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Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Lee HR, Shin HK, Park SY, Kim HY, Bae SS, Lee WS, Rhim BY, Hong KW, Kim CD - PLoS ONE (2015)

Bottom Line: We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly.Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy.In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

View Article: PubMed Central - PubMed

Affiliation: Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea; Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea.

ABSTRACT
Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

No MeSH data available.


Related in: MedlinePlus

A and D. Immunoprecipitation analysis. Whole cell lysates were obtained from N2aSwe cell lysates that were cultured in Tet- condition for 24 h with or without cilostazol (10 μM, A) or rSIRT1 (recombinant SIRT1, 300 nM; B). Upper panels (A and B): the effects of cilostazol and rSIRT1 on LC3-II expression were confirmed. Lower panels (A and B): cell lysates were immunoprecipitated with LC-3 antibody and then immunoblotted for acetylated LC3 LC3-1/II-Ac) using an anti-acetyl lysine antibody. The blot shown is representative of three experiments that produced similar results. C. Immunofluorescent assay of LC3 puncta in N2a cells treated with or without cilostazol (10 and 30 μM) after being pretreated with 3-methyladenine (2.5 mM), bafilomycin A1 (100 nM) or sirtinol (20 μM). D. Quantitative analysis was performed by counting numbers of LC3 puncta/cell. Results are the means ± SDs of 4 experiments.***P < 0.001 vs. DMSO. †††P < 0.001 vs. cilostazol (10 μM).
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pone.0134486.g006: A and D. Immunoprecipitation analysis. Whole cell lysates were obtained from N2aSwe cell lysates that were cultured in Tet- condition for 24 h with or without cilostazol (10 μM, A) or rSIRT1 (recombinant SIRT1, 300 nM; B). Upper panels (A and B): the effects of cilostazol and rSIRT1 on LC3-II expression were confirmed. Lower panels (A and B): cell lysates were immunoprecipitated with LC-3 antibody and then immunoblotted for acetylated LC3 LC3-1/II-Ac) using an anti-acetyl lysine antibody. The blot shown is representative of three experiments that produced similar results. C. Immunofluorescent assay of LC3 puncta in N2a cells treated with or without cilostazol (10 and 30 μM) after being pretreated with 3-methyladenine (2.5 mM), bafilomycin A1 (100 nM) or sirtinol (20 μM). D. Quantitative analysis was performed by counting numbers of LC3 puncta/cell. Results are the means ± SDs of 4 experiments.***P < 0.001 vs. DMSO. †††P < 0.001 vs. cilostazol (10 μM).

Mentions: To examine the effect of SIRT1 on autophagy components, we assessed and compared the deacetylations of LC3 by cilostazol or recombinant SIRT1 (rSIRT1) in the N2aSwe cells. Neuronal cells were exposed to Tet- for 24 h in the absence and presence of 10 μM of cilostazol or 300 nM of rSIRT1, and then one part of each whole cell lysates was immunoblotted for LC3. The other portions were immunoprecipitated with LC-3 antibody and immunoblotted for acetylated LC3 using an anti-acetyl lysine antibody. As shown in Fig 6A and 6B, LC3-II levels were markedly elevated by treatment with cilostazol or rSIRT1. However, after immunoprecipitating LC-3, the immunoblotted band intensity of acetylated LC3-II was markedly reduced (by ~30%) in samples treated with cilostazol or rSIRT1.


Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Lee HR, Shin HK, Park SY, Kim HY, Bae SS, Lee WS, Rhim BY, Hong KW, Kim CD - PLoS ONE (2015)

A and D. Immunoprecipitation analysis. Whole cell lysates were obtained from N2aSwe cell lysates that were cultured in Tet- condition for 24 h with or without cilostazol (10 μM, A) or rSIRT1 (recombinant SIRT1, 300 nM; B). Upper panels (A and B): the effects of cilostazol and rSIRT1 on LC3-II expression were confirmed. Lower panels (A and B): cell lysates were immunoprecipitated with LC-3 antibody and then immunoblotted for acetylated LC3 LC3-1/II-Ac) using an anti-acetyl lysine antibody. The blot shown is representative of three experiments that produced similar results. C. Immunofluorescent assay of LC3 puncta in N2a cells treated with or without cilostazol (10 and 30 μM) after being pretreated with 3-methyladenine (2.5 mM), bafilomycin A1 (100 nM) or sirtinol (20 μM). D. Quantitative analysis was performed by counting numbers of LC3 puncta/cell. Results are the means ± SDs of 4 experiments.***P < 0.001 vs. DMSO. †††P < 0.001 vs. cilostazol (10 μM).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526537&req=5

pone.0134486.g006: A and D. Immunoprecipitation analysis. Whole cell lysates were obtained from N2aSwe cell lysates that were cultured in Tet- condition for 24 h with or without cilostazol (10 μM, A) or rSIRT1 (recombinant SIRT1, 300 nM; B). Upper panels (A and B): the effects of cilostazol and rSIRT1 on LC3-II expression were confirmed. Lower panels (A and B): cell lysates were immunoprecipitated with LC-3 antibody and then immunoblotted for acetylated LC3 LC3-1/II-Ac) using an anti-acetyl lysine antibody. The blot shown is representative of three experiments that produced similar results. C. Immunofluorescent assay of LC3 puncta in N2a cells treated with or without cilostazol (10 and 30 μM) after being pretreated with 3-methyladenine (2.5 mM), bafilomycin A1 (100 nM) or sirtinol (20 μM). D. Quantitative analysis was performed by counting numbers of LC3 puncta/cell. Results are the means ± SDs of 4 experiments.***P < 0.001 vs. DMSO. †††P < 0.001 vs. cilostazol (10 μM).
Mentions: To examine the effect of SIRT1 on autophagy components, we assessed and compared the deacetylations of LC3 by cilostazol or recombinant SIRT1 (rSIRT1) in the N2aSwe cells. Neuronal cells were exposed to Tet- for 24 h in the absence and presence of 10 μM of cilostazol or 300 nM of rSIRT1, and then one part of each whole cell lysates was immunoblotted for LC3. The other portions were immunoprecipitated with LC-3 antibody and immunoblotted for acetylated LC3 using an anti-acetyl lysine antibody. As shown in Fig 6A and 6B, LC3-II levels were markedly elevated by treatment with cilostazol or rSIRT1. However, after immunoprecipitating LC-3, the immunoblotted band intensity of acetylated LC3-II was markedly reduced (by ~30%) in samples treated with cilostazol or rSIRT1.

Bottom Line: We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly.Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy.In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

View Article: PubMed Central - PubMed

Affiliation: Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea; Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea.

ABSTRACT
Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

No MeSH data available.


Related in: MedlinePlus