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Protective effects of SS31 on t‑BHP induced oxidative damage in 661W cells.

Ma W, Zhu X, Ding X, Li T, Hu Y, Hu X, Yuan L, Lei L, Hu A, Luo Y, Tang S - Mol Med Rep (2015)

Bottom Line: The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑Sen University, Guangzhou, Guangdong 510060, P.R. China.

ABSTRACT
The present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

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SS31 reduces protein and DNA peroxidation caused by t-BHP. The 661W cells were treated with 100 µM t-BHP, alone or with 100 nM SS31 for 24 h. The results revealed that treatment of the 661W cells with 100 µM t-BHP for 24 h increased the accumulation of nitrotyrosine (red) and 8-OHdG (red) in the nuclei and mitochondrial DNA. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG. Nuclei (blue) were stained using Hoechst. Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). 8-OHdG, 8-hydroxydeoxyguanosine.
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f2-mmr-12-04-5026: SS31 reduces protein and DNA peroxidation caused by t-BHP. The 661W cells were treated with 100 µM t-BHP, alone or with 100 nM SS31 for 24 h. The results revealed that treatment of the 661W cells with 100 µM t-BHP for 24 h increased the accumulation of nitrotyrosine (red) and 8-OHdG (red) in the nuclei and mitochondrial DNA. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG. Nuclei (blue) were stained using Hoechst. Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). 8-OHdG, 8-hydroxydeoxyguanosine.

Mentions: When ROS interact with proteins, lipids or DNA, cell dysfunction and death can occur. Certain sites of macromolecules are particularly susceptible to particular ROS, resulting in specific modifications that act as 'fingerprints', implicating oxidative damage in disease pathogenesis (19). The occurrence of nitrotyrosine residues in proteins is specific for peroxynitrite-induced protein oxidative damage (20). Hydroxyl radicals can also attack guanine at its C-8 position to yield 8-OHdG, which serves as another biomarker for DNA oxidative damage. The confocal microscopic images in the present study indicated that treatment of the 661W cells with 100 mM t-BHP for 24 h increased the production of nitrotyrosine and 8-OHdG. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG (Fig. 2).


Protective effects of SS31 on t‑BHP induced oxidative damage in 661W cells.

Ma W, Zhu X, Ding X, Li T, Hu Y, Hu X, Yuan L, Lei L, Hu A, Luo Y, Tang S - Mol Med Rep (2015)

SS31 reduces protein and DNA peroxidation caused by t-BHP. The 661W cells were treated with 100 µM t-BHP, alone or with 100 nM SS31 for 24 h. The results revealed that treatment of the 661W cells with 100 µM t-BHP for 24 h increased the accumulation of nitrotyrosine (red) and 8-OHdG (red) in the nuclei and mitochondrial DNA. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG. Nuclei (blue) were stained using Hoechst. Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). 8-OHdG, 8-hydroxydeoxyguanosine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581771&req=5

f2-mmr-12-04-5026: SS31 reduces protein and DNA peroxidation caused by t-BHP. The 661W cells were treated with 100 µM t-BHP, alone or with 100 nM SS31 for 24 h. The results revealed that treatment of the 661W cells with 100 µM t-BHP for 24 h increased the accumulation of nitrotyrosine (red) and 8-OHdG (red) in the nuclei and mitochondrial DNA. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG. Nuclei (blue) were stained using Hoechst. Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). 8-OHdG, 8-hydroxydeoxyguanosine.
Mentions: When ROS interact with proteins, lipids or DNA, cell dysfunction and death can occur. Certain sites of macromolecules are particularly susceptible to particular ROS, resulting in specific modifications that act as 'fingerprints', implicating oxidative damage in disease pathogenesis (19). The occurrence of nitrotyrosine residues in proteins is specific for peroxynitrite-induced protein oxidative damage (20). Hydroxyl radicals can also attack guanine at its C-8 position to yield 8-OHdG, which serves as another biomarker for DNA oxidative damage. The confocal microscopic images in the present study indicated that treatment of the 661W cells with 100 mM t-BHP for 24 h increased the production of nitrotyrosine and 8-OHdG. Concurrent treatment with 100 nM SS31 prevented the t-BHP-induced accumulation of nitrotyrosine and 8-OHdG (Fig. 2).

Bottom Line: The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑Sen University, Guangzhou, Guangdong 510060, P.R. China.

ABSTRACT
The present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

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