Activation of the Nrf2 signaling pathway and neuroprotection of nigral dopaminergic neurons by a novel synthetic compound KMS99220.

The transcription factor Nrf2 is known to induce gene expression of antioxidant enzymes and proteasome subunits. Because both oxidative stress and protein aggregation have damaging effects on neurons, activation of the Nrf2 signaling should be beneficial against neurodegeneration. In this study, we report a novel synthetic morpholine-containing chalcone KMS99220 that confers neuroprotection. It showed high binding affinity to the Nrf2 inhibitory protein Keap-1 and increased nuclear translocation of Nrf2 and gene expression of the antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and the catalytic and modifier subunits of glutamate-cysteine ligase in dopaminergic CATH.a cells. KMS99220 also increased expression of the proteasome subunits PSMB5, PSMB7, PSMB8 and PSMA1, and the respective chymotrypsin and trypsin-like proteasomal enzyme activities, and reduced α-synuclein aggregate in GFP-α-syn A53T-overexpressing cells. KMS99220 exhibited a favorable pharmacokinetic profile with excellent bioavailability and metabolic stability, did not interfere with activities of the cytochrome p450 isotypes, and showed no apparent in vivo toxicity when administered up to 2000 mg/kg. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 prevented degeneration of the nigral dopaminergic neurons, induced the Nrf2 target genes, and effectively prevented the associated motor deficits. These results suggest KMS99220 as a potential candidate for therapy against Parkinson's disease.

Potential repositioning of exemestane as a neuroprotective agent for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by selective degeneration of the nigral dopaminergic neurons, and neuroinflammation and oxidative stress are believed to be involved in its pathogenesis. In the present study, we provide data that the synthetic steroid exemestane, which is currently being used to treat breast cancer, may be useful for PD therapy. In BV-2 microglial cells, exemestane activated the transcription factor Nrf2 and induced expression of the Nrf2-dependent genes that encode the antioxidant enzymes NAD(P)H: quinone oxidoreductase 1, haem oxygenase-1, and glutamylcysteine ligase. It also downregulated gene expression of inducible nitric oxide (NO) synthase, lowered the levels of NO and reactive oxygen species, interleukin-1ß and tumour necrosis factor-α in lipopolysaccharide-activated microglial cells. In CATH.a dopaminergic neuronal cells, exemestane also induced the same set of Nrf2-dependent antioxidant enzyme genes and provided neuroprotection against oxidative damage. In vivo, the drug protected the nigral dopaminergic neurons, decreased microglial activation, and prevented motor deficits in C57Bl/6 male mice that had been administered with the dopaminergic neurotoxin MPTP. Taken together, the results suggested a utility of repositioning exemestane towards disease-modifying therapy for PD.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc.

Potential of Pseudoshikonin I Isolated from Lithospermi Radix as Inhibitors of MMPs in IL-1ß-Induced SW1353 Cells.

Pseudoshikonin I, the new bioactive constituent of Lithospermi radix, was isolated from this methanol extract by employing reverse-phase medium-pressure liquid chromatography (MPLC) using acetonitrile/water solvent system as eluents. The chemical structure was determined based on spectroscopic techniques, including 1D NMR (¹H, (13)C, DEPT), 2D NMR (gCOSY, gHMBC, gHMQC), and QTOF/MS data. In this study, we demonstrated the effect of pseudoshikonin I on matrix-metalloproteinase (MMPs) activation and expression in interleukin (IL)-1ß-induced SW1353 chondrosarcoma cells. MMPs are considered important for the maintenance of the extracellular matrix. Following treatment with PS, active MMP-1, -2, -3, -9, -13 and TIMP-2 were quantified in the SW1353 cell culture supernatants using a commercially available ELISA kit. The mRNA expression of MMPs in SW1353 cells was measured by RT-PCR. Pseudoshikonin I treatment effectively protected the activation on all tested MMPs in a dose-dependent manner. TIMP-2 mRNA expression was significantly upregulated by pseudoshikonin I treatment. Overall, we elucidated the inhibitory effect of pseudoshikonin on MMPs, and we suggest its use as a potential novel anti-osteoarthritis agent.

Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells.

UNLABELLED: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD-hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1-TRADD complex. Thus, this study identified a novel mechanism through which the hMYH-hTRADD interaction may affect the TNF-α signaling pathway. IMPLICATIONS: In HeLa cells, the hTRADD-hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

A novel compound VSC2 has anti-inflammatory and antioxidant properties in microglia and in Parkinson's disease animal model.

BACKGROUND AND PURPOSE: Neuroinflammation through microglial activation is involved in the pathogenesis of neurodegenerative diseases including Parkinson's disease (PD), a major neurodegenerative disorder characterized by dopaminergic neuronal death in the substantia nigra. We examined our novel synthetic compound VSC2 for its anti-inflammatory properties towards development of a PD therapy. EXPERIMENTAL APPROACH: We tested the effects of VSC2 on production of various NF-κB-dependent proinflammatory molecules and Nrf2-dependent antioxidant enzymes in BV-2 microglia and in vivo. KEY RESULTS: The vinyl sulfone compound, VSC2, most effectively suppressed the production of NO in LPS-activated microglia. It also down-regulated expression of inducible NOS (iNOS), COX-2, IL-1ß and TNF-α and inhibited nuclear translocalization and transcriptional activity of NF-κB. VSC2 increased total and nuclear Nrf2 levels, induced Nrf2 transcriptional activity and was bound to Keap1 with high affinity. Expression of the Nrf2-regulated antioxidant enzyme genes NAD(P)H quinone oxidoreducase-1 (NQO-1), haem oxygenase-1 (HO-1) and glutamylcysteine ligase (GCL) were up-regulated by VSC2. In the MPTP mouse model of PD, oral administration of VSC2 decreased the number of activated microglia in the substantia nigra, lowered the levels of iNOS, COX-2 and IL-1ß, and protected the dopaminergic neurons. VSC2 also elevated the levels of NQO1, HO-1, GCL and Nrf2 in the nigrostriatal area. CONCLUSIONS AND IMPLICATIONS: VSC2 has both anti-inflammatory and antioxidant properties and prevented neuroinflammation in microglia and in an animal model of PD. This suggests VSC2 as a potential candidate for PD therapy.

Human MutY homolog induces apoptosis in etoposide-treated HEK293 cells.

Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models.

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-alpha, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-alpha, IL-6, and IL-8 release from mast cells.