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1.
Int J Mol Sci ; 19(3)2018 Mar 19.
Article in English | MEDLINE | ID: mdl-29562668

ABSTRACT

Hydroquinone (HQ, 1,4-benzenediol) is a hydroxylated benzene metabolite with various biological activities, including anti-oxidative, neuroprotective, immunomodulatory, and anti-inflammatory functions. However, the anti-cancer activity of HQ is not well understood. In this study, the in vitro and in vivo anti-cancer activity of HQ was investigated in various cancer cells and tumor-bearing mouse models. HQ significantly induced the death of A431, SYF, B16F10, and MDA-MB-231 cells and also showed a synergistic effect on A431 cell death with other anti-cancer agents, such as adenosine-2',3'-dialdehyde and buthionine sulfoximine. In addition, HQ suppressed angiogenesis in fertilized chicken embryos. Moreover, HQ prevented lung metastasis of melanoma cells in mice in a dose-dependent manner without toxicity and adverse effects. HQ (10 mg/kg) also suppressed the generation of colon and reduced the thickness of colon tissues in azoxymethane/dextran sodium sulfate-injected mice. This study strongly suggests that HQ possesses in vitro and in vivo anti-cancer activity and provides evidence that HQ could be developed as an effective and safe anti-cancer drug.


Subject(s)
Antineoplastic Agents/pharmacology , Hydroquinones/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Azoxymethane , Benzoquinones/chemistry , Benzoquinones/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Shape/drug effects , Chickens , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Dextran Sulfate , Hydroquinones/chemistry , Hydroquinones/therapeutic use , Inhibitory Concentration 50 , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Male , Melanoma, Experimental/pathology , Mice, Inbred C57BL
3.
Oncoimmunology ; 4(1): e984550, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25949867

ABSTRACT

Dab2 is an adapter protein involved in receptor-mediated signaling, endocytosis, cell adhesion, hematopoietic cell differentiation, and angiogenesis. It plays a pivotal role in controlling cellular homeostasis. In the immune system, the Dab2 is a Foxp3 target gene and is required for regulatory T (Treg) cell function. Dab2 expression and its biological function in dendritic cells (DCs) have not been described. In this study, we found that Dab2 was significantly induced during the development of mouse bone marrow (BM)-derived DCs (BMDCs) and human monocyte-derived DCs (MoDCs). Even in a steady state, Dab2 was expressed in mouse splenic DCs (spDCs). STAT5 activation, Foxp3 expression, and hnRNPE1 activation mediated by PI3K/Akt signaling were required for Dab2 expression during GM-CSF-derived BMDC development regardless of TGF-ß signaling. Dab2-silencing was accompanied by enhanced IL-12 and IL-6 expression, and an improved capacity of DC for antigen uptake, migration and T cell stimulation, which generated strong CTL in vaccinated mice. Vaccination with Dab2-silenced DCs inhibited tumor growth more effectively than did vaccination with wild type DCs. Dab2-overexpression abrogated the efficacy of the DC vaccine in DC-based tumor immunotherapy. These data strongly suggest that Dab2 might be an intrinsic negative regulator of the immunogenicity of DCs, thus might be an attractive molecular target to improve DC vaccine efficacy.

4.
J Virol ; 89(8): 4262-80, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25653431

ABSTRACT

UNLABELLED: Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53(-/-) and eIF2α(CA) cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat-Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCE: HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.


Subject(s)
HIV Infections/immunology , HIV-1/immunology , Tumor Suppressor Protein p53/metabolism , Virus Replication/immunology , eIF-2 Kinase/metabolism , Base Sequence , Blotting, Western , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Cell Line , Gene Knockdown Techniques , HIV-1/physiology , Humans , Immunohistochemistry , Immunoprecipitation , Molecular Sequence Data , Phosphorylation , Sequence Analysis, DNA , Tandem Mass Spectrometry , Trans-Activators/metabolism , Tumor Suppressor Protein p53/genetics , Two-Hybrid System Techniques
5.
Mol Cells ; 38(2): 122-9, 2015.
Article in English | MEDLINE | ID: mdl-25623024

ABSTRACT

DBM-2198, a six-membered azasugar nucleotide (6-AZN)-containing phosphorothioate (P = S) oligonucleotide (AZPSON), was described in our previous publication [Lee et al. (2005)] with regard to its antiviral activity against a broad spectrum of HIV-1 variants. This report describes the mechanisms underlying the anti-HIV-1 properties of DBM-2198. The LTR-mediated reporter assay indicated that the anti-HIV-1 activity of DBM-2198 is attributed to an extracellular mode of action rather than intracellular sequence-specific antisense activity. Nevertheless, the antiviral properties of DBM-2198 and other AZPSONs were highly restricted to HIV-1. Unlike other P = S oligonucleo-tides, DBM-2198 caused no host cell activation upon administration to cultures. HIV-1 that was pre-incubated with DBM-2198 did not show any infectivity towards host cells whereas host cells pre-incubated with DBM-2198 remained susceptible to HIV-1 infection, suggesting that DBM-2198 acts on the virus particle rather than cell surface molecules in the inhibition of HIV-1 infection. Competition assays for binding to HIV-1 envelope protein with anti-gp120 and anti-V3 antibodies revealed that DBM-2198 acts on the viral attachment site of HIV-1 gp120, but not on the V3 region. This report provides a better understanding of the antiviral mechanism of DBM-2198 and may contribute to the development of a potential therapeutic drug against a broad spectrum of HIV-1 variants.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Oligodeoxyribonucleotides/pharmacology , Phosphorothioate Oligonucleotides/pharmacology , Piperidines/pharmacology , Virus Replication/drug effects , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Gene Expression Regulation, Viral , HeLa Cells , Humans , Jurkat Cells , Virus Attachment/drug effects
6.
Eur J Immunol ; 43(9): 2484-96, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23716134

ABSTRACT

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.


Subject(s)
Dendritic Cells/immunology , Early Growth Response Protein 2/genetics , Early Growth Response Protein 2/metabolism , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cancer Vaccines/immunology , Cell Movement , Cell Proliferation , Cells, Cultured , Dendritic Cells/metabolism , Female , Genes, MHC Class I , Genes, MHC Class II , Interleukin-12/biosynthesis , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , RNA Interference , RNA, Small Interfering , STAT5 Transcription Factor/metabolism , Spleen/cytology , Spleen/immunology , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins/biosynthesis , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology
7.
Free Radic Biol Med ; 57: 105-18, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23290930

ABSTRACT

The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also a common dietary component. Although numerous papers have addressed the potential role of HQ in tumorigenic responses, the immunosuppressive and anti-inflammatory effects of hydroquinone have also been considered. In this study, we characterized the mechanism of the induction of hemeoxygenase (HO)-1 and other phase 2 enzymes by HQ and its derivatives. HQ upregulated the mRNA and protein levels of HO-1 by increasing the antioxidant-response element-dependent transcriptional activation of Nrf-2. Src knockdown or deficiency induced via siRNA treatment and infection with a retrovirus expressing shRNA targeting Src, as well as exposure to PP2, a Src kinase inhibitor, strongly abrogated HO-1 expression. Interestingly, HQ directly targeted and bound to the sulfhydryl group of cysteine-483 (C483) and C400 residues of Src, potentially leading to disruption of intracellular disulfide bonds. Src kinase activity was dramatically enhanced by mutation of these cysteine sites, implying that these sites may play an important role in the regulation of Src kinase activity. Therefore, our data suggest that Src and, particularly, its C483 target site can be considered as prime molecular targets of the HQ-mediated induction of phase 2 enzymes, which is potentially linked to HO-1-mediated cellular responses such as immunosuppressive and anti-inflammatory actions.


Subject(s)
Heme Oxygenase-1/metabolism , Hydroquinones/pharmacology , Macrophages, Peritoneal/metabolism , src-Family Kinases/metabolism , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Cell Line , Cysteine/chemistry , Cysteine/metabolism , HEK293 Cells , Heme Oxygenase-1/genetics , Humans , Inflammation , Male , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , RNA Interference , RNA, Messenger/metabolism , Signal Transduction , Sulfhydryl Compounds/chemistry , Transcription, Genetic , Transcriptional Activation , p38 Mitogen-Activated Protein Kinases/metabolism , src-Family Kinases/genetics
8.
Mediators Inflamm ; 2012: 512926, 2012.
Article in English | MEDLINE | ID: mdl-23209344

ABSTRACT

Src kinase (Src) is a tyrosine protein kinase that regulates cellular metabolism, survival, and proliferation. Many studies have shown that Src plays multiple roles in macrophage-mediated innate immunity, such as phagocytosis, the production of inflammatory cytokines/mediators, and the induction of cellular migration, which strongly implies that Src plays a pivotal role in the functional activation of macrophages. Macrophages are involved in a variety of immune responses and in inflammatory diseases including rheumatoid arthritis, atherosclerosis, diabetes, obesity, cancer, and osteoporosis. Previous studies have suggested roles for Src in macrophage-mediated inflammatory responses; however, recently, new functions for Src have been reported, implying that Src functions in macrophage-mediated inflammatory responses that have not been described. In this paper, we discuss recent studies regarding a number of these newly defined functions of Src in macrophage-mediated inflammatory responses. Moreover, we discuss the feasibility of Src as a target for the development of new pharmaceutical drugs to treat macrophage-mediated inflammatory diseases. We provide insights into recent reports regarding new functions for Src that are related to macrophage-related inflammatory responses and the development of novel Src inhibitors with strong immunosuppressive and anti-inflammatory properties, which could be applied to various macrophage-mediated inflammatory diseases.


Subject(s)
Inflammation/etiology , Macrophages/physiology , src-Family Kinases/physiology , Animals , Heme Oxygenase-1/physiology , Humans , Kupffer Cells/physiology , NADPH Oxidases/physiology , Reactive Oxygen Species/metabolism , Signal Transduction , Toll-Like Receptors/physiology , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/chemistry
9.
Acta Pharmacol Sin ; 33(8): 1037-46, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22796759

ABSTRACT

AIM: The macrophage-mediated inflammatory response may contribute to the development of cancer, diabetes, atherosclerosis and septic shock. This study was to characterize several new compounds to suppress macrophage-mediated inflammation. METHODS: Peritoneal macrophages from C57BL/6 male mice and RAW264.7 cells were examined. Anti-inflammatory activity was evaluated in the cells exposed to lipopolysaccharide (LPS). The mechanisms of the anti-inflammatory activity were investigated via measuring transcription factor activation in response to specific signals and via assaying the activities of the target kinases. RESULTS: Of 7 candidate compounds tested, 8-(tosylamino)quinoline (8-TQ, compound 7) exhibited the strongest activities in suppressing the production of NO, TNF-α, and PGE(2) in LPS-activated RAW264.7 cells and peritoneal macrophages (the IC(50) values=1-5 µmol/L). This compound (1.25-20 µmol/L) dose-dependently suppressed the expression of the pro-inflammatory genes for iNOS, COX-2, TNF-α, and the cytokines IL-1ß and IL-6 at the level of transcription in LPS-activated RAW264.7 cells. 8-TQ (20 µmol/L) significantly suppressed the activation of NF-κB and its upstream signaling elements, including inhibitor of κB (IκBα), IκBα kinase (IKK) and Akt in LPS-activated RAW264.7 cells. In in vivo experiments, oral administration of 20 and 40 mg/kg 8-TQ for 3 d significantly alleviated the signs of LPS-induced hepatitis and HCl/EtOH-induced gastritis, respectively, in ICR mice. CONCLUSION: 8-TQ (compound 7) exerts significant anti-inflammatory activity through the inhibition of the Akt/NF-κB pathway, thus may be developed as a novel anti-inflammatory drug.


Subject(s)
Inflammation Mediators/antagonists & inhibitors , Macrophages, Peritoneal/drug effects , NF-kappa B/antagonists & inhibitors , Quinolines/pharmacology , Signal Transduction/drug effects , Tosyl Compounds/pharmacology , Animals , Cells, Cultured , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Signal Transduction/physiology
10.
Z Naturforsch C J Biosci ; 67(3-4): 222-32, 2012.
Article in English | MEDLINE | ID: mdl-22624339

ABSTRACT

Histone acetylation is linked to the control of chromatin remodeling, which is involved in cell growth, proliferation, and differentiation. It is not fully understood whether cyclic adenosine monophosphate (cAMP), a representative differentiation-inducing molecule, is able to modulate histone acetylation as part of its anticancer activity. In the present study, we aimed to address this issue using cell-permeable cAMP, i.e. dibutyryl cAMP (dbcAMP) and C6 glioma cells. As reported previously, under the conditions of our studies, treatment with dbcAMP clearly arrested C6 cell proliferation and altered their morphology. Its antiproliferative and differentiation-inducing activity in C6 glioma cells involved upregulation of p219WAF/CIP), p27(kip1), glial fibrillary acidic protein (GFAP), and Cx43, as well as downregulation of vimentin. Furthermore, dbcAMP modulated the phosphorylation of ERK and Akt in a time-dependent manner and altered the colocalization pattern of phospho-Src and the actin cytoskeleton. Interestingly, dbcAMP upregulated the enzyme activity of histone acetyltransferase (HAT) and, in parallel, enhanced cellular acetyllysine levels. Finally, the hyperacetylation-inducing compound, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, displayed similar anticancer activity to dbcAMP. Therefore, our data suggest that antiproliferative and differentiation-inducing activities of dbcAMP may be generated by its enhanced hyperacetylation function.


Subject(s)
Cell Differentiation , Cell Proliferation , Cyclic AMP/metabolism , Histones/metabolism , Acetylation , Animals , Cell Line, Tumor , Rats , Reverse Transcriptase Polymerase Chain Reaction
11.
Mediators Inflamm ; 2012: 732860, 2012.
Article in English | MEDLINE | ID: mdl-22474399

ABSTRACT

Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF-κB, AP-1, STAT-1, ATF-2, and CREB. According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK.


Subject(s)
Macrophage Activation/drug effects , Panax/chemistry , Polysaccharides/pharmacology , Activating Transcription Factor 2 , Animals , Cell Line , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Immunoblotting , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/metabolism , RGS Proteins/deficiency , RGS Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism
12.
Biochem Pharmacol ; 83(11): 1540-51, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22406106

ABSTRACT

Novel anti-inflammatory compounds were synthesised by derivatization of militarin, a compound isolated from Cordyceps militaris that is an ethnopharmacologically well-known herbal medicine with multiple benefits such as anti-cancer, anti-inflammatory, anti-obesity, and anti-diabetic properties. In this study, we explored the in vitro and in vivo anti-inflammatory potencies of these compounds during inflammatory responses, their inhibitory mechanisms, and acute toxicity profiles. To do this, we studied inflammatory conditions using in vitro lipopolysaccharide-treated macrophages and several in vivo inflammatory models such as dextran sodium sulphate (DSS)-induced colitis, EtOH/HCl-induced gastritis, and arachidonic acid-induced ear oedema. Methods used included real-time PCR, immunoblotting analysis, immunoprecipitation, reporter gene assays, and direct kinase assays. Of the tested compounds, compound III showed the highest nitric oxide (NO) inhibitory activity. This compound also inhibited the production of prostaglandin (PG)E(2) at the transcriptional level by suppression of Syk/NF-κB, IKKɛ/IRF-3, and p38/AP-1 pathways in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages. Consistent with these findings, compound III strongly ameliorated inflammatory symptoms in colitis, gastritis, and ear oedema models. In acute toxicity tests, there were no significant differences in body and organ weights, serum parameters, and stomach lesions between the untreated and compound III-treated mice. Therefore, this compound has the potential to be served as a lead chemical for developing a promising anti-inflammatory drug candidate with multiple kinase targets.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Enzyme Inhibitors/pharmacology , Inflammation/chemically induced , Inflammation/drug therapy , Phosphotransferases/antagonists & inhibitors , Propylene Glycols/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cell Line , Dinoprostone/genetics , Dinoprostone/metabolism , Gene Expression Regulation/drug effects , Humans , Macrophages/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Molecular Structure , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/genetics , Nitric Oxide/metabolism , Propylene Glycols/chemistry , Rats
13.
Mediators Inflamm ; 2012: 489810, 2012.
Article in English | MEDLINE | ID: mdl-22315508

ABSTRACT

Nanostructured, self-assembling peptides hold promise for a variety of regenerative medical applications such as 3D cell culture systems, accelerated wound healing, and nerve repair. The aim of this study was to determine whether the self-assembling peptide K5 can be applied as a carrier of anti-inflammatory drugs. First, we examined whether the K5 self-assembling peptide itself can modulate various cellular inflammatory responses. We found that peptide K5 significantly suppressed the release of tumor-necrosis-factor- (TNF-) α and prostaglandin E2 (PGE2) from RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS). Similarly, there was inhibition of cyclooxygenase- (COX-) 2 mRNA expression assessed by real-time PCR, indicating that the inhibition is at the transcriptional level. In agreement with this finding, peptide K5 suppressed the translocation of the transcription factors activator protein (AP-1) and c-Jun and inhibited upstream inflammatory effectors including mitogen activated protein kinase (MAPK), p38, and mitogen-activated protein kinase kinase 3/6 (MKK 3/6). Whether this peptide exerts its effects via a transmembrane or cytoplasmic receptor is not clear. However, our data strongly suggest that the nanostructured, self-assembling peptide K5 may possess significant anti-inflammatory activity via suppression of the p38/AP-1 pathway.


Subject(s)
Dinoprostone/biosynthesis , Peptides/pharmacology , Signal Transduction/drug effects , Transcription Factor AP-1/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Cell Line , Cells, Cultured , Cyclooxygenase 2/genetics , Cyclooxygenase 2/immunology , Dinoprostone/immunology , HEK293 Cells , Humans , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Male , Mice , Mice, Inbred C57BL , Nanostructures/chemistry , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/immunology , Signal Transduction/immunology , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/immunology , p38 Mitogen-Activated Protein Kinases/metabolism
14.
J Ethnopharmacol ; 139(2): 616-25, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-22182430

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum hydropiper L. (Polygonaceae) has been traditionally used to treat various inflammatory diseases such as rheumatoid arthritis. However, no systematic studies on the anti-inflammatory actions of Polygonum hydropiper and its inhibitory mechanisms have been reported. This study is therefore aimed at exploring the anti-inflammatory effects of 99% methanol extracts (Ph-ME) of this plant. MATERIALS AND METHODS: The effects of Ph-ME on the production of inflammatory mediators in RAW264.7 cells and peritoneal macrophages were investigated. Molecular mechanisms underlying the effects, especially inhibitory effects, were elucidated by analyzing the activation of transcription factors and their upstream signalling, and by evaluating the kinase activities of target enzymes. Additionally, a dextran sulphate sodium (DSS)-induced colitis model was employed to see whether this extract can be used as an orally available drug. RESULTS: Ph-ME dose-dependently suppressed the release of nitric oxide (NO), tumour necrosis factor (TNF)-α, and prostaglandin (PG)E(2), in RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS). Ph-ME inhibited mRNA expression of pro-inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and TNF-α by suppressing the activation of nuclear factor (NF)-κB, activator protein (AP-1), and cAMP responsive element binding protein (CREB), and simultaneously inhibited its upstream inflammatory signalling cascades, including cascades involving Syk, Src, and IRAK1. Consistent with these findings, the extract strongly suppressed the kinase activities of Src and Syk. Based on HPLC analysis, quercetin, which inhibits NO and PGE(2) activities, was found as one of the active ingredients in Ph-ME. CONCLUSION: Ph-ME exerts strong anti-inflammatory activity by suppressing Src/Syk/NF-κB and IRAK/AP-1/CREB pathways, which contribute to its major ethno-pharmacological role as an anti-gastritis remedy.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Colitis, Ulcerative/prevention & control , Macrophages/drug effects , Methanol/chemistry , Plant Extracts/pharmacology , Polygonum , Solvents/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/immunology , Colitis, Ulcerative/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Dextran Sulfate , Dinoprostone/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Interleukin-1 Receptor-Associated Kinases/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/pharmacology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plants, Medicinal , Polygonum/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/metabolism , Signal Transduction/drug effects , Syk Kinase , Time Factors , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transfection , Tumor Necrosis Factor-alpha/metabolism , src-Family Kinases/antagonists & inhibitors
15.
Pharmazie ; 66(4): 293-300, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21612158

ABSTRACT

Resveratrol, a stilbene type compound identified in wine and fruit juice, has been found to exhibit various pharmacological activities such as anti-oxidative, anti-cancerous, anti-inflammatory and anti-aging effects. Although numerous papers have explored the pharmacology of resveratrol in one particular cellular action, how this compound can have multiple effects simultaneously has not been fully addressed. In this study, therefore, we explored its broad-spectrum inhibitory mechanism using lipopolysaccharide (LPS)-mediated inflammatory responses and reporter gene assays involving overexpression of toll like receptor (TLR) adaptor molecules. Co-transfection of adaptor molecules such as (1) myeloid differentiation primary response gene 88 (MyD88), (2) Toll/4ll-1 Receptor-domain-containing adapter-inducing interferon-beta (TRIF), (3) TRIF-related adaptor molecule (TRAM), or (4) TANK-binding kinase (TBK) 1 strongly enhanced luciferase activity mediated by transcription factors including nuclear factor (NF)-KB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3. Of the adaptor proteins, TRIF and TBK1 but not MyD88 and IKK enhanced luciferase activity mediated by these transcription factors. Resveratrol dose-dependently suppressed LPS-induced NO production in macrophages. It also blocked the increases in levels of mRNA for IFN-1, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) that were induced by LPS. Resveratrol diminished the translocation or activation of IRF-3 at 90min, c-Jun, a subunit of AP-1, and STAT-1 at 120 min, and p50, a subunit of NF-KB, at 60 and 90 min. Resveratrol strongly suppressed the up-regulation of luciferase activity induced by these adaptor molecules with IC50 values of 5 to 65 microM. In particular, higher inhibitory effects of resveratrol were when TRIF or TBK1 were overexpressed following cotransfection of luciferase constructs with IRF-3 binding sequences. Taken together, our data suggest that the suppression of TRIF and TBK1, which mediates transcriptional activation of NF-kappaB, AP-1, and IRF-3, contributes to resveratrol's broad-spectrum inhibitory activity, and that this compound can be further developed as a lead anti-inflammatory compound.


Subject(s)
Adaptor Proteins, Vesicular Transport/drug effects , Adaptor Proteins, Vesicular Transport/physiology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Interferon Regulatory Factor-3/drug effects , Interferon Regulatory Factor-3/physiology , Protein Serine-Threonine Kinases/drug effects , Protein Serine-Threonine Kinases/physiology , Stilbenes/pharmacology , Animals , Blotting, Western , Cell Nucleus/drug effects , Cells, Cultured , Coloring Agents , Genes, Reporter/drug effects , Inflammation/chemically induced , Inflammation/prevention & control , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Male , Mice , Mice, Inbred C57BL , Nitric Oxide/metabolism , Plasmids/drug effects , Plasmids/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Resveratrol , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Tetrazolium Salts , Thiazoles
16.
Pharmazie ; 66(1): 58-62, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21391436

ABSTRACT

Cordyceps species have been known since long as a multi-utility ethnomedicinal herbal in Korea, China and Japan. It has been reported to exhibit a number of properties such as anti-oxidative, anti-cancer, antiinflammatory, anti-diabetic, and anti-obesity effects. In a previously conducted study, we had demonstrated that the ethanol extract of Cordyceps bassiana was able to suppress the production of interleukin (IL)-12 and interferon (IFN)-gamma in macrophages and T lymphocytes. In this study, we were able to further explore the molecular basis of its inhibitory mechanism using a butanol fraction of this herbal (Cb-BF) preparation. Similarly, this fraction also blocked the expression of cytokines such as IL-12 and tumor necrosis factor (TNF)-alpha as well as the proliferation of splenic lymphocytes and their production of IFN-gamma but not IL-4. Cb-BF suppressed the luciferase activities that are mediated by nuclear factor (NF)-kappaB, activator protein (AP)-1, and signal transducers and activators of transcription (STAT)-1. In agreement with this, these fractions diminished the translocation of the transcription factors into the nucleus. The study also demonstrated that the upstream signaling events for the activation of these factors such as spleen tyrosine kinase (Syk), janus kinase (JAK)-2, and extracellular signal-regulated kinase (ERK) were suppressed. Therefore, these results suggest that the butanol extract of Cordyceps bassiana may contain more than one active component capable of inhibiting the inflammatory signaling cascade and this can be considered as a potential candidate for treatment of diseases that require suppression of immune system.


Subject(s)
Cordyceps/chemistry , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Animals , Blotting, Western , Butanols , Coloring Agents , Genes, Reporter/drug effects , Humans , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-4/biosynthesis , Lipopolysaccharides/pharmacology , Lymphocytes/drug effects , Lymphocytes/immunology , Macrophages/drug effects , Macrophages/immunology , Mice , Plant Extracts/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Signal Transduction/drug effects , Solvents , Spleen/cytology , Spleen/drug effects , Tetrazolium Salts , Thiazoles , Transcription Factors , Tumor Necrosis Factor-alpha/biosynthesis , p38 Mitogen-Activated Protein Kinases/biosynthesis
17.
Acta Pharmacol Sin ; 32(3): 288-94, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21372823

ABSTRACT

AIM: To examine the role of protein L-isoaspartyl O-methyltransferase (PIMT; EC 2.1.1.77) on the secretion of Aß peptides. METHODS: HEK293 APPsw cells were treated with PIMT siRNA or adenosine dialdehyde (AdOX), a broad-spectrum methyltransferase inhibitor. Under the conditions, the level of Aß secretion and regulatory mechanism by PIMT were examined. RESULTS: Knock-down of PIMT and treatment with AdOX significantly increased Aß(40) secretion. Reductions in levels of PIMT decreased the secretion of soluble amyloid precursor protein alpha (sAPPα) without altering the total expression of APP or its membrane-bound C83 fragment. However, the levels of the C99 fragment generated by ß-secretase were enhanced. Moreover, the decreased secretion of sAPPα resulting from PIMT knock-down seemed to be linked with the suppression of the expression of α-secretase gene products, α-disintegrin and metalloprotease 10 (ADAM10) and ADAM17, as indicated by Western blot analysis. In contrast, ADAM10 was not down-regulated in response to treatment with the protein arginine methyltransferase (PRMT) inhibitor, AMI-1. CONCLUSION: This study demonstrates a novel role for PIMT, but not PRMT, as a negative regulator of Aß peptide formation and a potential protective factor in the pathogenesis of AD.


Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Membrane Proteins/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , ADAM10 Protein , ADAM17 Protein , Adenosine/analogs & derivatives , Adenosine/pharmacology , Alzheimer Disease/metabolism , HEK293 Cells , Humans , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , RNA Interference , RNA, Small Interfering , Transfection
18.
Front Biosci (Landmark Ed) ; 16(2): 517-30, 2011 01 01.
Article in English | MEDLINE | ID: mdl-21196185

ABSTRACT

Akt (protein kinase B) is a serine/threonine protein kinase that regulates cell metabolism, survival and proliferation. Recent studies of the role of Akt in phagocytosis, intracellular bacterial infections, LPS tolerance, production of inflammatory cytokines and mediators, and migration during macrophage-mediated innate immunity strongly suggest a pivotal role for this enzyme in the functional activation of macrophages. Considering that a variety of inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, diabetes, obesity, cancer and osteoporosis, are regulated by macrophage-mediated innate immunity, efforts should be more carefully focused on understanding the function of Akt in macrophage-mediated innate immunity. Although few studies have addressed this question, this review discusses recent findings that support an important role for Akt in macrophage-mediated innate immunity and underlines the need for trials to develop pharmaceutically useful drugs that target Akt for treatment of macrophage-mediated inflammatory diseases. The findings we review here suggest that a novel and safe Akt inhibitor with strong immunosuppressive and anti-inflammatory properties will be applied to various chronic inflammatory diseases in the near future.


Subject(s)
Immunity, Innate , Macrophages/immunology , Proto-Oncogene Proteins c-akt/immunology , Animals , Arthritis, Rheumatoid/drug therapy , Cell Movement , Chemotaxis/physiology , Gene Expression Regulation , Inflammation/drug therapy , Inflammation Mediators/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Phagocytosis/physiology , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/deficiency , Shock, Septic/drug therapy
19.
Immunopharmacol Immunotoxicol ; 33(1): 90-6, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20476843

ABSTRACT

Cordyceps species have been known as ethnopharmacologically valuable mushroom in Korea, China, and Japan. This plant has been reported to exhibit a variety of pharmacological activities such as antioxidative, anticancer, anti-inflammatory, antidiabetic, and antiobesity effects. Although numerous pharmacological potentials of Cordyceps spp. have been demonstrated, immunomodulatory effect of Cordyceps bassiana has not been published yet. To evaluate its immunomodulatory activity, macrophages activated by lipopolysaccharide (LPS) were employed and the production of interleukin-12 (IL-12) was explored in terms of understanding its molecular inhibitory mechanism. Seventy percent of ethanol extract from Cordyceps bassiana (Cb-EE) was able to suppress the expression of IL-12, a cytokine regulating interferon-γ (IFN-γ)-producing T helper type 1 (Th1) polarization response, at the transcriptional levels. The inhibitory effect of Cb-EE seemed to be due to activator protein-1 (AP-1) translocation inhibition, according to immunoblotting analysis with nuclear fraction and luciferase assay. In agreement with this, Cb-EE strongly suppressed the phosphorylation of p38, a prime signal to stimulate AP-1 translocation and IL-12 production, strongly suppressed by SB203580, a p38 inhibitor. Furthermore, this extract also suppressed IFN-γ production in both phytohemaglutinin A and LPS-activated splenocytes. Our results suggest that Cb-EE can be applied as a Th1 response regulatory herbal medicine.


Subject(s)
Cordyceps/chemistry , Immunologic Factors/pharmacology , Interleukin-12/biosynthesis , Macrophage Activation/drug effects , Macrophages/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/immunology , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/physiology , Ethanol , HEK293 Cells , Humans , Immunoblotting , Immunologic Factors/isolation & purification , Interferon-gamma/biosynthesis , Interleukin-12/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Luciferases/genetics , Macrophage Activation/immunology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/genetics , NF-kappa B/physiology , Reverse Transcriptase Polymerase Chain Reaction , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Transcription Factor AP-1/genetics , Transcription Factor AP-1/physiology , Transfection
20.
J Ethnopharmacol ; 134(2): 504-9, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21184821

ABSTRACT

ETHNOPHARMACOLOGICAL SIGNIFICANCE: The Cordyceps species are insect-borne mushrooms that have been ethnopharmacologically used for skin diseases such as eczema and dermatitis. AIM OF THE STUDY: In this study, we investigated the curative effects of the butanol fraction (CBBF) of Cordyceps bassiana on atopic dermatitis. MATERIALS AND METHODS: Dermatitis was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice. After a topical application of CBBF on the skin lesions, the dermatitis score, epidermal thickness, mast cell number, and interleukin (IL)-4 and interferon (IFN)-γ, as well as the levels of histamine and immunoglobulin E (IgE) in the serum, were measured. Moreover, effect of CBBF on histamine release was examined using RBL-2H3 under stimulation with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). RESULTS: CBBF inhibited atopic dermatitis symptoms and signs in the DNFB-treated NC/Nga mice. The suppressive activity of topically applied CBBF may be due to the dose-dependent blockade of a series of immunopathological events, including the release of histamine, the production of IgE, and the secretion of IL-4 and IFN-γ. However, this extract did not directly suppress the degranulation process, assessed by measuring ß-hexosaminidase release. CONCLUSIONS: Our results suggest that CBBF can be applied as an effective herbal remedy to treat atopic dermatitis.


Subject(s)
Biological Products/therapeutic use , Cordyceps , Dermatitis, Atopic/drug therapy , Immunologic Factors/blood , Phytotherapy , Skin/drug effects , Administration, Topical , Animals , Biological Products/pharmacology , Cattle , Cell Degranulation/drug effects , Cordyceps/chemistry , Dermatitis, Atopic/immunology , Dermatitis, Atopic/pathology , Dinitrofluorobenzene , Dose-Response Relationship, Drug , Epidermis/drug effects , Epidermis/pathology , Histamine/metabolism , Immunoglobulin E/biosynthesis , Interferon-gamma/metabolism , Interleukin-4/metabolism , Male , Mast Cells/drug effects , Mast Cells/metabolism , Mice , Mice, Inbred Strains , Serum Albumin , Skin/immunology , Skin/pathology , beta-N-Acetylhexosaminidases/blood
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