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Background Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Methods Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. Results All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. Conclusions Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-β-d-glucopyranosyl-(1→4)-β-d-xylopyranosyl)-28-β-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Background Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. Objectives The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. Methods To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FceRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. Results Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FceRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FceRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FceRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FceRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. Conclusions Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2-/- or Sirt1-/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2-/- and Sirt1-/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FceRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2-/- or Sirt1-/- mice. In conclusion, Tan IIA suppresses FceRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases.

Background In mast cells, induction of HSP70 expression during antigen stimulation has not been reported. Methods Mouse bone marrow derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression, and signaling protein phosphorylation, were evaluated by immunoblotting. Results HSP70 expression is induced in BMMC at an early stage of IgE/Ag-dependent stimulation, some of which is released from the cells in a granule-associated form. Induction of HSP70 expression was also observed with an IgE/Ag-stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, since a neutralizing anti-HSP70 antibody mitigated these activities, and since exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of Linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag stimulated BMMC, were not phosphorylated in HSP70-stimulated BMMC, whereas lymphocyte-specific-protein tyrosine kinase (Lck) was phosphorylated. Conclusion FceRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT. This article is protected by copyright. All rights reserved.

Adherent-invasive E. coli colonization and Toll-like receptor (TLR) expression are increased in the gut of inflammatory bowel disease (IBD) patients. However, the underlying mechanism of such changes has not been determined. In the current study, it was examined whether gut serotonin (5-hydroxytryptamine, 5-HT) can induce adherent-invasive E. coli colonization and increase TLR expression. In a co-culture system, commensal E. coli strain (BW25113, BW) adhered minimally to colon epithelial cells, but this was significantly enhanced by 5-HT to the level of a pathogenic strain (EDL933). Without inducing bacterial virulence, such as, biofilm formation, 5-HT enhanced BW-induced signaling in colon epithelial cells, that is, NADPH oxidase (NOX)-dependent superoxide production, the up-regulations of IL-8, TLR2, TLR4, and ICAM-1, and the down-regulations of E-cadherin and claudin-2. In a manner commensurate with these gene modulations, BW induced an increase in NF-κB and a decrease in GATA reporter signals in colon epithelial cells. However, 5-HT-enhanced BW adhesion and colon epithelial responses were blocked by knock-down of NOX2, TLR2, or TLR4. In normal mice, 5-HT induced the invasion of BW into gut submucosa, and the observed molecular changes were similar to those observed in vitro, except for significant increases in TNFα and IL-1β, and resulted in death. In dextran sulfate sodium-induced colitis mice (an IBD disease model), in which colonic 5-HT levels were markedly elevated, BW administration induced death in along with large amount of BW invasion into colon submucosa, and time to death was negatively related to the amount of BW injected. Taken together, our results demonstrate that 5-HT induces the invasion of commensal E. coli into gut submucosa by amplifying commensal bacteria-induced epithelial signaling (superoxide production and the inductions of NOX2 and TLR2/TLR4). The authors suggest that these changes may constitute the molecular basis for the pathogenesis of IBD.

Delphinidin, a polyphenol that belongs to the group of anthocyanidins and is abundant in many pigmented fruits and vegetables, possesses important antioxidant, anti‑inflammatory, anti-mutagenic and anticancer properties. In the present study, we investigated the inhibitory effects of delphinidin on vascular endothelial growth factor (VEGF) expression, an important factor involved in angiogenesis and tumor progression, in A549 human lung cancer cells. Delphinidin inhibited CoCl2- and epidermal growth factor (EGF)-induced VEGF mRNA expression and VEGF protein production. Delphinidin also decreased CoCl2- and EGF-stimulated expression of hypoxia‑inducible factor (HIF)‑1α, which is a transcription factor of VEGF. Delphinidin suppressed CoCl2- and EGF-induced hypoxia‑response element (HRE) promoter activity, suggesting that the inhibitory effects of delphinidin on VEGF expression are caused by the suppression of the binding of HIF-1 to the HRE promoter. We also found that delphinidin specifically decreased the CoCl2- and EGF-induced HIF-1α protein expression by blocking the ERK and PI3K/Akt/mTOR/p70S6K signaling pathways, whereas the p38-mediated pathways were not involved. In animal models, EGF-induced new blood vessel formation was significantly inhibited by delphinidin. Therefore, our results indicate that delphinidin has a potentially new role in anti‑angiogenic action by inhibiting HIF-1α and VEGF expression.

Emodin, one of the major compounds in the herb Reynoutria elliptica, is known to maintain immunosuppressive, anti-allergic, anti-cancer, and anti-inflammatory effects. In this study, we assessed the possibility of using emodin to induce apoptosis in stimulated immune cells in vitro. After treatment with emodin and concanavalin A (Con A), we observed DNA damage-induced apoptosis in splenocytes. Moreover, treatment with emodin and Con A increased the number of apoptotic splenocytes compared with untreated controls. Emodin also diminished the size of CD45R/B220+ cells, CD19+CD69+ cells, and cDC populations. These results indicate that emodin-induced apoptosis was involved in attenuating the immune activity promoted by DNA damage and in decreasing the number of CD45R/B220+ B cells and CD19+CD69+ activating B cells. This demonstration of emodin inducing apoptosis of Con A-stimulated immune cells indicates its potential utility as a therapy for diseases caused by abnormally activated immune cells.

Stressosome assembly mediated by the STAS domain was analyzed based on the crystal structure of RsbS and the cryo-EM structure of the RsbS–RsbRA complex., The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA–RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA–RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

As a master regulator for metabolic and energy homeostasis, AMPK controls the activity of metabolic enzymes and transcription factors in response to cellular ATP status. AMPK has been thus recognized as a main target for the regulation of cellular energy metabolism. Here, we report that AMPK can be down-regulated by the cullin-RING ubiquitin E3 ligase 4A (CRL4A) with cereblon (CRBN). CRL4A interacted with AMPK holoenzymes and mediated AMPKα-specific polyubiquitination for its proteasomal degradation through non-K48 polyubiquitin linkages. In the ubiquitination system, CRBN was required for efficient polyubiquitination of AMPKα subunits. Consistently, polyubiquitination of AMPKα subunits was reduced by inhibitors of CRL4A-CRBN. Physiologic function of AMPK down-regulation by CRL4-CRBN was also confirmed using mouse bone marrow-derived mast cells (BMMCs). The inactivation of CRL4A-CRBN in BMMC increased AMPK stability and suppressed secretion of allergic mediators via AMPK activation followed by MAPK inhibition. In addition, CRBN knockout of BMMC also decreased allergic responses in mice. Our results suggest that the CRL4A-CRBN axis could be a target for the regulation of AMPK-dependent responses.-Kwon, E., Li, X., Deng, Y., Chang, H. W., Kim, D. Y. AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

Nur77 (NR4A1) plays an important role in various inflammatory responses. Nur77 is rapidly degraded in cells and its protein level is critically controlled. Although few E3 ligases regulating the Nur77 protein have been defined, the deubiquitinase (DUB) responsible for Nur77 stability has not been reported to date. We identified ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) as a DUB that stabilizes Nur77 by preventing its proteasomal degradation. We found that OTUB1 interacted with Nur77 to deubiquitinate it, thereby stabilizing Nur77 in an Asp88-dependent manner. This suggests that OTUB1 targets Nur77 for deubiquitination via a non-canonical mechanism. Functionally, OTUB1 inhibited TNFα-induced IL-6 production by promoting Nur77 protein stability. OTUB1 modulated the stability of Nur77 as a counterpart of tripartite motif 13 (Trim13). That is, OTUB1 reduced the ubiquitination and degradation of Nur77 potentiated by Trim13. In addition, this DUB also inhibited IL-6 production, which was further amplified by Trim13 in TNFα-induced responses. These findings suggest that OTUB1 is an important regulator of Nur77 stability and plays a role in controlling the inflammatory response.

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A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 μM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1β and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Fourteen compounds were isolated from the flowers of Inula japonica THUNB. (Asteraceae), including two new compounds, (1S,2S,4S,5S,8S,10R)-2-acetoxy-4,3-dihydroxy-pseudoguai-7(11)-en-12,8-olide (1) and (1S,2S,4S,5S,8S,10R)-2,4,13-trihydroxy-pseudoguai-7(11)-en-12,8-olide (2), and twelve known compounds, budlein B (3), 6β-hydroxytomentosin (4), 6-deacetoxybritanin (5), 4-epipulchellin (6), britanin (7), tomentosin (8), (+)-dihydroquercetin (9), (-)-syringaresinol (10), quercetagetin 3,4'-dimethyl ether (11), luteolin (12), britanin G (13) and inuchinenolide C (14). Structures of 1 and 2 were determined based on one and two dimensional (1D)- and (2D)-NMR data and Mosher's esterification method. Compounds 9 and 12 showed inhibitory activities toward DNA topoisomerase I with IC50 values of 55.7 and 37.0 µM, respectively, compared to camptothecin (CPT) with an IC50 of 24.5 µM. Compounds 7-9 and 11-14 exhibited more potent inhibitory activity against topoisomerases II with IC50 values of 6.9, 3.8, 3.0, 6.9, 10.0, 14.7 and 13.8 µM, respectively, than that of etoposide (VP-16) with an IC50 of 26.9 µM. Compounds 4-7 and 10-14 exhibited weak cytotoxicities to the selected cancer cell lines.

Alcohol consumption exacerbates alcoholic liver disease by attenuating the activity of AMP-activated protein kinase (AMPK). AMPK is activated by fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, and inhibited by direct interaction with cereblon (CRBN), a component of an E3 ubiquitin ligase complex. Based on these preliminary findings, we investigated that CRBN would be up-regulated in the liver by alcohol consumption and that CRBN deficiency would ameliorate hepatic steatosis and pro-inflammatory responses in alcohol-fed mice by increasing AMPK activity. Wild-type, CRBN and PPARα null mice were fed an alcohol-containing liquid diet and administered with fenofibrate. Gene expression profiles and metabolic changes were measured in the liver and blood of these mice. Expression of CRBN, cytochrome P450 2E1 (CYP2E1), lipogenic genes, pro-inflammatory cytokines, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were increased in the Lieber–DeCarli alcohol-challenged mice. Fenofibrate attenuated the induction of CRBN and reduced hepatic steatosis and pro-inflammatory markers in these mice. Ablation of the gene encoding CRBN produced the same effect as fenofibrate. The increase in CRBN gene expression by alcohol and the reduction of CRBN expression by fenofibrate were negated in PPARα null mice. Fenofibrate increased the recruitment of PPARα on CRBN gene promoter in WT mice but not in PPARα null mice. Silencing of AMPK prevented the beneficial effects of fenofibrate. These results demonstrate that activation of PPARα by fenofibrate alleviates alcohol-induced hepatic steatosis and inflammation by reducing the inhibition of AMPK by CRBN. CRBN is a potential therapeutic target for the alcoholic liver disease.

Background AMP-activated protein kinase (AMPK) exerts its anti-inflammatory effects by suppressing redox-sensitive nuclear factor kappa B (NF-κB) and pro-inflammatory cytokines including TNF-α. However, it is unclear whether AMPK regulates anti-inflammatory cytokine expressions in the presence of oxidative stress-induced inflammation. We sought to elucidate the mechanisms whereby AMPK regulates inflammatory cytokine expressions under NADPH oxidase (NOX)-induced oxidative stress. Methods HT-29 human colonic epithelial cells transfected with AMPKα shRNA and mouse models with AMPKα knocked out in epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) were used to examine the effects of AMPK and NOX on signaling pathways and cytokine expressions. Results In HT-29 cells, 5-hydroxytryptamine (5-HT)-induced NOX activity was enhanced by AMPKα silencing, and resulted in inflammatory cell death. AMPKα deletion specific for colon epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) intensified 5-HT- or dextran sulfate sodium (DSS)-induced upregulations of NOX2, TNF-α, and IL-6, but completely abolished basal and 5-HT- or DSS-induced upregulation of IL-10 in colon epithelium. Furthermore, 5-HT- and DSS-induced changes were accompanied by marked upregulations of increased inflammatory signaling pathways linked to NF-κB, AP-1, and STAT3 transcription factors, and to GATA, a cell fate-directing signaling. In addition, AMPKα deletion significantly fortified 5-HT- or DSS-induced downregulations of cytoprotective signaling pathways (Nrf2, HIF-1α, and KLF4). Conclusion Basal AMPKα maintains an anti-inflammatory state by inhibiting NOX, balancing pro−/anti-inflammatory signaling pathways, and directing IL-10 production. When these regulatory roles of AMPK are diminished by oxidative stress, colon epithelium undergoes inflammation despite IL-10 production.

Nur77 is a member of the NR4A subfamily of nuclear receptors and has been shown to regulate various biological processes such as apoptosis and inflammation. Here, we show that Nur77 ubiquitination is mediated by the tripartite motif 13 (Trim13), a RING-type E3 ubiquitin ligase. The interaction between Nur77 and Trim13 was confirmed by co-immunoprecipitation. Moreover, we found that Lys539 in Nur77 ubiquitination is targeted for Trim13, which leads to Nur77 degradation. The Trim13-mediated ubiquitination of Nur77 was optimal in the presence of the E2 enzyme UbcH5. Importantly, in addition to Trim13-mediated ubiquitination, the stability of Nur77 was also regulated by casein kinase 2α (CK2α). Pharmacological inhibition of CK2 markedly increased Nur77 levels, whereas overexpression of CK2α, but not its inactive mutant, dramatically decreased Nur77 levels by promoting Nur77 ubiquitination. CK2α phosphorylated Ser154 in Nur77 and thereby regulated Nur77 protein levels by promoting its ubiquitin-mediated degradation. Importantly, we also show that degradation of Nur77 is involved in TNFα-mediated IL-6 production via CK2α and Trim13. Taken together, these results suggest that the sequential phosphorylation and ubiquitination of Nur77 controls its degradation, and provide a therapeutic approach for regulating Nur77 activity through the CK2α-Trim13 axis as a mechanism to control the inflammatory response.

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.

Background IgE/Ag-stimulated mast cells release various pro-allergic inflammatory mediators, including histamine, eicosanoids, and pro-inflammatory cytokines. NecroX-5, a cell permeable necrosis inhibitor, showed cytoprotective effects in both in vitro and in vivo models. However, the anti-allergic effect of NecroX-5 has not yet been investigated. The aims of this study were to evaluate the anti-allergic activity of NecroX-5 in vivo and to investigate the underlying mechanism in vitro. Methods The anti-allergic activity of NecroX-5 was evaluated in vitro using bone marrow-derived mast cells (BMMCs) and IgE receptor-bearing RBL-2H3 or KU812 cells and in vivo using a mouse model of passive anaphylaxis. The levels of histamine, eicosanoids (PGD2 and LTC4), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using enzyme immunoassay kits. The mechanism underlying the action of NecroX-5 was investigated using immunoblotting, immunoprecipitation, and gene knockdown techniques. Results NecroX-5 markedly inhibited mast cell degranulation and the synthesis of eicosanoids, TNF-α, and IL-6 by suppressing the activation of Syk, LAT, phospholipase Cγ1, MAP kinases, the Akt/NF-κB pathway, and intracellular Ca2+ mobilization via the activation of phosphatase SHP-1. Oral administration of NecroX-5 effectively suppressed mast cell-dependent passive cutaneous and systemic anaphylactic reactions in a dose-dependent manner. Conclusions NecroX-5 might be a potential candidate for the development of a novel anti-allergic agent that suppresses IgE-dependent mast cells signaling.

Isothiocyanates (ITCs) derived from cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit preventative effects against various types of cancers. Yet, the inhibitory effects of ITCs on C6 glioma cell invasion and migration have not been reported. Thus, we aimed to analyze ITC-regulated MMP-9 activation, a crucial enzyme of cancer metastasis that degrades the extracellular matrix, in C6 glioma cells to investigate the inhibitory effects on cancer invasion and migration by ITCs. In the present study, we found that ITCs specifically suppressed PMA-induced MMP-9 secretion and protein expression. The inhibitory effects of ITCs on PMA-induced MMP-9 expression were found to be associated with the inhibition of MMP-9 transcription levels through suppression of nuclear translocation of NF-κB and activator protein-1 (AP-1). It was also confirmed that ITCs decreased MMP-9-mediated signaling such as FAK and JNK, whereas they had no effect on the phosphorylation of ERK and p38. Moreover, wound-healing and Τranswell invasion assays showed that ITCs inhibited the migration and invasion of C6 glioma cells. These results suggest that ITCs could be potential agents for the prevention of C6 glioma cell migration and invasion by decreasing FAK/JNK-mediated MMP-9 expression.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.

The effect of butanol extracts of the skin of Anguilla japonica (BESA) on endoplasmic reticulum (ER) stress-induced insulin resistance in L6 myotubes was investigated. Western blotting revealed that BESA increased phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and stimulated glucose uptake in L6 myotubes. Stimulation of AMPK and glucose uptake by BESA were significantly (p

Rutaecarpine is a pentacyclic indolopyridoquinazolinone alkaloid found in Evodia rutaecarpa and other related herbs. It has a variety of intriguing biological properties, which continue to attract the academic and industrial interest. Studies on rutaecarpine have included isolation from new natural sources, development of new synthetic methods for its total synthesis, the discovery of new biological activities, metabolism, toxicology, and establishment of analytical methods for determining rutaecarpine content. The present review focuses on the synthesis, biological activities, and structure-activity relationships of rutaecarpine derivatives, with respect to their antiplatelet, vasodilatory, cytotoxic, and anticholinesterase activities.

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells.

Hypoglycemic effects of ethylacetate extracts of Anguilla japonica (EMA) muscles in db/db mice were investigated. To understand the mechanism responsible for the hypoglycemic effects of EMA, the effects of EMA on AMP-activated protein kinase (AMPK) activation in L6 myotubes and in vivo using type II diabetic db/db mice were analyzed. In L6 myotubes, the phosphorylation degrees of AMPK and acetyl-CoA carboxylase (ACC) were markedly increased and glucose uptake was significantly (p

Sauchinone, the biologically active lignan of Saururus chinensis, has been reported to have anti-inflammatory, antitumor, antioxidant, and hepatoprotective properties. However, little is known about the effect of sauchinone on FceRI-mediated mast cell activation. The aim of this study was to evaluate the anti-allergic activity of sauchinone and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and the mast cell-mediated passive cutaneous anaphylaxis (PCA) model. Sauchinone markedly suppressed FceRI-mediated activation of positive signaling mediators, including Syk, linker for activation of T cells (LAT), phospholipase C (PLC)γ, mitogen-activated protein (MAP) kinases, Akt, IκB kinase (IKK), and intracellular Ca2+, and increased the activation of negative signaling mediators, including liver kinase B (LKB)1/AMP-activated protein kinase (AMPK) and Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1. Interestingly, sauchinone increased the interaction between SHP-1 and Syk. Consequently, sauchinone significantly suppressed FceRI-mediated BMMC degranulation and synthesis of eicosanoids and cytokines. These inhibitory effects of sauchinone were diminished in BMMCs treated with siRNAs targeting LKB1, AMPKα2, or SHP-1, and in BMMCs isolated from AMPKα2-deficient mice. In addition, administration of sauchinone markedly suppressed the IgE-mediated PCA reaction in wild-type mice, and this inhibitory effect was significantly reduced in AMPKα2-/- mice. Taken together, these data suggest that sauchinone suppresses FceRI-mediated mast cell activation and anaphylaxis through modulation of the LKB1/AMPK and SHP-1/Syk pathways. Therefore, sauchinone might be a potential therapeutic agent for the treatment of allergic inflammatory diseases.

Emodin, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, has several beneficial pharmacologic effects, which include anti-cancer, anti-diabetic, and anti-inflammatory activities. In this study, the authors examined the effect of emodin on the production of proinflammatory cytokines, such as, tumor necrosis factor (TNF)-α and interleukin (IL)-6, in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the mechanism responsible for the regulation of pro-inflammatory cytokine production by emodin, the authors assessed its effects on the activations of transcriptional factor nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Emodin attenuated the nuclear translocation of (NF)-κB p65 and its DNA-binding activity by reducing the phosphorylation and degradation of IκBα and the phosphorylation of IκB kinase B (IKK). Furthermore, emodin dose-dependently attenuated the phosphorylations of MAPKs, such as, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase, and the stress-activated protein kinases (SAPK)/c-Jun-N-terminal kinase (JNK). Taken together, the findings of this study suggest that the anti-inflammatory effects of emodin on PMA plus A23187-stimulated BMMCs are mediated via the inhibition of NF-κB activation and of the MAPK pathway.

The aim of this study was to investigate the effect of saucerneol F (SF) on the productions of the pro-inflammatory cytokines, TNF-α and IL-6, in IgE/Ag-induced mouse bone marrow-derived mast cells (BMMCs). SF dose-dependently suppressed the transcriptions of these pro-inflammatory cytokines. To identify the molecular mechanisms responsible for these suppressions, we examined the effect of SF on three important transcription factors; activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and STAT5. It was found that SF inhibited the nuclear translocation of the p65 subunit of NF-κB to the nucleus and its DNA-binding ability. SF also attenuated mitogen-activated protein kinase (MAPK)-mediated AP-1 activation and STAT5 activation. Biochemical analysis of FcεRI-mediated signaling pathways demonstrated that SF inhibited the phosphorylation of Fyn and multiple downstream signaling processes, including Syk, Gab2, and the Akt/IKK/IκB and MAPK pathways. Taken together, our results suggest that SF inhibits the production of pro-inflammatory cytokines by suppressing Fyn kinase-dependent signaling events.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a crucial role in the maintenance of cellular energy homeostasis, and several natural compounds that activate AMPK possibly enhance glucose uptake by muscle cells. In this study, we found that pinusolide stimulated AMPK phosphorylation and glucose uptake and these effects were significantly reduced by siRNA LKB1 or compound C, suggesting that enhanced glucose uptake by pinusolide is predominantly accomplished via an LKB1-mediated AMPK activation pathway. An insulin resistance state was induced by exposing cells to 30mM glucose, as indicated by reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake. Under these conditions, the phosphorylation of AMPK and ACC were decreased. Surprisingly, disrupted insulin signaling and decreased AMPK activity by high glucose concentrations were prevented by pinusolide. Moreover, this treatment increased insulin-stimulated glucose uptake via AMPK activation. Taken together, our findings suggest a link between high glucose and insulin resistance in muscle cells, and provide further evidence that pinusolide attenuates blockade of insulin signaling by enhancing IRS-1 tyrosine phosphorylation by the activating the AMPK pathway. In addition, this study indicates the targeting of AMPK represents a new therapeutic strategy for hyperglycemia-induced insulin resistance and type 2 diabetes.

The purpose of this study is to investigate the effects of n-hexane extracts from bones and internal organs of Japanese eel, Anguilla japonica (HEE), on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in stem cell factor (SCF), IL-10, plus LPS-induced mouse bone marrow-derived mast cells (BMMCs) and on passive cutaneous anaphylaxis (PCA) in mice. HEE suppressed SCF/IL-10/LPS-induced PGD2 generation, and concomitantly reduced COX-2 protein expression dose-dependently. To understand the mechanistic basis for the inhibition of PGD2 generation by HEE, we examined the effects of HEE on upstream signaling pathways essential for COX-2 induction. HEE was found to inhibit the translocation of nuclear factor-κB (NF-κB) p65 subunit to the nucleus and its DNA-binding ability through the inhibition of TAK1, IKK and IκB phosphorylation. Furthermore, HEE also attenuated mitogen-activated protein kinase (MAPK)-mediated regulation of DNA binding of activator protein-1 (AP-1). Moreover, oral administration of HEE inhibited anti-dinitrophenyl (DNP) IgE-induced PCA in a dose dependent manner. Taken together, the present study provides new insights into the anti-inflammatory activity of HEE, which could be a promising candidate to be used for an inflammatory therapy.

Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH2-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca(2+)/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance.

Background and Purpose Endoplasmic reticulum (ER) stress has been implicated in the pathogeneses of insulin resistance and type 2 diabetes, and extracellular signal-regulated kinase (ERK) antagonist is an insulin sensitizer that can restore muscle insulin responsiveness in both tunicamycin-treated muscle cells and type 2 diabetic mice. The present study was undertaken to determine whether the chemical or genetic inhibition ER stress pathway targeting by ERK results in metabolic benefits in muscle cells.

The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S. chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man B suppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FceRI-mediated mast cells.

Previously we reported that saucerneol D (SD), a naturally occurring sesquilignan isolated from Saururus chinensis ( S. chinensis ) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. The aim of this study was to elucidate whether SD modulates the generation of other inflammatory mediators in activated mast cells. We investigated the effects of SD on cyclooxygenase-2 (COX-2)-dependent prostaglandin D 2 (PGD 2 ) and 5-lipoxygenase (5-LO)-dependent leukotriene C 4 (LTC 4 ) generations as well as degranulation in cytokine-stimulated mouse bone marrow-derived mast cells (BMMCs). Biochemical analyses of the cytokine-mediated signaling pathways showed that SD suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes including phospholipase Cγ1 (PLCγ1)-mediated intracellular Ca 2+ influx and activation of mitogen-activated protein kinases (MAPKs; including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH 2 -terminal kinase (JNK) and p38) and the nuclear factor-κB (NF-κB) pathway. Taken together, the present study suggests that SD suppresses eicosanoid generation and degranulation through Syk-dependent pathway in BMMCs.

Isothiocyanates, which are present as glucosinolate precursors in cruciferous vegetables, have strong activity against various cancers. Here, we compared the anti-metastatic effects of isothiocyanates (benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN)) by examining how they regulate MMP-9 expression. Isothiocyanates, particularly PEITC, suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 activity and invasion in various cancer cell lines. By contrast, N-methyl phenethylamine, a PEITC analog without an isothiocyanate functional group, had no effect. A reporter gene assay demonstrated that BITC, PEITC, and SFN suppressed TAP-induced MMP-9 expression by inhibiting AP-1 and NF-κB in U20S osteosarcoma cells. All three compounds reduced phosphorylation of FAK, ERK1/2, and Akt. In addition, MMP-9 expression was downregulated by inhibiting FAK, ERK1/2, and Akt. Isothiocyanates-mediated inhibition of FAK phosphorylation suppressed phosphorylation of ERK1/2 and Akt in U2OS and A549 cells, along with the translocation of p65 and c-Fos, suggesting that isothiocyanates inhibit MMP-9 expression and cell invasion by blocking phosphorylation of FAK. Furthermore, isothiocyanates, abolished MMP-9 expression and tumor metastasis in vivo with the following efficacy: PEITC>BITC>SFN. Thus, isothiocyanates act as anti-metastatic compounds that suppress MMP-9 activity/expression by inhibiting NF-κB and AP-1 via suppression of the FAK/ERK and FAK/Akt signaling pathways.

The inability to monitor the in vivo dynamics of mast cells (MCs) limits the better understanding of its role in cancer progression. Here, we report on noninvasive imaging of MC migration to tumor lesions in mice and evaluation of the effects of migrated MCs on tumor progression through reporter gene-based in vivo optical imaging and glucose metabolism monitoring in cancer with 18F-fluorodeoxyglucose (18F-FDG) in vitro and in vivo. Murine MCs (MC-9) and Lewis lung cancer cells (LLC) expressing an enhanced firefly luciferase (effluc) gene were established, termed MC-9/effluc and LLC/effluc, respectively. MC-9/effluc cell migration to LLC tumor lesions was initially detected within 1 h post-transfer and distinct bioluminescence imaging signals emitted from MC-9/effluc cells were observed at tumor sites until 96 h. In vivo optical imaging as well as a biodistribution study with 18F-FDG demonstrated more rapid tumor growth and upregulated glucose uptake potentially associated with MC migration to tumor lesions. These results suggest that the combination of a reporter gene-based optical imaging approach and glucose metabolism status monitoring with 18F-FDG represents a promising tool to better understand the biological role of MCs in tumor microenvironments and to develop new therapeutic drugs to regulate their involvement in enhanced tumor growth.

Ethnopharmacology relevance Oldenlandia diffusa (OD) has long been known as an apoptotic inducer in breast tumors in ethnomedicine. Aim of the study To scientifically confirm the anti-breast cancer effects of water, methanol (MeOH) and butanol (BuOH) extracts of O. diffusa on cell apoptosis, matrix metalloproteinases (MMPs), intercellular adhesion molecule (ICAM)−1 and intracellular signaling in MCF-7 breast cancer cells. Materials and methods MeOH extracts (MOD) and BuOH extracts (BOD) were prepared and examined for their ability to inhibit phorbol myristate acetate (PMA)-induced matrix metalloproteinase (MMP)−9 and intercellular adhesion molecule (ICAM)−1 expressions in MCF-7 human breast cancer cells. Additionally, transwell migration, invasion and transcriptional activity were assessed. Results of immunofluorescence confocal microscopy for translocation of NF-κB and p-ERK and p-p38 were also checked. Finally, apoptotic signals including processed caspase-8, caspase-7, poly ADP-ribose polymerase, Bax and Bcl-2 were examined. Results MOD and BOD specifically inhibited PMA-induced MMP-9 expression as well as invasive and migration potential via ICAM-1. The inhibitory activity was also based on the suppressed transcriptional activity in MCF-7 breast cancer cells. Results of immunofluorescence confocal microscopy showed that translocation of NF-κB decreased upon BOD and MOD treatments, with a decreased level of p-ERK and p-p38 phosphorylation. In addition, treatment of MCF-7 cells with MOD and BOD activated apoptosis-linked proteins including enzymatically active forms of processed caspase-8, caspase-7 and poly ADP-ribose polymerase, together with increased expression of mitochondrial apoptotic protein, Bax and decreased expression of Bcl-2. Conclusion The results indicate that OD as an anti-metastatic agent suppresses the metastatic response by targeting p-ERK, p-38 and NF-κB, thus reducing the invasion capacity of MCF-7 breast cancer cells through inhibition of MMP-9 and ICAM-1 expression and plays an important role in the regulation of breast cancer cell apoptosis.

Fourteen compounds were isolated from the flowers of Inula japonica THUNB. (Asteraceae), including two new compounds, (1S,2S,4S,5S,8S,10R)-2-acetoxy-4,3-dihydroxy-pseudoguai-7(11)-en-12,8-olide (1) and (1S,2S,4S,5S,8S,10R)-2,4,13-trihydroxy-pseudoguai-7(11)-en-12,8-olide (2), and twelve known compounds, budlein B (3), 6β-hydroxytomentosin (4), 6-deacetoxybritanin (5), 4-epipulchellin (6), britanin (7), tomentosin (8), (+)-dihydroquercetin (9), (-)-syringaresinol (10), quercetagetin 3,4'-dimethyl ether (11), luteolin (12), britanin G (13) and inuchinenolide C (14). Structures of 1 and 2 were determined based on one and two dimensional (1D)- and (2D)-NMR data and Mosher's esterification method. Compounds 9 and 12 showed inhibitory activities toward DNA topoisomerase I with IC50 values of 55.7 and 37.0 µM, respectively, compared to camptothecin (CPT) with an IC50 of 24.5 µM. Compounds 7-9 and 11-14 exhibited more potent inhibitory activity against topoisomerases II with IC50 values of 6.9, 3.8, 3.0, 6.9, 10.0, 14.7 and 13.8 µM, respectively, than that of etoposide (VP-16) with an IC50 of 26.9 µM. Compounds 4-7 and 10-14 exhibited weak cytotoxicities to the selected cancer cell lines.

Rutaecarpine is a pentacyclic indolopyridoquinazolinone alkaloid found in Evodia rutaecarpa and other related herbs. It has a variety of intriguing biological properties, which continue to attract the academic and industrial interest. Studies on rutaecarpine have included isolation from new natural sources, development of new synthetic methods for its total synthesis, the discovery of new biological activities, metabolism, toxicology, and establishment of analytical methods for determining rutaecarpine content. The present review focuses on the synthesis, biological activities, and structure-activity relationships of rutaecarpine derivatives, with respect to their antiplatelet, vasodilatory, cytotoxic, and anticholinesterase activities.

The stem and root barks of Ulmus davidiana var. japonica (Ulmaceae) have been used to treat inflammatory diseases including mastitis, rhinitis, sinusitis, and enteritis. In an ongoing study focused on the discovery of natural anti-inflammatory compounds from natural products, a methanol extract of the stem and root barks of U. davidiana var. japonica showed anti-inflammatory activities. Activity-guided fractionation of the methanol extract yielded a new trihydroxy fatty acid, 9,12,13-trihydroxyoctadeca-10(Z),15(Z)-dienoic acid (1), and a known compound, pinellic acid (2). These two trihydroxy fatty acids 1 and 2 inhibited prostaglandin D2 production with IC50 values of 25.8 and 40.8 μM, respectively. These results suggest that 9,12,13-trihydroxyoctadeca-10(Z),15(Z)-dienoic acid (1) and pinellic acid (2) are among the anti-inflammatory principles in this medicinal plant. Copyright © 2012 John Wiley & Sons, Ltd.

Matrix metalloproteinases-9 (MMP-9) plays an important role in the pathogenesis of atherosclerosis and migration of vascular smooth muscle cells (VSMCs) after an arterial injury. In this study, we investigated the potential molecular mechanisms underlying the anti-atheroscleroic effects of melittin, a major component of bee venom, in human aortic smooth muscle cells (HASMCs). Melttin significantly suppressed MMP-9 and MMP-2 secretion, as well as TNF-α-induced MMP-9 expression in the HASMCs. In addition, we found that the inhibitory effects of melittin on TNF-α-induced MMP-9 protein expression are associated with the inhibition of MMP-9 transcription levels. Mechanistically, Melittin suppressed TNF-α-induced MMP-9 activity by inhibiting the phosphorylation of p38 and ERK1/2, but did not affect the phosphorylation of JNK and Akt. Reporter gene and western blotting assays showed that melittin inhibits MMP-9 transcriptional activity by blocking the activation of NF-κB via IκBα signaling pathway. Moreover, the matrigel migration assay showed that melittin reduced TNF-α-induced HASMC migration. These results suggest that melittin suppresses TNF-α-induced HASMC migration through the selective inhibition of MMP-9 expression and provide a novel role of melittin in the anti-atherosclerotic action.

In this study, we examined the effects of citreorosein (CIT), an anthraquinone component of Polygoni cuspidati radix (P. cuspidati, Polygonaceae), on cyclooxygenase (COX)-2 dependent prostaglandin (PG)D2 generation in mast cells, central effector cells of allergy and other inflammatory diseases. CIT strongly inhibited COX-2-dependent PGD2 generation in a concentration-dependent manner in mouse bone marrow-derived mast cells (BMMCs) stimulated with stem cell factor (SCF)/IL-10/LPS. In an effort to identify the mechanisms underlying the inhibition of COX-2-dependent PGD2 generation by CIT, we examined the effects of this compound on MAP kinases, Akt and NF-κB signaling pathways, which are essential for COX-2 induction. CIT inhibited nuclear translocation of the nuclear factor (NF)-κB p65 subunit and its cognate DNA-binding activity, which correlated with its inhibitory effects on the phosphorylation of Akt and IKK and subsequent phosphorylation and degradation of IκB. Furthermore, CIT significantly attenuated the DNA binding of activator protein (AP)-1 that regulates COX-2 expression through the reduction of the phosphorylation of c-Jun. Moreover, inhibition of PGD2 generation by CIT was accompanied by a decrease in phosphorylation of cytosolic phospholipase A2α. Taken together, the present study suggests that CIT represents a potential therapeutic approach for the treatment of inflammatory diseases.

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati (P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C4 (LTC4) generation in mast cells. Cit suppresses both degranulation and the generation of LTC4 in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca2+, phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A2 (PLA2) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC4 generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation.

This study was designed to evaluate the role of mammalian target of rapamycin (mTOR)/p70S61 kinase (S6K1) pathways in ER stress-induced insulin resistance in L6 myotubes. Pretreatment with 5μg/ml of tunicamycin or 600nM thapsigargin for 3h decreased insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake, and increased the level of mTOR/S6K1 phosphorylation in L6 myotubes. However, the inhibition of mTOR activity by rapamycin (inhibitor of several intracellular pathways including S6K1 pathways) reversed the ER stress-reduced tyrosine phosphorylation of IRS-1 and glucose uptake. Furthermore, pretreatment of cells with rapamycin decreased ER stress-induced phosphorylation of mTOR and S6K1. Interestingly, inhibition of mTOR by rapamycin did not affect ER stress markers such as PERK and JNK activity under the ER stress condition. Similar results were obtained with or without pretreatment with tunicamycin in the absence or presence of S6K1 RNAi. Moreover, S6K1 RNAi-mediated knockdown preserved insulin-stimulated Akt phosphorylation and glucose uptake in ER-stressed L6 myotubes, which was blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Taken together, these results suggest that rapamycin improved ER stress-induced insulin resistance via inhibition of mTOR/S6K1 hyperphosphorylation in L6 myotubes.