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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

Bcl-2, Bax, p53, cPKC and PKC-δ were differentially expressed in euploid and DS neurons which were given various treatments in the following fashion: untreated control, incubation of neurons with H2O2 for 24 hours (H2O2), incubation of neurons with γT3 (10 μM) for 24 hours (γT3), one hour of γT3 (10 μM) pre-treatment in neurons followed by H2O2 incubation for 24 hours (γT3 + H2O2), incubation of neurons with αT (10 μM) for 24 hours (αT) and one hour of αT (10 μM) pre-treatment in neurons, followed by H2O2 incubation for 24 hours (αT + H2O2). (a) Western blot of Bcl-2, Bax, p53, cPKC and PKC in DS neurons; (b) Western blot of the same proteins in euploid neurons.
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Figure 5: Bcl-2, Bax, p53, cPKC and PKC-δ were differentially expressed in euploid and DS neurons which were given various treatments in the following fashion: untreated control, incubation of neurons with H2O2 for 24 hours (H2O2), incubation of neurons with γT3 (10 μM) for 24 hours (γT3), one hour of γT3 (10 μM) pre-treatment in neurons followed by H2O2 incubation for 24 hours (γT3 + H2O2), incubation of neurons with αT (10 μM) for 24 hours (αT) and one hour of αT (10 μM) pre-treatment in neurons, followed by H2O2 incubation for 24 hours (αT + H2O2). (a) Western blot of Bcl-2, Bax, p53, cPKC and PKC in DS neurons; (b) Western blot of the same proteins in euploid neurons.

Mentions: From our investigation, euploid neurons showed a completely different protein expression profile compared to DS neurons. In the control group, DS neurons were shown to have lower Bcl-2/Bax ratio and p53 expression compared to euploid neurons, while cPKC and PKC-δ expressions were higher in DS neurons. Figure 5 (a) (i), Figure 5 (a) (ii) and Figure 6a revealed that for DS neurons, pre-treatment of γT3 followed by H2O2 has significantly lower Bcl-2/Bax ratio than the controls, whereas other treatment showed changes which were not statistically significant. This result suggests that γT3 does not contribute to the survival of DS neurons under H2O2 assault via the Bcl-2/Bax heterodimer complex formation. However, the analysis of Western blot for the euploid neurons in Figure 5 (b) (i), Figure 5 (b) (ii) and Figure 6 (a) showed a different picture: Bcl-2/Bax ratio increased significantly in euploid neurons when neurons were pre-treated with either γT3 or αT followed by H2O2 which suggested that γT3 pre-treatment attenuated apoptosis and improved cell survival of normal euploid neurons. However, p53 expression was not significantly different across various treatments in both DS and euploid neurons, as depicted in Figure 5 (a) (iii), Figure 5 (b) (iii) and Figure 6 (b). Nevertheless, the comparison of p53 expression between DS and euploid neurons showed lower p53 expression in DS neurons for these treatment groups: control, H2O2, γT3 followed by H2O2 and γT3 treatments only. Taking it all together, these results show that lower Bcl-2/Bax ratio DS neurons in the untreated groups, was in agreement with the results from previous study which reported that fetal DS neurons had increased Bax and p53 expressions mediated by the transcription factor est-2 when treated with H2O2 [37]. On the other hand, another study reported that APO-1, caspase-3 and Bcl-2 protein expression levels were unaltered in the fetal DS neurons [38]. A previous study also stated that incubation of αT induced the up-regulation of Bcl-2 as preventive effects from neuronal cell death [39]. Thus, treatment of only αT and γT3 without the presence of H2O2 in human neuron did not show pro-apoptosis tendency (from the Bcl-2/Bax ratio and p53 expression) compared to rat cerebellar culture as reported previously [21].


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)

Bcl-2, Bax, p53, cPKC and PKC-δ were differentially expressed in euploid and DS neurons which were given various treatments in the following fashion: untreated control, incubation of neurons with H2O2 for 24 hours (H2O2), incubation of neurons with γT3 (10 μM) for 24 hours (γT3), one hour of γT3 (10 μM) pre-treatment in neurons followed by H2O2 incubation for 24 hours (γT3 + H2O2), incubation of neurons with αT (10 μM) for 24 hours (αT) and one hour of αT (10 μM) pre-treatment in neurons, followed by H2O2 incubation for 24 hours (αT + H2O2). (a) Western blot of Bcl-2, Bax, p53, cPKC and PKC in DS neurons; (b) Western blot of the same proteins in euploid neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Bcl-2, Bax, p53, cPKC and PKC-δ were differentially expressed in euploid and DS neurons which were given various treatments in the following fashion: untreated control, incubation of neurons with H2O2 for 24 hours (H2O2), incubation of neurons with γT3 (10 μM) for 24 hours (γT3), one hour of γT3 (10 μM) pre-treatment in neurons followed by H2O2 incubation for 24 hours (γT3 + H2O2), incubation of neurons with αT (10 μM) for 24 hours (αT) and one hour of αT (10 μM) pre-treatment in neurons, followed by H2O2 incubation for 24 hours (αT + H2O2). (a) Western blot of Bcl-2, Bax, p53, cPKC and PKC in DS neurons; (b) Western blot of the same proteins in euploid neurons.
Mentions: From our investigation, euploid neurons showed a completely different protein expression profile compared to DS neurons. In the control group, DS neurons were shown to have lower Bcl-2/Bax ratio and p53 expression compared to euploid neurons, while cPKC and PKC-δ expressions were higher in DS neurons. Figure 5 (a) (i), Figure 5 (a) (ii) and Figure 6a revealed that for DS neurons, pre-treatment of γT3 followed by H2O2 has significantly lower Bcl-2/Bax ratio than the controls, whereas other treatment showed changes which were not statistically significant. This result suggests that γT3 does not contribute to the survival of DS neurons under H2O2 assault via the Bcl-2/Bax heterodimer complex formation. However, the analysis of Western blot for the euploid neurons in Figure 5 (b) (i), Figure 5 (b) (ii) and Figure 6 (a) showed a different picture: Bcl-2/Bax ratio increased significantly in euploid neurons when neurons were pre-treated with either γT3 or αT followed by H2O2 which suggested that γT3 pre-treatment attenuated apoptosis and improved cell survival of normal euploid neurons. However, p53 expression was not significantly different across various treatments in both DS and euploid neurons, as depicted in Figure 5 (a) (iii), Figure 5 (b) (iii) and Figure 6 (b). Nevertheless, the comparison of p53 expression between DS and euploid neurons showed lower p53 expression in DS neurons for these treatment groups: control, H2O2, γT3 followed by H2O2 and γT3 treatments only. Taking it all together, these results show that lower Bcl-2/Bax ratio DS neurons in the untreated groups, was in agreement with the results from previous study which reported that fetal DS neurons had increased Bax and p53 expressions mediated by the transcription factor est-2 when treated with H2O2 [37]. On the other hand, another study reported that APO-1, caspase-3 and Bcl-2 protein expression levels were unaltered in the fetal DS neurons [38]. A previous study also stated that incubation of αT induced the up-regulation of Bcl-2 as preventive effects from neuronal cell death [39]. Thus, treatment of only αT and γT3 without the presence of H2O2 in human neuron did not show pro-apoptosis tendency (from the Bcl-2/Bax ratio and p53 expression) compared to rat cerebellar culture as reported previously [21].

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