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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—C. Inhibition of Tet- condition-induced reductions in beclin-1 (A), Atg5 (B) and SIRT1 expressions (C) by cilostazol (10 or 30 μM) in N2aSwe cells. D. Inhibition of Tet- condition-induced endogenous increases in Aβ level by cilostazol as determined by Western blotting (D) and of intracellular Aβ accumulation as determined by ELISA (E, F). The inhibitory effects of cilostazol were blocked by KT5720 (1 μM), sirtinol (20 μM) (E), bafilomycin A1 (BAF, 100 ng/ml), or 3-methyladenine (3-MA, 2.5 mM) (F).Results are presented as means ± SDs (N = 4–5). ##P < 0.01, ###P < 0.001 vs. Tet+ condition (as control), **P < 0.01 ***P < 0.001 vs. DMSO; †P < 0.05, †††P < 0.001 vs. cilostazol alone (CSZ, 10 μM). PBS, phosphate-buffered saline.
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pone.0134486.g004: A—C. Inhibition of Tet- condition-induced reductions in beclin-1 (A), Atg5 (B) and SIRT1 expressions (C) by cilostazol (10 or 30 μM) in N2aSwe cells. D. Inhibition of Tet- condition-induced endogenous increases in Aβ level by cilostazol as determined by Western blotting (D) and of intracellular Aβ accumulation as determined by ELISA (E, F). The inhibitory effects of cilostazol were blocked by KT5720 (1 μM), sirtinol (20 μM) (E), bafilomycin A1 (BAF, 100 ng/ml), or 3-methyladenine (3-MA, 2.5 mM) (F).Results are presented as means ± SDs (N = 4–5). ##P < 0.01, ###P < 0.001 vs. Tet+ condition (as control), **P < 0.01 ***P < 0.001 vs. DMSO; †P < 0.05, †††P < 0.001 vs. cilostazol alone (CSZ, 10 μM). PBS, phosphate-buffered saline.

Mentions: We further confirmed cilostazol significantly inhibited endogenously overproduced Aβin N2aSwe cells induced by using the model of Tet+ and Tet- conditions. As shown in Fig 4A, 4B and 4C, the protein expressions of beclin-1, Atg5, and SIRT1 were significantly reduced by 76.1 ± 6.8%, 85.3 ± 3.2% and 76.8 ± 6.4%, respectively (each, P < 0.01) when N2aSwe cells were exposed to Tet-. Interestingly, by pretreatment with cilostazol (10 or 30 μM), these attenuated levels of SIRT1, beclin-1, and Atg5 were largely overexpressed (10 μM cilostazol: by 155.1 ± 18.1%, 136.6 ± 11.7%, and 125.7 ± 8.0%, respectively). Intriguingly, increased Aβ level under Tet- condition (by 139.5 ± 9.8%, P < 0.001) was significantly inhibited by cilostazol pretreatment (10 or 30 μM) to 89.4 ± 2.4 (P < 0.001) and 75.5 ± 16.0% (P < 0.001), respectively (Fig 4D). These results were further evaluated by measuring Aβ levels by ELISA. When N2aSwe cells were exposed to Tet- condition, intracellular Aβ1–42 levels significantly increased by 362.2 ± 7.7 ng/ml (P < 0.001), and this increase was markedly reduced to 186.4 ± 10.9 ng/ml (P < 0.001) and 165.9 ± 10.9 ng/ml (P < 0.001) by pretreating cilostazol at 10 and 30 μM, respectively. Furthermore, this cilostazol-induced inhibition was significantly blocked by pretreatment with KT5720 (1 μM, a cAMP-dependent protein kinase inhibitor) or sirtinol (20 μM, a SIRT1 inhibitor) (Fig 4E). In addition, cilostazol-induced inhibition of Aβ1–42 level was also blocked by bafilomycin A1 (100 nM, P < 0.001) or 3-methyladenine (2.5 mM, P < 0.05), respectively (Fig 4F). Overall, these results indicate cilostazol stimulates beclin-1, Atg5 and SIRT1 expression even under Aβ-enriched conditions, and that cilostazol inhibits intracellular Aβ accumulation by elevating autophagy.


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—C. Inhibition of Tet- condition-induced reductions in beclin-1 (A), Atg5 (B) and SIRT1 expressions (C) by cilostazol (10 or 30 μM) in N2aSwe cells. D. Inhibition of Tet- condition-induced endogenous increases in Aβ level by cilostazol as determined by Western blotting (D) and of intracellular Aβ accumulation as determined by ELISA (E, F). The inhibitory effects of cilostazol were blocked by KT5720 (1 μM), sirtinol (20 μM) (E), bafilomycin A1 (BAF, 100 ng/ml), or 3-methyladenine (3-MA, 2.5 mM) (F).Results are presented as means ± SDs (N = 4–5). ##P < 0.01, ###P < 0.001 vs. Tet+ condition (as control), **P < 0.01 ***P < 0.001 vs. DMSO; †P < 0.05, †††P < 0.001 vs. cilostazol alone (CSZ, 10 μM). PBS, phosphate-buffered saline.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526537&req=5

pone.0134486.g004: A—C. Inhibition of Tet- condition-induced reductions in beclin-1 (A), Atg5 (B) and SIRT1 expressions (C) by cilostazol (10 or 30 μM) in N2aSwe cells. D. Inhibition of Tet- condition-induced endogenous increases in Aβ level by cilostazol as determined by Western blotting (D) and of intracellular Aβ accumulation as determined by ELISA (E, F). The inhibitory effects of cilostazol were blocked by KT5720 (1 μM), sirtinol (20 μM) (E), bafilomycin A1 (BAF, 100 ng/ml), or 3-methyladenine (3-MA, 2.5 mM) (F).Results are presented as means ± SDs (N = 4–5). ##P < 0.01, ###P < 0.001 vs. Tet+ condition (as control), **P < 0.01 ***P < 0.001 vs. DMSO; †P < 0.05, †††P < 0.001 vs. cilostazol alone (CSZ, 10 μM). PBS, phosphate-buffered saline.
Mentions: We further confirmed cilostazol significantly inhibited endogenously overproduced Aβin N2aSwe cells induced by using the model of Tet+ and Tet- conditions. As shown in Fig 4A, 4B and 4C, the protein expressions of beclin-1, Atg5, and SIRT1 were significantly reduced by 76.1 ± 6.8%, 85.3 ± 3.2% and 76.8 ± 6.4%, respectively (each, P < 0.01) when N2aSwe cells were exposed to Tet-. Interestingly, by pretreatment with cilostazol (10 or 30 μM), these attenuated levels of SIRT1, beclin-1, and Atg5 were largely overexpressed (10 μM cilostazol: by 155.1 ± 18.1%, 136.6 ± 11.7%, and 125.7 ± 8.0%, respectively). Intriguingly, increased Aβ level under Tet- condition (by 139.5 ± 9.8%, P < 0.001) was significantly inhibited by cilostazol pretreatment (10 or 30 μM) to 89.4 ± 2.4 (P < 0.001) and 75.5 ± 16.0% (P < 0.001), respectively (Fig 4D). These results were further evaluated by measuring Aβ levels by ELISA. When N2aSwe cells were exposed to Tet- condition, intracellular Aβ1–42 levels significantly increased by 362.2 ± 7.7 ng/ml (P < 0.001), and this increase was markedly reduced to 186.4 ± 10.9 ng/ml (P < 0.001) and 165.9 ± 10.9 ng/ml (P < 0.001) by pretreating cilostazol at 10 and 30 μM, respectively. Furthermore, this cilostazol-induced inhibition was significantly blocked by pretreatment with KT5720 (1 μM, a cAMP-dependent protein kinase inhibitor) or sirtinol (20 μM, a SIRT1 inhibitor) (Fig 4E). In addition, cilostazol-induced inhibition of Aβ1–42 level was also blocked by bafilomycin A1 (100 nM, P < 0.001) or 3-methyladenine (2.5 mM, P < 0.05), respectively (Fig 4F). Overall, these results indicate cilostazol stimulates beclin-1, Atg5 and SIRT1 expression even under Aβ-enriched conditions, and that cilostazol inhibits intracellular Aβ accumulation by elevating autophagy.

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