Sang-Youel Park

Autophagy induced by resveratrol prevents human prion protein-mediated neurotoxicity.

Our previous study revealed that resveratrol blocks prion protein peptide PrP(106-126)-induced neurotoxicity. However, the mechanism of resveratrol-mediated neuroprotection in prion diseases is not clear. Resveratrol initiates neuroprotective effects via the activation of autophagy, which protects organelles, cells, and organisms against misfolded protein-disorders, including Alzheimers disease and Parkinsons disease via regulation of mitochondrial homeostasis. Thus, we focused on elucidating the mechanisms responsible for resveratrol-mediated neuroprotection related to mitochondrial homeostasis as a result of autophagy activation. Resveratrol prevented PrP(106-126)-induced neuronal cell death by activating autophagy. Moreover, resveratrol-induced autophagy prevented the PrP(106-126)-induced reduction in mitochondrial potential and translocation of Bax to the mitochondria and cytochrome c release. Our results indicate that treatment with resveratrol appears to protect against neurotoxicity caused by prion protein peptides and the neuroprotection is induced by resveratrol-mediated autophagy signals.

Autophagy induced by the class III histone deacetylase Sirt1 prevents prion peptide neurotoxicity

Sirtuin 1 (Sirt1) is a class III histone deacetylase that mediates the protective effects of neurons in neurodegenerative disorders, including Alzheimers and prion disease. However, the mechanism directly involved in neuroprotection is still poorly understood. Recent evidence has demonstrated that activating Sirt1 induces autophagy, and that activating autophagy protects neurons against neurodegenerative disorders by regulating mitochondrial homeostasis. Thus, we focused on the mechanism of the Sirt1-mediated neuroprotective effect that was associated with regulating mitochondrial homeostasis via autophagy. Adenoviral-mediated Sirt1 overexpression prevented prion protein (PrP)(106-126)-induced neurotoxicity via autophagy processing. Moreover, Sirt1-induced autophagy protected against the PrP(106-126)-mediated decrease in the mitochondrial membrane potential value. Additionally, Sirt1 overexpression decreased PrP(106-126)-induced Bax translocation to the mitochondria and cytochrome c release into the cytosol. Sirt1 knockdown using small interfering (si) RNAs induced downregulation of Sirt1 protein expression and sensitized neuron cells to PrP(106-126)-induced cell death and mitochondrial dysfunction. Knockdown of autophagy-related 5 (ATG5) using small interfering RNA decreased autophagy-related 5 and autophagy marker microtubule-associated protein 1 light chain 3-II protein levels and blocked the effect of a Sirt1 activator against PrP(106-126)-induced mitochondrial dysfunction and neurotoxicity. Taken together, this study is the first report demonstrating that autophagy induced by Sirt1 activation plays a pivotal role protecting against prion-induced neuron cell death and also suggests that regulating autophagy including which by Sirt1 activation may be a therapeutic target for neurodegenerative disorders including the prion disease.

SIRT1, a class III histone deacetylase, regulates TNF-α-induced inflammation in human chondrocytes

OBJECTIVE The present study was performed to elucidate the possible role of SIRT1 signaling in joint inflammation in human articular chondrocytes. DESIGN Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect gene products and proteins involved in tumor necrosis factor α (TNF-α)-induced inflammation and cartilage degradation in human primary chondrocytes. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated by gelatin zymography. Overexpression and knockdown of SIRT1 were also performed to investigate whether SIRT1 is associated with the anti-inflammatory activity of resveratrol in chondrocytes. RESULTS Resveratrol dose-dependently inhibited TNF-α-induced cyclooxygenase-2 (COX-2), MMP-1, MMP-3, MMP-13 and PGE(2) production in human chondrocytes. Moreover, MMP-2 and MMP-9 activity was increased by treatment with TNF-α; however, SIRT1 activation decreased the proinflammatory effects induced by TNF-α. In addition, treatment of SIRT1 activator and overexpression of SIRT1 inhibited the expression and activation of the main proinflammatory regulator NF-κB, which was increased by TNF-α. When SIRT1 was overexpressed in chondrocytes, the anti-inflammatory action of SIRT1 was similar to that exerted by resveratrol. CONCLUSIONS SIRT1 activation deacetylates and inactivates NF-κB, and thereby, exerts an anti-inflammatory effect on chondrocytes, suggesting that SIRT1 activators could be explored as potential treatments for arthritis.

SIRT1, a histone deacetylase, regulates prion protein-induced neuronal cell death

Prion diseases associated with the conversion of the cellular prion protein (PrP(C)) to the misfolded isoform (PrP(Sc)), affect the central nervous system (CNS) of humans and animals. Resveratrol, an activator of class III histone deacetylase SIRT1, is important in attenuating cellular injury and oxidative stress. The present study investigated the effects of SIRT1 activation on prion protein-mediated neuronal cell death and examined its possible signals in intracellular apoptotic pathways. Resveratrol treatment significantly increased both SIRT1 protein expression and SIRT1 activity and protected neuronal cells against PrP (106-126)-induced cell death. Resveratrol-mediated SIRT1 activation decreased the acetylation of p53 and p65 induced by prion protein and SIRT1 inhibitor. SIRT1 activation also inhibited PrP (106-126)-mediated p38 mitogen-activating protein kinase (MAPK) activation and caspase-3 cleavage, and increased the expression of anti-apoptotic Bcl-xL protein. Furthermore, SIRT1 overexpression by using adenoviral vector protected neuronal cells against PrP (106-126). These results indicate that resveratrol inhibits PrP (106-126)-induced neuronal cell death by regulating SIRT1 activity and SIRT-related signaling, and suggest that prion-related disease may be attenuated by SIRT1 activation or by intake of SIRT1-activating molecules.

Melatonin‐induced autophagy protects against human prion protein‐mediated neurotoxicity

Abstract: Melatonin has neuroprotective effects in the models of neurodegenerative disease including Alzheimer’s and Parkinson’s disease. Several studies have shown that melatonin prevents neurodegeneration by regulation of mitochondrial function. However, the protective action of melatonin has not been reported in prion disease. We investigated the influence of melatonin on prion‐mediated neurotoxicity. Melatonin rescued neuronal cells from PrP(106–126)‐induced neurotoxicity by prevention of mitochondrial dysfunction. Moreover, the protective effect of melatonin against mitochondrial dysfunction was related with autophagy activation. Melatonin‐treated cells were dose‐dependently increased in LC3‐II, an autophagy marker. Melatonin‐induced autophagy prevented a PrP(106–126)‐induced reduction in mitochondrial potential and translocation of Bax to the mitochondria and cytochrome c release. On the other hand, downregulation of autophagy protein 5 with Atg5 siRNA or the autophagy blocker 3‐methyladenine prevented the melatonin‐mediated neuroprotective effects. This is the first report demonstrating that treatment with melatonin appears to protect against prion‐mediated neurotoxicity and that the neuroprotection is induced by melatonin‐mediated autophagy signals. The results of this study suggest that regulation of melatonin is a therapeutic strategy for prion peptide‐induced apoptosis.

Endogenous MMP-9 and not MMP-2 promotes rheumatoid synovial fibroblast survival, inflammation and cartilage degradation

OBJECTIVE The aim of this study was to investigate the effect of endogenous matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) on the invasive characteristics of RA synovial fibroblasts. METHODS Synovial fibroblasts isolated from patients with RA or OA were treated with MMP small interfering RNA (siRNA), inhibitors and recombinant proteins or TNF-α, with or without cartilage explants. Cell viability and proliferation were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and 5-bromo-2-deoxyuridine (BrdU) proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelect invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; and inflammatory mediators and MMPs by ELISA, western blot and zymography. RESULTS MMP-2 was expressed by both OA and RA synovial fibroblasts, whereas only RA synovial fibroblasts expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RA synovial fibroblast proliferation equally. However, MMP-9 siRNA had greater effects compared with MMP-2 siRNA on promoting apoptosis and suppressing RA synovial fibroblast viability, migration and invasion. Suppression/inhibition of MMP-9 also decreased the production of IL-1β, IL-6, IL-8 and TNF-α, inactivated nuclear factor κB (NF-κB), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and suppressed RA synovial fibroblast-mediated cartilage degradation. In contrast, suppression/inhibition of MMP-2 stimulated TNF-α and IL-17 secretion and activated NF-κB, while recombinant MMP-2 (rMMP-2) inactivated NF-κB and suppressed RA synovial fibroblast-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results. CONCLUSION Endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RA synovial fibroblasts may directly contribute to joint destruction in RA.

Hypoxia-inducible factor-1 alpha regulates prion protein expression to protect against neuron cell damage

The human prion protein fragment, PrP (106-126), may contain a majority of the pathological features associated with the infectious scrapie isoform of PrP, known as PrP(Sc). Based on our previous findings that hypoxia protects neuronal cells from PrP (106-126)-induced apoptosis and increases cellular prion protein (PrP(C)) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate PrP(C) expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes. Under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by PrP (106-126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of PrP(C) mRNA and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In PrP(C) knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length Prnp gene (Ad-Prnp) were utilized for overexpression of the Prnp gene in PrP(C) knockout hippocampal neuron cells. Adenoviral transfection of PrP(C) knockout cells with Prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal PrP(C) is regulated by HIF-1α, and PrP(C) overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases.

Sulforaphane induced adipolysis via hormone sensitive lipase activation, regulated by AMPK signaling pathway

Sulforaphane, an aliphatic isothiocyanate derived from cruciferous vegetables, is known for its antidiabetic properties. The effects of sulforaphane on lipid metabolism in adipocytes are not clearly understood. Here, we investigated whether sulforaphane stimulates lipolysis. Mature adipocytes were incubated with sulforaphane for 24h and analyzed using a lipolysis assay which quantified glycerol released into the medium. We investigated gene expression of hormone-sensitive lipase (HSL), and levels of HSL phosphorylation and AMP-activated protein kinase on sulforaphane-mediated lipolysis in adipocytes. Sulforaphane promoted lipolysis and increased both HSL gene expression and HSL activation. Sulforaphane suppressed AMPK phosphorylation at Thr-172 in a dose-dependent manner, which was associated with a decrease in HSL phosphorylation at Ser-565, enhancing the phosphorylation of HSL Ser-563. Taken together, these results suggest that sulforaphane promotes lipolysis via hormone sensitive lipase activation mediated by decreasing AMPK signal activation in adipocytes.

Hypoxia protects neuronal cells from human prion protein fragment-induced apoptosis.

J. Neurochem. (2010) 112, 715–722.

Preparation, characterization, in-vitro drug release and cellular uptake of poly(caprolactone) grafted dextran copolymeric nanoparticles loaded with anticancer drug

Biodegradable and biocompatible polymers that are engineered to nanostructures play a key role in providing solution for sustained chemotherapy. This study is focused on preparation, drug encapsulation efficiency, in-vitro drug release, in-vitro cellular uptake and cell viability of poly(caprolactone) grafted dextran (PGD) nanoparticles (NPs) formulation containing vinblastine as the anticancer drug. Drug-loaded PGD NPs were prepared by a modified oil/water emulsion method and characterized by laser light scattering, atomic force microscopy (AFM), and zeta potential. The drug encapsulation efficiency was determined spectrophotometrically and in-vitro drug release was estimated using dialysis bag. Breast cancer cell line (MCF-7) was used to image and measure the cellular uptake of fluorescent PGD NPs. Cancer cell viability was assessed by treating MCF-7 cells with vinblastine-loaded PGD NPs by crystal violet staining method. Result showed that the vinblastine-loaded PGD NPs were superior in properties such as drug encapsulation efficiency, the cellular uptake and the cancer cell mortality.