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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

Effect of cilostazol on Aβ-induced cytotoxicity.Decrease in cell viability in response to exogenous Aβ1–42 in N2a cells (A) and to endogenously overproduced Aβ in the N2aSwe cells (B), and the recovery by cilostazol in the absence and presence of 3-methyladenine (3-MA, 2.5 mM). Results are the means ± SDs of three experiments. ###P < 0.001 vs. PBS (A) and Tet- condition (B); ***P < 0.001 vs. DMSO; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. 10 or 30 μM cilostazol alone. PBS, phosphate-buffered saline. C. Hypothetical model: Neuroprotective effect of cilostazol against Aβ-induced neurotoxicity is ascribable to the increased induction of autophagy by increasing the cAMP/PKA coupled SIRT1 activation, thereby enhances Aβ and CTFβclearance and increases cell viability.
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pone.0134486.g007: Effect of cilostazol on Aβ-induced cytotoxicity.Decrease in cell viability in response to exogenous Aβ1–42 in N2a cells (A) and to endogenously overproduced Aβ in the N2aSwe cells (B), and the recovery by cilostazol in the absence and presence of 3-methyladenine (3-MA, 2.5 mM). Results are the means ± SDs of three experiments. ###P < 0.001 vs. PBS (A) and Tet- condition (B); ***P < 0.001 vs. DMSO; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. 10 or 30 μM cilostazol alone. PBS, phosphate-buffered saline. C. Hypothetical model: Neuroprotective effect of cilostazol against Aβ-induced neurotoxicity is ascribable to the increased induction of autophagy by increasing the cAMP/PKA coupled SIRT1 activation, thereby enhances Aβ and CTFβclearance and increases cell viability.

Mentions: The cytotoxic effects of exogenously applied Aβ1–42 in N2a cells and of endogenously released Aβ in N2aSwe cells were assessed using an MTT assay. Exposure of N2a cells to 10 μM of Aβ1–42 for 24 h resulted in a significant decline in cell viability by 51.6 ± 2.7% (P < 0.001). The decreased viability induced by Aβ1–42 was largely recovered by 10 or 30 μM of cilostazol to 81.7 ± 2.6% (P < 0.001) and 88.0 ± 3.7% (P < 0.001), respectively. Furthermore, this effect of cilostazol was significantly blocked by pretreating cells with 3-methyladenine (2.5 mM, a chemical inhibitor of autophagy) (Fig 7A).


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)

Effect of cilostazol on Aβ-induced cytotoxicity.Decrease in cell viability in response to exogenous Aβ1–42 in N2a cells (A) and to endogenously overproduced Aβ in the N2aSwe cells (B), and the recovery by cilostazol in the absence and presence of 3-methyladenine (3-MA, 2.5 mM). Results are the means ± SDs of three experiments. ###P < 0.001 vs. PBS (A) and Tet- condition (B); ***P < 0.001 vs. DMSO; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. 10 or 30 μM cilostazol alone. PBS, phosphate-buffered saline. C. Hypothetical model: Neuroprotective effect of cilostazol against Aβ-induced neurotoxicity is ascribable to the increased induction of autophagy by increasing the cAMP/PKA coupled SIRT1 activation, thereby enhances Aβ and CTFβclearance and increases cell viability.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134486.g007: Effect of cilostazol on Aβ-induced cytotoxicity.Decrease in cell viability in response to exogenous Aβ1–42 in N2a cells (A) and to endogenously overproduced Aβ in the N2aSwe cells (B), and the recovery by cilostazol in the absence and presence of 3-methyladenine (3-MA, 2.5 mM). Results are the means ± SDs of three experiments. ###P < 0.001 vs. PBS (A) and Tet- condition (B); ***P < 0.001 vs. DMSO; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. 10 or 30 μM cilostazol alone. PBS, phosphate-buffered saline. C. Hypothetical model: Neuroprotective effect of cilostazol against Aβ-induced neurotoxicity is ascribable to the increased induction of autophagy by increasing the cAMP/PKA coupled SIRT1 activation, thereby enhances Aβ and CTFβclearance and increases cell viability.
Mentions: The cytotoxic effects of exogenously applied Aβ1–42 in N2a cells and of endogenously released Aβ in N2aSwe cells were assessed using an MTT assay. Exposure of N2a cells to 10 μM of Aβ1–42 for 24 h resulted in a significant decline in cell viability by 51.6 ± 2.7% (P < 0.001). The decreased viability induced by Aβ1–42 was largely recovered by 10 or 30 μM of cilostazol to 81.7 ± 2.6% (P < 0.001) and 88.0 ± 3.7% (P < 0.001), respectively. Furthermore, this effect of cilostazol was significantly blocked by pretreating cells with 3-methyladenine (2.5 mM, a chemical inhibitor of autophagy) (Fig 7A).

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