Limits...
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.

Show MeSH
Effect of SS31 on the t-BHP-induced release of cytochrome c. The 661W cells were subjected to double immunofluorescence antibody staining using mouse anti-cytochrome c antibody (red) and rabbit anti-HSP60 antibody (green). Nuclei were demarcated using Hoechst staining (blue). The overlay of all three types of staining (yellow indicates combination of red and green) is shown in the Merged column. Immunofluorescence was analyzed using confocal microscopy (magnification, ×1,000). Arrows indicate the releasing of cytochrome c from the mitochondria in the apoptotic cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581771&req=5

f6-mmr-12-04-5026: Effect of SS31 on the t-BHP-induced release of cytochrome c. The 661W cells were subjected to double immunofluorescence antibody staining using mouse anti-cytochrome c antibody (red) and rabbit anti-HSP60 antibody (green). Nuclei were demarcated using Hoechst staining (blue). The overlay of all three types of staining (yellow indicates combination of red and green) is shown in the Merged column. Immunofluorescence was analyzed using confocal microscopy (magnification, ×1,000). Arrows indicate the releasing of cytochrome c from the mitochondria in the apoptotic cell.

Mentions: As it is well known that excessive ROS and decreased ΔΨm can induce apoptotic death, the present study examined the release of mitochondrial cytochrome c, an important signaling molecule in apoptosis. This included examining whether oxidative stress induced the release of cytochrome c from the mitochondria and whether the addition of SS31 prevented this release. As shown in Fig. 6, the 661W cells in the control group exhibited an exact overlap of anti-cytochrome c and HSP60-labeled mitochondrial fluorescence in the confocal micrographs, indicating the co-localization of cytochrome c and mitochondria in the cells. No cytochrome c release was identified from the mitochondria prior to treatment with t-BHP. Following treatment with 100 µM t-BHP for 24 h, cytochrome c was observed in the cytoplasm of the 661W cells, and this was not coincident with the mitochondrial labeling, indicating that treatment with t-BHP induced the release of cytochrome c from the mitochondria in the 661W cells (Fig. 6, arrows). By contrast, treatment with 100 nM SS31 inhibited the release of cytochrome c into the cytosol (Fig. 6).


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)

Effect of SS31 on the t-BHP-induced release of cytochrome c. The 661W cells were subjected to double immunofluorescence antibody staining using mouse anti-cytochrome c antibody (red) and rabbit anti-HSP60 antibody (green). Nuclei were demarcated using Hoechst staining (blue). The overlay of all three types of staining (yellow indicates combination of red and green) is shown in the Merged column. Immunofluorescence was analyzed using confocal microscopy (magnification, ×1,000). Arrows indicate the releasing of cytochrome c from the mitochondria in the apoptotic cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-mmr-12-04-5026: Effect of SS31 on the t-BHP-induced release of cytochrome c. The 661W cells were subjected to double immunofluorescence antibody staining using mouse anti-cytochrome c antibody (red) and rabbit anti-HSP60 antibody (green). Nuclei were demarcated using Hoechst staining (blue). The overlay of all three types of staining (yellow indicates combination of red and green) is shown in the Merged column. Immunofluorescence was analyzed using confocal microscopy (magnification, ×1,000). Arrows indicate the releasing of cytochrome c from the mitochondria in the apoptotic cell.
Mentions: As it is well known that excessive ROS and decreased ΔΨm can induce apoptotic death, the present study examined the release of mitochondrial cytochrome c, an important signaling molecule in apoptosis. This included examining whether oxidative stress induced the release of cytochrome c from the mitochondria and whether the addition of SS31 prevented this release. As shown in Fig. 6, the 661W cells in the control group exhibited an exact overlap of anti-cytochrome c and HSP60-labeled mitochondrial fluorescence in the confocal micrographs, indicating the co-localization of cytochrome c and mitochondria in the cells. No cytochrome c release was identified from the mitochondria prior to treatment with t-BHP. Following treatment with 100 µM t-BHP for 24 h, cytochrome c was observed in the cytoplasm of the 661W cells, and this was not coincident with the mitochondrial labeling, indicating that treatment with t-BHP induced the release of cytochrome c from the mitochondria in the 661W cells (Fig. 6, arrows). By contrast, treatment with 100 nM SS31 inhibited the release of cytochrome c into the cytosol (Fig. 6).

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.

Show MeSH