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Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

Wang C, Zeng Z, Liu Q, Zhang R, Ni J - Int J Mol Sci (2014)

Bottom Line: Its biological function is also associated with cell apoptosis.Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability.The results hereby imply the potentiality of Clu and Se in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. raulw2003@163.com.

ABSTRACT
Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

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Altered expression levels of apoptosis-related genes Bax, Bcl-2 and Bad in the Clu-knockdown N2a cells. (A) Real-time PCR detection of Bax, Bcl-2 and Bad mRNA expression; (B) Western blot analyses of Bax and Bcl-2 protein expression. Cells transfected with pGPU–Clu–mus–shSc (Sc) were used as the control. **p < 0.01 and ***p < 0.001 vs. the control.
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ijms-15-21331-f003: Altered expression levels of apoptosis-related genes Bax, Bcl-2 and Bad in the Clu-knockdown N2a cells. (A) Real-time PCR detection of Bax, Bcl-2 and Bad mRNA expression; (B) Western blot analyses of Bax and Bcl-2 protein expression. Cells transfected with pGPU–Clu–mus–shSc (Sc) were used as the control. **p < 0.01 and ***p < 0.001 vs. the control.

Mentions: Bax, Bcl-2 and Bad belong to the family of Bcl-2 proteases, which mediate cell apoptosis. In the Clu-knockdown N2a cells, altered levels of Bax, Bcl-2 and Bad were detected at both mRNA and protein levels by real-time PCR and western blot analyses, respectively. β-Actin served as an internal standard. At the mRNA level, the expression of Bcl-2 decreased to 75.69% (p < 0.01), while the expression of Bax and Bad increased to 202.67% (p < 0.001) and 147.46% (p < 0.001) respectively, compared to the Sc control (Figure 3A). At the protein level, the expression of Bcl-2 was also significantly decreased (Figure 3B), and the expression of Bax was significantly increased (Figure 3B). The results from both mRNA and protein levels showed the same trends in the alteration of apoptosis-related factors.


Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

Wang C, Zeng Z, Liu Q, Zhang R, Ni J - Int J Mol Sci (2014)

Altered expression levels of apoptosis-related genes Bax, Bcl-2 and Bad in the Clu-knockdown N2a cells. (A) Real-time PCR detection of Bax, Bcl-2 and Bad mRNA expression; (B) Western blot analyses of Bax and Bcl-2 protein expression. Cells transfected with pGPU–Clu–mus–shSc (Sc) were used as the control. **p < 0.01 and ***p < 0.001 vs. the control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4264228&req=5

ijms-15-21331-f003: Altered expression levels of apoptosis-related genes Bax, Bcl-2 and Bad in the Clu-knockdown N2a cells. (A) Real-time PCR detection of Bax, Bcl-2 and Bad mRNA expression; (B) Western blot analyses of Bax and Bcl-2 protein expression. Cells transfected with pGPU–Clu–mus–shSc (Sc) were used as the control. **p < 0.01 and ***p < 0.001 vs. the control.
Mentions: Bax, Bcl-2 and Bad belong to the family of Bcl-2 proteases, which mediate cell apoptosis. In the Clu-knockdown N2a cells, altered levels of Bax, Bcl-2 and Bad were detected at both mRNA and protein levels by real-time PCR and western blot analyses, respectively. β-Actin served as an internal standard. At the mRNA level, the expression of Bcl-2 decreased to 75.69% (p < 0.01), while the expression of Bax and Bad increased to 202.67% (p < 0.001) and 147.46% (p < 0.001) respectively, compared to the Sc control (Figure 3A). At the protein level, the expression of Bcl-2 was also significantly decreased (Figure 3B), and the expression of Bax was significantly increased (Figure 3B). The results from both mRNA and protein levels showed the same trends in the alteration of apoptosis-related factors.

Bottom Line: Its biological function is also associated with cell apoptosis.Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability.The results hereby imply the potentiality of Clu and Se in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. raulw2003@163.com.

ABSTRACT
Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

Show MeSH
Related in: MedlinePlus