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Upregulation of genes and coexpression networks related to immune function and inflammation have been repeatedly reported in the brain of individuals with schizophrenia. However, a causal relationship between the abnormal immune/inflammation-related gene expression and schizophrenia has not been determined. We conducted co-expression networks using publicly available RNA-seq data from prefrontal cortex (PFC) and hippocampus (HP) of 64 individuals with schizophrenia and 64 unaffected controls from the SMRI tissue collections. We identified proinflammatory cytokine, transmembrane tumor necrosis factor-α (tmTNFα), as a potential regulator in the module of co-expressed genes that we find related to the immune/inflammation response in endothelial cells (ECs) and/or microglia of the brain of individuals with schizophrenia. The immune/inflammation-related modules associated with schizophrenia and the TNF signaling pathway that regulate the network were replicated in an independent cohort of brain samples from 68 individuals with schizophrenia and 135 unaffected controls. To investigate the association between the overexpression of tmTNFα in brain ECs and schizophrenia-like behaviors, we induced short-term overexpression of the uncleavable form of (uc)-tmTNFα in ECs of mouse brain for 7 weeks. We found schizophrenia-relevant behavioral deficits in these mice, including cognitive impairment, abnormal sensorimotor gating, and sensitization to methamphetamine (METH) induced locomotor activity and METH-induced neurotransmitter levels. These uc-tmTNFα effects were mediated by TNF receptor2 (TNFR2) and induced activation of TNFR2 signaling in astrocytes and neurons. A neuronal module including neurotransmitter signaling pathways was down-regulated in the brain of mice by the short-term overexpression of the gene, while an immune/inflammation-related module was up-regulated in the brain of mice after long-term expression of 22 weeks. Our results indicate that tmTNFα may play a direct role in regulating neurotransmitter signaling pathways that contribute to the clinical features of schizophrenia.

Background: Histone Deacetylase (HDAC) inhibitors represent an extensive class of targeted anticancer agents. Among the most explored structure moieties, hydroxybenzamides and hydroxypropenamides have been demonstrated to have potential HDAC inhibitory effects. Several compounds of these structural classes have been approved for clinical uses to treat different types of cancer, such as givinostat (ITF2357) and belinostat (PXD-101). Aims: This study aims at developing novel HDAC inhibitors bearing N-hydroxybenzamides and Nhydroxypropenamides scaffolds with potential cytotoxicity against different cancer cell lines. Methods: Two new series of N-hydroxybenzamides and N-hydroxypropenamides analogues (4a-j, 6a-j) designed based on the structural features of nexturastat A, AR-42, and PXD-101, were synthesized and evaluated for HDAC inhibitory potency as well as cytotoxicity against three human cancer cell lines (SW620 (colorectal adenocarcinoma), PC3 (prostate adenocarcinoma), and NCI-H23 (adenocarcinoma, non-small cell lung cancer). Molecular simulations were finally carried out to gain more insight into the structure-activity relationships. Results: It was found that the N-hydroxypropenamides (6a-e) displayed very good HDAC inhibitory potency and cytotoxicity. Various compounds, e.g. 6a-e, especially compound 6e, were up to 5-fold more potent than suberanilohydroxamic acid (SAHA) in terms of cytotoxicity. These compounds also comparably inhibited HDACs with IC50 values in the sub-micromolar range. Docking experiments showed that these compounds bound to HDAC2 at the enzyme active binding site with the same binding mode of SAHA, but with higher binding affinities. Conclusions: The two series of N-hydroxybenzamides and N-hydroxypropenamides designed and synthesized were potential HDAC inhibitors and antitumor agents. Further development of these compounds should be warranted.

Background: Histone deacetylases (HDAC) enzymes are emerging as potential targets for cancer treatments. In this study, several series of novel hydroxamic acids incorporating 1-((1H- 1,2,3-triazol-4-yl)methyl)-3-substituted-2-oxoindolines were explored. Methods: The compounds were designed using Autodock Vina program, then synthesized and evaluated in vitro and in silico for their inhibitory activity against HDACs. The cytotoxicity was measured by SRB method. The enzyme inhibitory effects of the compounds were evaluated by the fluorescent assay. Results: Biological evaluation showed that these hydroxamic acids were generally cytotoxic against four human cancer cell lines (SW620, colon; PC-3, prostate; AsPC-1, pancreas; NCI-H23, lung). Several compounds, e.g. 7g, 11c, and 11g, displayed up to 10-fold more potent than SAHA (suberoylanilide hydroxamic acid, vorinostat) in term of cytotoxicity. The synthesized compounds were also comparably potent to SAHA in inhibiting HDAC2. In particular, compound 11c displayed potential inhibitory effects against HDAC1, HDAC2, HDAC6, and HDAC8 with comparable or slightly higher potency than SAHA. Docking results on four class I and IIB isoenzymes indicated that these compounds tightly bound to HDACs at the active site with binding affinities much higher than that of SAHA. Finally, chemo-informatics approaches were employed to assess the pharmacokinetic and toxicity profiles of 7g and 11c. We identified degradation via phase II metabolism and toxicity two of the most serious problems that need further optimization. Conclusion: Taking altogether our findings are encouraging and current hydroxamate derivatives are worth being considered as potential HDAC inhibitors and could be useful for further research on the development of new anti-cancer agents.

Background: Combrestastatin A-4 (CA-4) is a potent anticneoplastic and antiangiogenesis natural substance isolated from Combretum caffrum. Over the past two decades, numerous derivatives of CA-4 have been discovered. However, none of these derivatives has reached the clinical stage. Thus, continuing effort is needed in developing CA-4 analogues with improved pharmacological properties. Methods: In this study, two series of thiazolidine-2,4-dione and 2-oxoindoline derivatives incorporating 3,4,5-trimethoxybenzyl scaffold were designed and synthesized as CA-4 analogues. Results: Numerous CA-4 analogues bearing thiazolidine-2,4-dione/2-oxoindoline have been synthesized. These compounds were evaluated against several human cancer cell lines. It was found that a series of 5/7-substituted-1-(3,4,5-trimethoxy)benzylindoline-2,3-diones (2a-g) exhibited significant cytotoxicity. Especially compound 2d bearing a 5-bromo substituent showed the best activity with IC50 values in sub-microgram/mL scale in four human cancer cell lines tested. This compound also exhibited potent tubulin polymerization inhibitory activity. A series of (Z)-5-arylidene- 3-(3,4,5-trimethoxybenzyl)thiazolidine-2,4-diones (6a-j), on the other hand, displayed only moderate cytotoxic activities with only compound 6a showing comparable cytotoxicity to 2d. Finally, in silico molecular modeling and drug-likeness profiling revealed that five compounds 2b, 2d, 2e, 3e and 6a bound to tubulin active binding sites with strong binding affinities. Conclusion: This study discovered some novel CA-4 analogues with cytotoxic potency and antitubulin activity acceptable to be further developed as effective anticancer drug candidates.

In our search for novel small molecules targeting histone deacetylases, we have designed and synthesized a series of novel hydroxamic acids incorporating indole moiety as a cap group (3a–l). Biological evaluation showed that these hydroxamic acids potently inhibited HDAC2 with IC50values in submicromolar range and up to tenfold (compound 3j) better than that of SAHA (also known as suberoylanilide hydroxamic acid). In four human cancer cell lines [SW620 (colon), PC-3 (prostate), AsPC-1 (pancreatic), NCI-H23 (lung)], the synthesized compounds that exhibited potent cytotoxicity with several compounds (3k, 3l) were found to be 12- to 77-fold more cytotoxic than SAHA. Docking experiments indicated that the compounds tightly bound to HDAC2 at the active binding site with binding affinities much higher than that of SAHA. Our present results demonstrate that these novel hydroxamic acids are potential for further development as anticancer agents.

ABSTRACTAutophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis(Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3′-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

Bioactivity-guided isolation of a methanolic extract of Euphorbia fischerianaled to the isolation of four new abietane-type diterpenoids, fischeriolides A–D (1–4), together with 11 known diterpenoids. Their structures were elucidated based on the interpretation of 1D and 2D NMR spectroscopic and HRESIMS data. The absolute configuration of compound 3was determined by single-crystal X-ray diffraction analysis and electronic circular dichroism methods. Compounds 5–9exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50values in the range 4.9–12.6 μM.

Objective We investigated whether purpurin inhibits various pathways of inflammation leading to atopic dermatitis.Introduction 1,2,4-Trihydroxyanthraquinone, commonly called purpurin, is an anthraquinone that is a naturally occurring red/yellow dye. Purpurin is a highly antioxidative anthraquinone and previous studies have reported antibacterial, anti-tumor, and anti-oxidation activities in cells and animals. However, the skin inflammatory inhibition activity mechanism study of purpurin has not been elucidated in vitro.Methods In this study, we investigated the anti-inflammatory activity of purpurin in HaCaT (human keratinocyte) cell lines stimulated with a mixture of tumor necrosis factor-alpha (TNF-α)/Interferon-gamma (IFN-γ). The inhibitory effect of Purpurin on cytokines (IL-6, IL-8, and IL-1β) and chemokine (TARC, MDC, and RANTES) was confirmed by ELISA and RT-qPCR. We investigated each signaling pathway and the action of inhibitors through western blots.Results The expression levels of cytokines and chemokines were dose-dependently suppressed by purpurin treatment in TNF-α/IFN-γ-induced HaCaT cells from ELISA and real-time PCR. Purpurin also inhibited protein kinase B (AKT), mitogen-activated protein kinase (MAPKs), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) activation in TNF-α/IFN-γ-stimulated HaCaT cells. Additionally, there was a synergistic effect when purpurin and inhibitor were applied together, and inflammation was dramatically reduced.Conclusion Therefore, these results demonstrate that purpurin exhibits anti-inflammatory and anti-atopic dermatitis activity in HaCaT cells.

Histone deacetylases (HDAC) are currently a group of validated targets for anticancer drug discovery and development. In our research program to find novel small molecules targeting these enzymes, we designed and synthesized two series of 3-hydroxyimino-2-oxoindoline- and 3- methoxyimino-2-oxoindoline-based N-hydroxypropenamides (3a-g, 6a-g). The results show that these propenamides potently inhibited HDAC2 with IC50 values in sub-micromolar range, approximately 10-fold lower than that of SAHA (also known as suberoylanilohydroxamic acid). Evaluation of cytotoxicity of these compounds in three human cancer cell lines revealed that most of the synthesized compounds were up to 5-fold more cytotoxic than SAHA. Docking studies showed that the compounds bound to HDAC2 at the binding site with higher binding affinities compared to SAHA. Our present results demonstrate that these novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides are potential for further development as anticancer agents.

The search for newer histone deacetylase (HDAC) inhibitors has attracted a great deal of interest of medicinal chemists worldwide, especially after the first HDAC inhibitor (Zolinza®, widely known as SAHA or Suberoylanilide hydroxamic acid) was approved by the FDA for the treatment of Tcell lymphoma in 2006. As a continuity of our ongoing research in this area, we designed and synthesized a series of 5-aryl-1,3,4-thiadiazole-based hydroxamic acids as analogues of SAHA and evaluated their biological activities. Most of the compounds in this series, e.g. compounds with 5-aryl moiety being 2- furfuryl (5a), 5-bromofuran-2-yl (5b), 5-methylfuran-2-yl (5c), thiophen-2-yl (5d), 5-methylthiophen-2-yl (5f) and pyridyl (5g-i), were found to have potent anticancer cytotoxicity with IC50values of generally 5- to 10-fold lower than that of SAHA in 4 human cancer cell lines assayed. Those compounds with potent cytotoxicity were also found to have strong HDAC inhibition effects. Docking studies revealed that compounds 5a and 5d displayed high affinities towards HDAC2 and 8.

Two new 2-phenoxychromones 1and 2and two prenylflavonoids 3and 4along with 12 known compounds (5–16) were isolated from the CH2Cl2-soluble fraction of a methanol extract of Epimedium koreanum. Compounds 1, 4, 6, 7, 9, 10, 12, and 15exhibit inhibitory effects on nitric oxide production with IC50values ranging from 16.8 to 49.3 μM. Compounds 1, 4, 7, and 12also showed inhibitory effects on interleukin-1β production with IC50values ranging from 8.6 to 38.9 μM in RAW 264.7 macrophages.

In this study, we investigated the hepatoprotective effects of aged black garlic (ABG) in rodent models of liver injury. ABG inhibited carbon tetrachloride-induced elevation of aspartate transaminase (AST) and alanine transaminase (ALT), which are markers of hepatocellular damage, in SD rats. D-galactosamine-induced hepatocellular damage was also suppressed by ABG treatment. However, ABG does not affect the elevation of alkaline phosphatase (ALP), a marker of hepatobilliary damage, in rats treated with carbon tetrachloride or D-galactosamine. We also examined the effect of ABG on high-fat diet (HFD)-induced fatty liver and subsequent liver damage. ABG had no significant effect on body weight increase and plasma lipid profile in HFD-fed mice. However, HFD-induced increase in AST and ALT, but not ALP, was significantly suppressed by ABG treatment. These results demonstrate that ABG has hepatoprotective effects and suggest that ABG supplementation might be a good adjuvant therapy for the management of liver injury.

Results from clinical studies have demonstrated that inhibitors of histone deacetylase (HDAC) enzymes possess promise for the treatment of several types of cancer. Zolinza® (widely known as SAHA) has been approved by the FDA for the treatment of T-cell lymphoma. As a continuity of our ongoing research to find novel small molecules to target these important enzymes, we synthesized a series of benzothiazole-containing analogues of SAHA and found several compounds with very potent anticancer cytotoxicity. In this study, three more compounds of this type, including N1-(6- chlorobenzo[d]thiazol-2-yl)-N8-hydroxyoctanediamide (3a), N1-[6-(trifluoromethyl)benzo[d]thiazol-2-yl]-N8-hydroxyoctanediamide (3b) and N1-(thiazol-2-yl)-N8-hydroxyoctanediamide (6) were synthesized and evaluated for HDAC inhibition and cytotoxic activities. All three compounds showed very potent HDAC inhibitory effects. Docking revealed that both two compounds 3a, 3b showed higher affinities towards HDAC8 compared to SAHA. In vitro, compound 3a exhibited cytotoxicity equipotent to SAHA against five human cancer cell lines. In term of in vivo activity, compound 3a demonstrated equivalent efficacy to SAHA in mouse xenograft model.

In this study, we investigated the effect of methanolic extract isolated from the root of Lycoris aurea(LA) on the growth of cancer cells and the tube formation activity of endothelial cells. Various cancer cells were treated with LA at doses of 0.3, 1, 3, 10 or 30 μg/mland LA significantly suppressed the growth of several cancer cell lines, including ACHN, HCT-15, K-562, MCF-7, PC-3 and SK-OV-3, in a dose-dependent manner. We also found that LA induced cell cycle arrest at G2/M phase in ACHN renal cell adenocarcinoma cells. Further study demonstrated that LA concentration-dependently inhibited the tube formation, which is a widely used in vitromodel of reorganization stage of angiogenesis, in human umbilical vein endothelial cells. Collectively, these results show that LA inhibits the growth of cancer cells and tube formation of endothelial cells and the growth-inhibitory effect of LA might be mediated, at least in part, by blocking cell cycle progression.

PPARγ ligands have been reported to reduce proliferation of human prostate cancer cells. However, the molecular mechanism of PPARγ agonist-induced cell growth inhibition of prostate cancer cells is not clear. GSK-3β expression and NFκB activity have important roles in prostate cancer development. To investigate the mechanisms of the PPARγ agonist-induced prostate cancer cell growth inhibition, we examined the effect of troglitazone on the expression of PPARγ, GSK-3β and activity of NFκB as well as on the prostate cancer cell growth. Troglitazone induced the expression of PPARγ in the nuclear of PC-3 cells, but not in LNCaP cells. Troglitazone (0-16 uM) inhibited cancer cell growth in a similar extend between both cells accompanied by the induction of cell cycle arrest in G0/G1 phase and an increased in the similar extent of apoptotic cell death in concentration dependent manner. Troglitazone inhibited the constitutive expression of GSK-3β and activation of NFκB. Co-treatment of troglitazone with a GSK-3β inhibitor (AR-a014418) or GSK-3β siRNA significantly augmented the inhibitory effect of troglitazone on the NFκB activity and on prostate cancer cell growth inhibition and apoptotic cell death. However, overexpression of GSK-3β hindered troglitazone-induced cell growth inhibition and NFκB inactivation. These results suggest that PPARγ agonist, troglitazone, inhibits prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression.

Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory cytokine that plays an important role in the development of various inflammatory diseases. TNF-alpha has been considered as a potential therapeutic target for the treatment of chronic inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In this study, we report that cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (DBM1285) is a novel inhibitor of TNF-alpha production. DBM1285 concentration-dependently inhibited lipopolysaccharide (LPS)-induced TNF-alpha secretion in various cells of macrophage/monocyte lineage, including mouse bone marrow macrophages, THP-1 cells, and RAW 264.7 cells. However, LPS-induced mRNA expression of TNF-alpha was not affected by DBM1285 in these cells. Further studies demonstrated that the inhibitory effect of DBM1285 on TNF-alpha production might be mediated by post-transcriptional regulation through the modulation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) signaling pathway. We also confirmed that DBM1285 directly inhibits p38 MAPK enzymatic activity. In vivo administration of DBM1285 inhibited LPS-induced increase in the plasma level of TNF-alpha in mice. Whole-blood in vivo target inhibition assay also revealed that DBM1285 attenuates p38 MAPK activity after oral administration in mice. Moreover, DBM1285 suppressed zymosan-induced inflammation and adjuvant-induced arthritis in murine models. Collectively, these results suggest that DBM1285 inhibits TNF-alpha production, at least in part, by blocking the p38 MAPK/MK2 pathway. Furthermore, in vivo results suggest that DBM1285 might be a possible therapeutic candidate for the treatment of TNF-alpha-related chronic inflammatory diseases.

α-Viniferin, an oligostilbene of trimeric resveratrol, has been reported to have antiinflammatory potential in carrageenin-induced paw edema or adjuvant-induced arthritis in animal models. However, little is known about the molecular basis. In this study, α-viniferin at 3 – 10 μM dose-dependently inhibited interferon (IFN)-γ–induced Ser727phosphorylation of the signal transducer and activation of transcription-1 (STAT-1), a pivotal transcription factor controlling IFN-γ–targeted genes, in RAW 264.7 macrophages, and also IFN-γ–induced activation of the extracellular signal-regulated kinase (ERK)-1, a protein kinase upstream of the Ser727phosphorylation of STAT-1. However, α-viniferin, only at a higher concentration of 10 μM, inhibited Janus kinase 2–mediated Tyr701phosphorylation of STAT-1 in the cells. To understand STAT-1–dependent inflammatory responses, we quantified nitric oxide (NO) or chemokines. α-Viniferin at 3 – 10 μM dose-dependently inhibited IFN-γ–induced production of NO, IFN-γ–inducible protein-10 (IP-10), or the monokine induced by IFN-γ(MIG) in RAW 264.7 cells and also that of NO in primary macrophages-derived from C57BL/6 mice. Furthermore, α-viniferin diminished IFN-γ–induced protein levels of inducible NO synthase (iNOS), attenuated mRNA levels of iNOS, IP-10, or MIG as well as inhibited promoter activity of the iNOS gene. In conclusion, this study proposes an antiinflammatory mechanism of α-viniferin, down-regulating STAT-1–inducible inflammatory genes via inhibiting ERK-mediated STAT-1 activation in IFN-γ–stimulated macrophages.

Manassantin A (MSA) inhibits nitric oxide production by macrophages through the inhibition of NF-κB activation, but the effect of MSA on dendritic cells has not been elucidated yet. Here we investigated the inhibitory effects of MSA on immune functions of dendritic cells (DCs). MSA inhibited lipopolysaccharide (LPS)-induced phenotypic maturation of DCs, which was proved by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. MSA also inhibited functional maturation of DCs, that is, decreased the gene expression of IL-12, IL-1β, TNF-α, and IFN-α/β; enhanced antigen capture capacity of DCs; and impaired induction of allogenic T cell activation. As a mechanism of action, MSA inhibited LPS-induced activation of NF-κB, ERK, p38, and JNK, which played pivotal roles in toll-like receptor 4–mediated DC maturation. Collectively, these results suggested that MSA might be used for the treatment of DC-related immune diseases.

Nuclear transcription factor-κB (NF-κB) is constitutively activated in prostate and colon cancers and is related with the resistance of cancer cells against chemotherapeutics. Previously, we found that obovatol, an active compound isolated from Magnolia obovata, inhibited cancer cell growth through inhibition of NF-κB activity. We investigated here whether obovatol could sensitize cancer cells against docetaxel through inhibition of NF-κB activity in prostate cancer (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. The combination treatment with each drug at one half the respective IC50dose (5 μM obovatol + 5 nM docetaxel) was more effective and significant (60% – 70%) in the inhibition of cancer cell growth than single treatment by each drug (20% – 40%); inhibition was exerted through a significant increase of apoptosis induction (60% – 80%) by the combination treatment compared to the single treatment (10% – 30%). Correlating well with the synergistic inhibition (combination indices are less than 1 in all cell types), the combination significantly inhibited NF-κB activities as well as expression of NF-κB target apoptotic cell death proteins, but decreased anti-apoptotic cell death proteins. Similar combination effects of obovatol with other chemotherapeutic agents (paclitaxel, cisplatin, and doxorubicin) on the inhibition of cell growth and NF-κB activity were also found. These results indicate that obovatol augments cell growth inhibition by chemotherapeutics through inactivation of NF-κB and suggest that obovatol may have therapeutic advantages in the combination treatment with other chemotherapeutics.

Diarctigenin was previously isolated as an inhibitor of nitric oxide (NO) production in macrophages from the seeds of Arctium lappa used as an alternative medicine for the treatment of inflammatory disorders. However, little is known about the molecular basis of these effects. Here, we demonstrated that diarctigenin inhibited the production of NO, prostaglandin E(2), tumor necrosis factor-alpha, and interleukin (IL)-1beta and IL-6 with IC(50) values of 6 to 12 miciroM in zymosan- or lipopolysaccharide-(LPS) activated macrophages. Diarctigenin attenuated zymosan-induced mRNA synthesis of inducible NO synthase (iNOS) and also inhibited promoter activities of iNOS and cytokine genes in the cells. Because nuclear factor (NF)-kappaB plays a pivotal role in inflammatory gene transcription, we next investigated the effect of diarctigenin on NF-kappaB activation. Diarctigenin inhibited the transcriptional activity and DNA binding ability of NF-kappaB in zymosan-activated macrophages but did not affect the degradation and phosphorylation of inhibitory kappaB (IkappaB) proteins. Moreover, diarctigenin suppressed expression vector NF-kappaB p65-elicited NF-kappaB activation and also iNOS promoter activity, indicating that the compound could directly target an NF-kappa-activating signal cascade downstream of IkappaB degradation and inhibit NF-kappaB-regulated iNOS expression. Diarctigenin also inhibited the in vitro DNA binding ability of NF-kappaB but did not affect the nuclear import of NF-kappaB p65 in the cells. Taken together, diarctigenin down-regulated zymosan- or LPS-induced inflammatory gene transcription in macrophages, which was due to direct inhibition of the DNA binding ability of NF-kappaB. Finally, this study provides a pharmacological potential of diarctigenin in the NF-kappaB-associated inflammatory disorders.

In light of the anti-inflammatory properties of histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), we examined a new HDAC inhibitor KBH-A42 for its anti-inflammatory activities. KBH-A42 showed noteworthy anti-inflammatory properties in vitro via suppression of the production of TNF-a, a proinflammatory cytokine, and nitric oxide (NO), a proinflammatory effector molecule, in LPS-stimulated RAW264.7 cells and peritoneal macrophages. It also inhibited TNF-a production in vivo as demonstrated in a LPS-induced mouse endotoxemia model. The levels of TNF-a, IL-1ß, IL-6 and iNOS mRNAs determined by RT-PCR propose that the inhibition of these pro-inflammatory mediators by KBH-A42 resulted from inhibiting expression of these genes. However, the EMSA study to see the effect of KBH-A42 on the binding of NF-?B, a transcription factor, to a specific DNA sequence showed that the binding of NF-?B to DNA was not changed regardless of increasing the concentration of KBH-A42 in the presence and absence of LPS stimulation. Interestingly, DNA binding of another transcription factor AP-1 dose-dependently increased by KBH-A42. KBH-A42 differentially regulated the phosphorylation of MAP kinases. While the phosphprylation of ERK1/2 and SAPK/JNK was not affected by KBH-A42, the phosphorylation of p38 decreased by KBH-A42. These results showed that KBH-A42 inhibits production of proinflammatory cytokines in macrophages by decreasing their mRNA levels, and p38 kinase is involved in the KBH-A42-mediated inhibition.

Abstract: The objective of this research is to draw the optimal cycle length and evaluate the effect of the fraction of anoxic and anaerobic phases in a cycle maximizing the nutrients removal in a modified temporal and spatial phase separated process. A pilot-scale system operated at HRTs of 10–21h, SRTs of 16–34 d, cycle times of 2–8h, and mixed liquor temperature from 9 to 30°C showed average removals of BOD, TN, and TP as high as 93, 79, and 86%, respectively. Higher nitrogen removal could be achieved for shorter cycle time, while better phosphorus removal could be accomplished for longer cycle time. Optimal cycle time for simultaneous removal of nitrogen and phosphorus conflicted with each other. The effect of ratio of cycle time to system HRT on system performance was also shown to have the same tendency as that of cycle time. Higher TN removal of phased isolation technology could be achieved relative to that of SBR as the cycle length became longer in the same HRT. As the fraction of anoxic/anaerobic phase in a cycle became larger, the removal efficiency of TN and TP simultaneously decreased because of the discharge of untreated ammonia nitrogen and released phosphorus. [Copyright &y& Elsevier]

Benzoxathiole derivatives have been used in the treatment of acne and have shown cytostatic, antipsoriatic, and antibacterial properties. However, little is known about the molecular basis for these pharmacological properties, although nuclear factor (NF)-κB activation is closely linked to inflammation and cell proliferation. Here, we demonstrate that the novel small-molecule benzoxathiole 6,6-dimethyl-2-(phenylimino)-6,7-dihydro-5H-benzo-[1,3]oxathiol-4-one (BOT-64) inhibits NF-κB activation with an IC50value of 1 µM by blocking inhibitory κB(IκB) kinase β (IKKβ), and suppresses NF-κB-regulated expression of inflammatory genes in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. BOT-64 inhibits IKKβ-mediated IκBα phosphorylation in LPS-activated macrophages, resulting in sequential prevention of downstream events, including proteolytic degradation of IκBα, DNA binding ability, and transcriptional activity of NF-κB. BOT-64 inhibits LPS-inducible IKKβ activity in the cells and catalytic activity of highly purified IKKβ. Moreover, the effect of BOT-64 on cell-free IKKβ was abolished by substitution of Ser-177 and Ser-181 residues in the activation loop of IKKβ to glutamic acid residues, indicating a direct interaction site of benzoxathiole. BOT-64 attenuates NF-κB-regulated expression of inflammatory genes such as inducible nitric-oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in LPS-activated or expression vector IKKβ-transfected macrophages. Furthermore, BOT-64 dose-dependently increases the survival rates of endotoxin LPS-shocked mice.

In our previous studies, we showed that angelan, a polysaccharide purified from AngelicagigasNakai, is a potent LPS-mimetic in murine macrophages [Jeon, Y.J., Han, S.B., Ahn, K.S., Kim, H.M., 1999. Activation of NF-kB/Rel in angelan-stimulated macrophages. Immunopharmacology 43, 1–9]. Angelan stimulates murine macrophage to produce cytokines including iNOS and activate NF-κB/Rel. In the present study, we investigated the role of CD14 and complement receptor type 3 (CR3) in mediating NO production and NF-κB/Rel activation induced by angelan and LPS. Three major differences between angelan and LPS were observed. First, angelan does not require serum proteins for NO response and NF-κB/Rel activation, while the activation by LPS requires serum proteins. Second, blocking of either CD14 or CR3 decreased angelan-induced NO response, while LPS-mediated NO production was inhibited by anti-CD14 mAb only. Third, angelan induced strong NF-κB/Rel and slight AP-1 DNA binding, whereas LPS potently activated both NF-κB/Rel and AP-1. Both angelan and LPS degraded IκB proteins and subsequently induced the mobilization of NF-κB/Rel proteins (p65, c-rel and p50) into nucleus. This suggests that macrophages display a common signaling machinery leading to the NF-κB/Rel activation in response to different stimulants. In conclusion, angelan and LPS use the membrane receptor CD14 and CR3 differentially for signaling NF-κB/Rel activation and NO production.

In our previous studies we showed that the primary target cell of angelan, a polysaccharide purified from Angelica gigasNakai, is a macrophage (Han et al., 1998). In the present study we examined the effect of angelan on iNOS, IL-1β, and TNF-α transcription in mouse macrophage line RAW 264.7. We show that angelan produces a marked induction of iNOS, IL-1β, and TNF-α transcription by RAW 264.7 cells. Since these gene transcriptions have been recently shown to be under the control of NF-κB/Rel family of transcription factors, we assessed the effect of angelan on NF-κB/Rel using a electrophoretic mobility shift assay. Treatment of RAW 264.7 cells with angelan produced strong induction of NF-κB/Rel binding. Treatment of RAW 264.7 cells with angelan slightly induced AP-1 binding activity, whereas Oct binding was not affected by angelan. Angelan stimulated macrophages to activate NF-κB/Rel, whereas neither B-cells nor T-cells were affected by the angelan. In conclusion, we demonstrate that the stimulation effect of angelan on macrophage is mediated by specific activation of NF-κB/Rel.

The relevance of MDR‐1 gene expression to the multidrug resistance phenotype was investigated. Drug‐resistant cells, KB‐V1 and MCF7/ADR, constantly expressed mRNA of the MDR‐1 gene and were more resistant to vinblastine and adriamycin than drug‐sensitive cells, KB‐3–1 and MCF7. The drug efflux rate of KB‐V1 was the same as KB‐3–1 although the MDR‐1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB‐3–1 than KB‐V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P‐glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR‐1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms.

The three fractions of inulase differing in isoelectric pH were isolated from a strain ofAspergillusnigerby isoelectric focussing and characterized in their temperature stability. These fractions had distinguishable thermal stability. Among them, one fraction (pI 5.2) having main activity showed a high thermal stability comparable to that of the Novozyme 230 (Novo A/S, Denmark) at 60°C.

The immunopharmacological characteristics of angelan, a polysaccharide purified from Angelica gigasNakai, were investigated in relation to the specificity to immune cells. The treatment of angelan increased the expression of IL-2, IL-4, IL-6, and IFN-γ. The expression of IL-6 and IFN-γ was rapidly augmented but that of IL-2 responded later. In the case of IL-4, angelan stimulated at early time after exposure but down-regulated thereafter. These results suggested that macrophages and natural killer cells involved in nonspecific immunity were primarily activated and helper T cells were secondarily affected by angelan. Angelan also had lympho-proliferative potential to B cells, specifically. The specificity of angelan was also elucidated in a cell fractionation experiment. The activated B cells by angelan also increased antibody production. The direct activation of B cells, macrophages, and accessory cells and the indirect activation of helper T cells coordinately increased immune functions such as in vitro and in vivo T-dependent immunization and antibody production. The experiment of host resistance to syngeneic tumors also showed that angelan potentiated the immune functions. In conclusion, angelan, a purified polysaccharide from an oriental herbal drug, showed characteristic immunostimulation, which was different from clinically used polysaccharides such as lentinan and PSK.

Mice deficient in the toll-like receptor (TLR) or the myeloid differentiation factor 88 (MyD88) are resistant to acute liver failure (ALF) with sudden death of hepatocytes. Chalcone derivatives from medicinal plants protect from hepatic damages including ALF, but their mechanisms remain to be clarified. Here, we focused on molecular basis of piperidylmethyloxychalcone (PMOC) in the treatment of TLR/MyD88-associated ALF. C57BL/6J mice were sensitized with D-galactosamine (GalN) and challenged with Escherichia coli lipopolysaccharide (LPS, TLR4 agonist) or oligodeoxynucleotide containing unmethylated CpG motif (CpG ODN, TLR9 agonist) for induction of ALF. Post treatment with PMOC sequentially ameliorated hepatic inflammation, apoptosis of hepatocytes, severe liver injury and shock-mediated death in ALF-induced mice. As a mechanism, PMOC inhibited the catalytic activity of TGF-β-activated kinase 1 (TAK1) in a competitive manner with respect to ATP, displaced fluorescent ATP probe from the complex with TAK1, and docked at the ATP-binding active site on the crystal structure of TAK1. Moreover, PMOC inhibited TAK1 auto-phosphorylation, which is an axis in the activating pathways of nuclear factor-κB (NF-κB) or activating protein 1 (AP1), in the liver with ALF in vivo or in primary liver cells stimulated with TLR agonists in vitro. PMOC consequently suppressed TAK1-inducible NF-κB or AP1 activity in the inflammatory injury, an early pathogenesis leading to ALF. The results suggested that PMOC could contribute to the treatment of TLR/MyD88-associated ALF with the ATP-binding site of TAK1 as a potential therapeutic target.

ABSTRACTCD226 is an activating receptor expressed on natural killer (NK) cells, CD8+T cells, and other immune cells. Upon binding to its ligands expressed on target cells, CD226 activates intracellular signaling that triggers cytokine production and degranulation in NK cells. However, the role of CD226 in contact dynamics between NK and cancer cells has remained unclear. Our time-lapse images showed that individual wild-type CD226+NK cells contacted B16F10 melanoma cells for 23.7 min, but Cd226−/−NK cells only for 12.8 min, although both NK cell subsets showed equal contact frequency over 4 h. On the surface of B16F10 cells, CD226+cells stayed at the same site with oscillating movement (named stable contact), while Cd226−/−NK cells moved around at a velocity of 4 μm/min (named unstable contact). Consequently, Cd226−/−NK cells did not kill B16F10 cells in vitroand did not inhibit their metastasis into the lung in vivo. Taken together, our data demonstrate that CD226 enables prolonged stable interaction between NK and cancer cells, which is needed for efficient killing of cancer cells.

Senile plaques composed of β-amyloid (Aβ) are a pathological hallmark of Alzheimer disease. Presenilin 2 (PS2) mutations increase Aβ generation in the brains of Alzheimer disease patients, but the underlying mechanism of Aβ generation by PS2 mutations remains to be clarified. The Aβ is generated through the sequential cleavage of amyloid precursor protein by β- and γ-secretases. Here, we show that the PS2 mutation N141I enhances the activity of β-secretase and expression of the β-site amyloid precursor protein cleavage enzyme 1, a major neuronal β-secretase in the brains of PS2 transgenic mice and in PC12 cells overexpressing mutant PS2. In parallel with the increased activity of β-secretase, activation of extracellular signal–regulated kinase (ERK), Aβ1-40 and Aβ1-42 levels, generation of reactive oxygen species, and lipid peroxidation were higher in the mutant mouse neurons and the PC12 cells. Colocalization of phosphorylated ERK (phospho-ERK) and β-site amyloid precursor protein cleavage enzyme 1 with hydroxynonenal-histidine was found in the mutant brains. An ERK inhibitor U0126 and an antioxidant N-acetylcysteine prevented the expression and activity of β-secretase, ERK activation, and reactive oxygen species generation in both neurons and PC12 cells expressing mutant PS2 in a dose-dependent manner. Together, these data suggest that oxidative stress–mediated ERK activation contributes to increases in β-secretase and, thus, an increase of Aβ generation in neuronal cells expressing mutant PS2.

Inhibition of adipocytes differentiation is suggested to be an important strategy for prevention and/or treatment of obesity. In our present study, Cordyceps militarisshowed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of cordycepin (1), guanosine (2) and tryptophan (3) as active compounds. All the three compounds were more effective in the prevention of early stage of adipogenesis than in lipolysis. In addition, combinational treatment of three compounds significantly increased anti-adipogenic activity.