T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS-induced murine macrophage cells.

Mycobacterium tuberoculosis (Mtb) is a contagious pathogen that causes human tuberculosis (TB). TB is a major global health threat that causes 9.6 million illnesses and 1.5 million deaths per year. Recent studies have suggested Mtb-secreted proteins as new candidates for therapeutic drugs and vaccines. LprG is a Mtb-secreted surface glycolipoprotein encoded by lprG (Rv1411c), which forms an operon with Rv1410c, where Rv1410c encodes P55, an efflux pump membrane protein. Various in vitro and in vivo studies have reported on the target-binding activity, cell envelope biosynthesis, and mycobacterial virulence of LprG. However, the anti-inflammatory effect of LprG in macrophages has not yet been investigated. In this study, we demonstrated that LprG can suppress lipopolysaccharide (LPS)-induced inflammation in a macrophage model. LprG inhibited LPS-stimulated nitric oxide (NO) production. LprG also suppressed expression of inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) at the transcriptional and protein levels. In addition, LprG decreased mRNA expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, LprG attenuated nuclear factor kappa-B (NF-κB) translocation and IκB phosphorylation. Moreover, LprG specifically inhibited phosphorylated kinases such as c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinase 1/2 (p-ERK1/2), but not p-p38. Taken together, these results suggest that LprG inhibits LPS-stimulated inflammation via downregulation of NO, COX-2, iNOS, and pro-inflammatory cytokines through the NF-κB, AP-1, and MAPK signaling pathways. The present study will aid in the development of anti-inflammatory medications using Mtb. The organism, which has long been regarded as a human pathogenic or human health-threating agent, can be utilized as a future medical resource.

Inhibitory Effect of Avenanthramides (Avn) on Tyrosinase Activity and Melanogenesis in α-MSH-Activated SK-MEL-2 Cells: In Vitro and In Silico Analysis.

Melanin causes melasma, freckles, age spots, and chloasma. Anti-melanogenic agents can prevent disease-related hyperpigmentation. In the present study, the dose-dependent tyrosinase inhibitory activity of Avenanthramide (Avn)-A-B-C was demonstrated, and 100 µM Avn-A-B-C produced the strongest competitive inhibition against inter-cellular tyrosinase and melanin synthesis. Avn-A-B-C inhibits the expression of melanogenesis-related proteins, such as TRP1 and 2. Molecular docking simulation revealed that AvnC (-7.6 kcal/mol) had a higher binding affinity for tyrosinase than AvnA (-7.3 kcal/mol) and AvnB (-6.8 kcal/mol). AvnC was predicted to interact with tyrosinase through two hydrogen bonds at Ser360 (distance: 2.7 Å) and Asn364 (distance: 2.6 Å). In addition, AvnB and AvnC were predicted to be skin non-sensitizers in mammals by the Derek Nexus Quantitative Structure-Activity Relationship system.

Avenanthramide C Suppresses Matrix Metalloproteinase-9 Expression and Migration Through the MAPK/NF- κB Signaling Pathway in TNF-α-Activated HASMC Cells.

In oat ingredients, flavonoids and phenolic acids are known to be the most important phenolic compounds. In phenolic compounds, wide-ranging biological responses, including antioxidative, anti-inflammatory, anti-allergic, and anti-cancer properties, were reported. Avenanthramide C (Avn C), a component of the phenolic compound of oats, has been reported to be highly antioxidant and anti-inflammatory, but its role in an anti-atherosclerosis response is unknown. The aim of this research was to assess the effect of Avn C on expression of MMP-9 on TNF-α-activated human arterial smooth-muscle cells (HASMC) and signaling involved in its anti-atherosclerosis activity. HASMC cells are known to produce inflammatory cytokines involving IL-6, IL-1ß, and TNF-α during arteriosclerosis activity. Avn C specifically reduced IL-6 secretion in HASMC cells. Furthermore, we investigated whether Avn C could inhibit NF-κB nuclear protein translocation. Avn C suppressed nuclear protein translocation of NF-κB in TNF-α-stimulated HASMCs. The MMP-9 enzyme activity and expression are controlled through the MAPKs signaling path during the Avn C treatment. We confirmed that the levels of wound healing (p-value = 0.013, *p < 0.05) and migration (p-value = 0.007, **p < 0.01) are inhibited by 100 ng/ml TNF-α and 100 µM Avn C co-treated. Accordingly, Avn C inhibited the expression of MMP-9 and cell migration through the MAPK/NF-κB signaling pathway in TNF-α-activated HASMC. Therefore, Avn C can be identified and serve as disease prevention material and remedy for atherosclerosis.

4-O-methylascochlorin attenuates inflammatory responses induced by lipopolysaccharide in RAW 264.7 macrophages.

Inflammation is implicated in various diseases, such as inflammatory bowel disease and cancer. Ascochlorin (ASC) and its derivatives have been shown to modulate inflammatory responses in many previous studies. However, the effects of 4-O-methylascochlorin (MAC), one of the ASC derivatives, on inflammatory responses have yet to be reported. In addition, the consequences of chemical modification of ASC on protein signaling and immunity have yet to be fully understood. The fourth carbon in MAC is methylated, which may result in modulation of immune response differently compared with ASC. Hence, we have investigated the role of MAC in inflammatory response induced by lipopolysaccharide in murine macrophage cells. Here, we found that MAC treatment decreased the inflammatory response by murine macrophages. When murine macrophages were treated with MAC, the transcription and translation of various pro-inflammatory indicators such as iNOS and COX-2 decreased. In addition, the ELISA results showed that the expression of TNF-α, IL-6, and IL-1ß, which are pro-inflammatory cytokines, was successfully decreased by MAC. Such effects of MAC appear to be mediated via downregulation of MAPK signaling and the transactivational activity of NF-κB. Lipopolysaccharide upregulates MAPK protein phosphorylation and NF-κB translocation, which in turn enhances the transactivation of genes related to NF-κB. Such results of lipopolysaccharide were attenuated by MAC. Collectively, our results indicate that MAC alleviated the inflammatory responses induced by lipopolysaccharide in murine macrophages successfully by modulating MAPK signaling pathway and NF-κB-related genes. This study shows that MAC, similar to other ASC derivatives, can potentially be used therapeutically to reduce the harmful damage induced by prolonged inflammation. In addition, the structural differences between ASC and its derivatives as well as their effect on intracellular signaling will also be discussed.

4-O-Carboxymethylascochlorin Inhibits Expression Levels of on Inflammation-Related Cytokines and Matrix Metalloproteinase-9 Through NF-κB/MAPK/TLR4 Signaling Pathway in LPS-Activated RAW264.7 Cells.

Toll-like receptor 4 (TLR4) and matrix metalloproteinase-9 (MMP-9) are known to play important roles in inflammatory diseases such as arteriosclerosis and plaque instability. The purpose of this study was to perform the effect of 4-O-carboxymethylascochlorin (AS-6) on MMP-9 expression in lipopolysaccharide (LPS)-induced murine macrophages and signaling pathway involved in its anti-inflammatory effect. Effect of AS-6 on MAPK/NF-κB/TLR4 signaling pathway in LPS-activated murine macrophages was examined using ELISA, Western blotting, reverse transcription polymerase chain reaction (RT-PCR) and fluorescence immunoassay. MMP-9 enzyme activity was examined by gelatin zymography. AS-6 significantly suppressed MMP-9 and MAPK/NF-κB expression levels in LPS-stimulated murine macrophages. Expression levels of inducible nitric oxide synthase (iNOS), COX2, MMP-9, JNK, ERK, p38 phosphorylation, and NF-κB stimulated by LPS were also decreased by AS-6. Moreover, AS-6 suppressed TLR4 expression and dysregulated LPS-induced activators of transcription signaling pathway. The results of this study showed that AS-6 can inhibit LPS-stimulated inflammatory response by suppressing TLR4/MAPK/NF-κB signals, suggesting that AS-6 can be used to induce the stability of atherosclerotic plaque and prevent inflammatory diseases in an in vitro model.

4-O-carboxymethylascochlorin protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS-induced neuroinflammation and death by inhibiting MAPK, NF-κB, and Akt pathways.

In our previous studies, structurally similar compounds of ascochlorin and ascofuranone exhibited anti-inflammatory activity. Neural inflammation plays a significant role in the commence and advancement of neurodegenerative diseases. It is not known whether 4-O-carboxymethylascochlorin (AS-6) regulates the initial stage of inflammatory responses at the cellular level in BV2 microglia cells. We here investigated the anti-inflammatory effects of AS-6 treatment in microglia cells with the microglial protection in neurons. We found that the lipopolysaccharide (LPS)-stimulated production of nitric oxide, a main regulator of inflammation, is suppressed by AS-6 in BV2 microglial cells. In addition, AS-6 dose-dependently suppressed the increase in COX-2 protein and messenger RNA levels in LPS-stimulated BV2 cells. Moreover, AS-6 inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. At the intracellular level, AS-6 inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells. AS-6 negatively affected mitogen-activated protein kinases (MAPK) and Akt phosphorylation: Phosphorylated forms of ERK, JNK, p38, and Akt decreased. To check whether AS-6 protects against inflammatory inducer-mediated neurotoxicity, neuronal SH-SY5Y cells were coincubated with BV2 cells in conditioned medium. AS-6 exerted a neuroprotective effect by suppressing microglial activation by LPS or amyloid-ß peptide. AS-6 is a promising suppressor of inflammatory responses in LPS-induced BV2 cells by attenuating NF-κB and MAPKs signaling. AS-6 protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS-induced neuroinflammation and death via inhibiting MAPK, NF-κB, and Akt pathways.