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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. Analysis of the effects of SIRT1-knockdown in N2aSwe cells. Proteins (30 μg) from a negative control and SIRT1- knockdown samples were loaded onto 10 ~ 15% SDS-polyacrylamide gels. In N2aSwe cells transfected with 200 nM of SIRT1 siRNA, SIRT1 protein levels were at ~ 40% of the level in negative controls (A). Cilostazol failed to elevate the expressions of SIRT1 (B), beclin1 (C), Atg5 (D), and (E) LC3 in SIRT1-siRNA treated N2a cells, as contrasted to the levels of all four in the negative control cells.Results are the means ± SDs of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO. ††P < 0.01 vs. cilostazol alone (CSZ, 10 μM).
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pone.0134486.g005: A. Analysis of the effects of SIRT1-knockdown in N2aSwe cells. Proteins (30 μg) from a negative control and SIRT1- knockdown samples were loaded onto 10 ~ 15% SDS-polyacrylamide gels. In N2aSwe cells transfected with 200 nM of SIRT1 siRNA, SIRT1 protein levels were at ~ 40% of the level in negative controls (A). Cilostazol failed to elevate the expressions of SIRT1 (B), beclin1 (C), Atg5 (D), and (E) LC3 in SIRT1-siRNA treated N2a cells, as contrasted to the levels of all four in the negative control cells.Results are the means ± SDs of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO. ††P < 0.01 vs. cilostazol alone (CSZ, 10 μM).

Mentions: To confirm cilostazol-induced increases in beclin-1, Atg5, and LC3-II levels are mediated via SIRT1 activation, N2aSwe cells were transfected with SIRT1 siRNA or scrambled siRNA duplex (negative control) (both at 100 or 200 nM). In N2aSwe cells subjected to SIRT1-gene silencing, SIRT1 protein expression was reduced to ~ 40% by 200 nM of SIRT1 siRNA oligonucleotide (Fig 5A). In SIRT1- silenced cells, cilostazol failed to elevate SIRT1 protein expression, whereas in cells transfected with scrambled siRNA duplex (negative control), cilostazol at 10 or 30 μM significantly elevated the expression of SIRT1 to 159.2 ± 11.2% and 139.8 ± 13.2%, respectively (Fig 5B). Similarly, beclin-1, Atg5, and LC3-II levels were not increased by cilostazol (10 or 30 μM) in cells transfected with SIRT1 siRNA, whereas levels of these proteins were increased in cells transfected with scrambled siRNA duplex (negative control) (Fig 5C, 5D and 5E). These results show cilostazol increases the expressions of beclin-1, Atg5, and LC3-II by up-regulating SIRT1.


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. Analysis of the effects of SIRT1-knockdown in N2aSwe cells. Proteins (30 μg) from a negative control and SIRT1- knockdown samples were loaded onto 10 ~ 15% SDS-polyacrylamide gels. In N2aSwe cells transfected with 200 nM of SIRT1 siRNA, SIRT1 protein levels were at ~ 40% of the level in negative controls (A). Cilostazol failed to elevate the expressions of SIRT1 (B), beclin1 (C), Atg5 (D), and (E) LC3 in SIRT1-siRNA treated N2a cells, as contrasted to the levels of all four in the negative control cells.Results are the means ± SDs of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO. ††P < 0.01 vs. cilostazol alone (CSZ, 10 μM).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526537&req=5

pone.0134486.g005: A. Analysis of the effects of SIRT1-knockdown in N2aSwe cells. Proteins (30 μg) from a negative control and SIRT1- knockdown samples were loaded onto 10 ~ 15% SDS-polyacrylamide gels. In N2aSwe cells transfected with 200 nM of SIRT1 siRNA, SIRT1 protein levels were at ~ 40% of the level in negative controls (A). Cilostazol failed to elevate the expressions of SIRT1 (B), beclin1 (C), Atg5 (D), and (E) LC3 in SIRT1-siRNA treated N2a cells, as contrasted to the levels of all four in the negative control cells.Results are the means ± SDs of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO. ††P < 0.01 vs. cilostazol alone (CSZ, 10 μM).
Mentions: To confirm cilostazol-induced increases in beclin-1, Atg5, and LC3-II levels are mediated via SIRT1 activation, N2aSwe cells were transfected with SIRT1 siRNA or scrambled siRNA duplex (negative control) (both at 100 or 200 nM). In N2aSwe cells subjected to SIRT1-gene silencing, SIRT1 protein expression was reduced to ~ 40% by 200 nM of SIRT1 siRNA oligonucleotide (Fig 5A). In SIRT1- silenced cells, cilostazol failed to elevate SIRT1 protein expression, whereas in cells transfected with scrambled siRNA duplex (negative control), cilostazol at 10 or 30 μM significantly elevated the expression of SIRT1 to 159.2 ± 11.2% and 139.8 ± 13.2%, respectively (Fig 5B). Similarly, beclin-1, Atg5, and LC3-II levels were not increased by cilostazol (10 or 30 μM) in cells transfected with SIRT1 siRNA, whereas levels of these proteins were increased in cells transfected with scrambled siRNA duplex (negative control) (Fig 5C, 5D and 5E). These results show cilostazol increases the expressions of beclin-1, Atg5, and LC3-II by up-regulating SIRT1.

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