose and isomer type. Figure 4 shows the morphology of H2O2-treated DS neurons as stained by Neu-N, a marker for differentiated neurons [33] which had undergone apoptosis; pre-treatment with 10 μM of αT or γT3 retained some of the neurons' viability but cell morphologies were not fully maintained. Reduced Neu-N expression in differentiated neurons indicated perturbed cell morphology induced by H2O2 assault, which pre-treatment of either αT or γT3 were not substantial enough to protect cells from oxidative assault. However, comparatively αT seems to be more protective to the DS neurons than γT3 as more cells were stained with Neu-N, although the staining was dim. This is not surprising since the previous study has shown that in vitro DS cortical neuron culture had higher sensitivity to H2O2-induced oxidative damage compared to euploid neurons [27]. H2O2 directly induces cellular damage and has been reported to induce parallel apoptosis and autophagy [34], making it more difficult for αT and γT3 to protect the cells especially if the pre-treatment incubation period was short. Since our previous studies have shown that high dose of vitamin E compounds the toxic effect of H2O2, here we further investigate whether non-lethal doses of high concentration αT and γT3 will further exacerbate the detrimental effects of H2O2 via the mitochondrial Bcl-2 family pathway or the PKC signalling pathway as both cPKC and PKC-δ are redox sensitive and were reported to be involved in the initiation of apoptosis signalling [35,36].


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

Then SM, Sanfeliu C, Top GM, Wan Ngah WZ, Mazlan M - Nutr Metab (Lond) (2012)

The effects of α-tocopherol (αT) and γ-tocotrienol (γT3) 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 αT and γT3 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285086&req=5

Figure 3: The effects of α-tocopherol (αT) and γ-tocotrienol (γT3) 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 αT and γT3 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).
Mentions: In both DS and euploid neurons, the pre-treatment of γT3 and αT at concentration ≤ 50 μM was able to reduce cell death induced by H2O2 [Figure 2 (a) and Figure 3 (a)] and increase cell viability [Figure 2 (b) and Figure 3 (b)]. Figure 2 (c) and Figure 3 (c) shows that both pre-treatments of up to 10 μM γT3 and 50 μM αT were able to attenuate apoptosis in H2O2-induced DS and euploid neurons respectively. However, the effectiveness of γT3 and αT pre-treatment was more pronounced in euploid neurons compared to DS neurons, as two-way ANOVA analysis showed that vitamin E isomer type and concentration contributed significantly to cell viability and apoptosis rate in euploid neurons but not in DS neurons. This indicated that the protective effect of the vitamin E in euploid neurons is dose and isomer type dependent, while protective effects of vitamin E in DS neurons is not dependent on dose and isomer type. Figure 4 shows the morphology of H2O2-treated DS neurons as stained by Neu-N, a marker for differentiated neurons [33] which had undergone apoptosis; pre-treatment with 10 μM of αT or γT3 retained some of the neurons' viability but cell morphologies were not fully maintained. Reduced Neu-N expression in differentiated neurons indicated perturbed cell morphology induced by H2O2 assault, which pre-treatment of either αT or γT3 were not substantial enough to protect cells from oxidative assault. However, comparatively αT seems to be more protective to the DS neurons than γT3 as more cells were stained with Neu-N, although the staining was dim. This is not surprising since the previous study has shown that in vitro DS cortical neuron culture had higher sensitivity to H2O2-induced oxidative damage compared to euploid neurons [27]. H2O2 directly induces cellular damage and has been reported to induce parallel apoptosis and autophagy [34], making it more difficult for αT and γT3 to protect the cells especially if the pre-treatment incubation period was short. Since our previous studies have shown that high dose of vitamin E compounds the toxic effect of H2O2, here we further investigate whether non-lethal doses of high concentration αT and γT3 will further exacerbate the detrimental effects of H2O2 via the mitochondrial Bcl-2 family pathway or the PKC signalling pathway as both cPKC and PKC-δ are redox sensitive and were reported to be involved in the initiation of apoptosis signalling [35,36].

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia. suemian@ppukm.ukm.my.

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 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.

Methods: Primary 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.

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 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.

Conclusion: Our study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.

No MeSH data available.


Related in: MedlinePlus