NF-kappaB DNA Binding Activity Research Articles

Dark chocolate consumption buffers intracellular pro-inflammatory reactivity to acute psychosocial stress in healthy men

Flavanoid-rich dark chocolate consumption has beneficial effects on cardiovascular health, but underlying mechanisms are elusive. We investigated the effect of acute dark chocolate consumption on inflammatory measures before and after stress. Healthy men aged 20–50 years were randomly assigned to a single intake of either 50 g of flavonoid-rich dark chocolate (n = 31) or 50 g of optical identical flavonoid-free placebo chocolate (n = 34). Two hours after chocolate intake, both groups underwent the Trier Social Stress-Test. We measured DNA binding activity of the pro-inflammatory transcription factor NF-kappaB (NF-kappaB-BA) in peripheral blood mononuclear cells, and plasma and whole blood mRNA levels of the pro-inflammatory cytokines IL-1beta and IL-6, and the anti-inflammatory cytokine IL-10 prior to chocolate intake, before and several times after stress cessation. We also repeatedly measured the flavonoid epicatechin. Compared to the placebo chocolate group, the dark chocolate group revealed a marginal increase in IL-10 mRNA prior to stress (p = .065), and a significantly blunted stress reactivity of NF-kappaB-BA, IL-1beta mRNA, and IL-6 mRNA ( p ′ s . 037 ) with higher epicatechin levels relating to lower pro-inflammatory stress reactivity ( p ′ s . 034 ). None of the other measures showed a significant chocolate effect ( p ′ s > . 18 ). Our findings indicate that acute flavonoid-rich dark chocolate exerts anti-inflammatory effects both by increasing mRNA expression of the anti-inflammatory cytokine IL-10 and by attenuating the intracellular pro-inflammatory stress response. This mechanism may add to beneficial effects of dark chocolate on cardiovascular health.

A Novel Synthetic Compound, YH-1118, Inhibited LPS-Induced Inflammatory Response by Suppressing I��B Kinase/NF-��B Pathway in Raw 264.7 Cells

For the search of a potent first-in-class compound to inactivate macrophages responsible for inflammatory responses, in the present study, we investigated the anti-inflammatory effects of YH-1118, a novel synthetic compound, in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, Raw 264.7. YH-1118 inhibited LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression at both the protein and mRNA levels. The suppression of LPS-induced iNOS expression by YH-1118 was mediated via nuclear factor kappa B (NF-κB), but not activator protein-1 (AP-1) transcription factor. This was supported by the finding that YH-1118 attenuated the phosphorylation of inhibitor of κBα (IκBα) and nuclear translocation and DNA binding activity of NF-kappaB. Through the mechanisms that YH- 1118 inhibited the activation of IkappaB kinases (IKKs), upstream activators of NF-κB, or p38 MAPK, YH-1118 significantly suppressed LPS-induced production of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 (p < 0.05). In conclusion, our results suggest that YH-1118 inhibits LPS-induced inflammatory responses by blocking IKK and NF-κB activation in macrophages, and may be a therapeutic candidate for the treatment of various inflammatory diseases.

SAGE components enhance cell death through nuclear factor kappa-B signaling

The sage components linalyl acetate (Ly) and alpha-terpineol (Te) exhibit synergistic anti-proliferative effects. We investigated the effects of Ly and Te on NF-kappaB signaling in HCT-116 colon cancer cells. Ly and Te combinations dose-dependently reduced HCT-116 viability at non-cytotoxic concentrations. Combination treatment induced 30%-60% increase in PreG1 through induction of apoptosis and necrosis. DNA binding assays revealed that combination treatment suppressed both basal and TNF-alpha-induced NF-kappaB activation. This suppression correlated with the inhibition of p65 nuclear translocation and IkappaB-alpha degradation. The lack of change in IKK expression levels or inhibition in IkappaB-alpha phosphorylation suggest the involvement of an IKK-independent mechanism. Ly and Te combination was found to downregulate the expression of NF-kappaB-regulated antiapoptotic and proliferative gene products. Separate treatments and drug combinations significantly decreased DNA binding activity of NF-kappaB which led to the potentiation of cell death induced by the colon cancer drugs oxaliplatin and 5-FU. These results indicate that Ly and Te anticancer activities are partly mediated through the suppression of NF-kappaB activation, suggesting their use in combination with chemotherapeutic agents to induce apoptosis.

Attenuation of pain and inflammation in adjuvant‐induced arthritis by the proteasome inhibitor MG132

In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-kappaB, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-kappaB DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-kappaB subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry. Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-kappaB/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-kappaB activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

Hepatitis C virus core protein induces malignant transformation of biliary epithelial cells by activating nuclear factor‐κB pathway

In an earlier study, we found that hepatitis C virus core protein, HCV-C, participated in the malignant transformation of HCV-C transfected normal human biliary epithelial (hBE) cells by activating telomerase. Here we further investigated the signaling of the malignant transformation. Reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunoprecipitation were used to analyze the expression of HCV-C, human telomerase reverse transcriptase (hTERT), nuclear factor-kappaB (NF-kappaB) and NF-kappaB inhibitor alpha (IkappaBalpha) genes and the phosphorylation level of IkappaBalpha protein. Electrophoretic mobility shift assays (EMSA) and NF-kappaB-linked luciferase reporter assays were carried out to measure NF-kappaB activity. The expression of HCV-C and hTERT was detected only in HCV-C-transfected hBE (hBE-HCV-C) cells but not in vector-transfected or parental hBE cells. More NF-kappaB protein accumulated in nuclear extracts of hBE-HCV-C cells rather than in those of control cells, though total NF-kappaB protein level showed no difference among these cells. DNA binding activity of NF-kappaB and the NF-kappaB-linked luciferase activity were much higher in HCV-C-transfected hBE cells than those in vector- or non-transfected hBE cells. In addition, the IkappaBalpha phosphorylation level, but not the IkappaBalpha mRNA or protein levels, was increased after HCV-C transfection. Hepatitis C virus core protein activates NF-kappaB pathway in hBE cells by increasing the phosphorylation of IkappaBalpha. The pathway may be responsible for HCV-C-induced malignant transformation of hBE cells.

Acute stress enhances contact dermatitis by promoting nuclear factor‐κB DNA‐binding activity and interleukin‐18 expression in mice

Psychological stress adversely affects the immune system, and aggravates various skin diseases, such as psoriasis, alopecia areata and atopic dermatitis. However, the precise underlying mechanisms remain to be elucidated. The goal of this study was to use a murine restraint stress model to determine the mechanisms by which psychological stress modulates immune response in contact dermatitis. In the present study, mice were sensitized and challenged on the skin with 2,4-dinitrofluorobenzene. Acute restraint stress was administrated to healthy or sensitized mice before challenge, and nuclear factor (NF)-kappaB DNA-binding activation of nuclear protein and expression of interleukin (IL)-18 mRNA in murine spleen lymphocytes was detected. Chemical sympathectomy was performed using the neurotoxin 6-hydroxy-dopamine to determine the effect of the sympathetic nervous system. The experiment showed that restraint stress induced a series of changes which include increasing of NF-kappaB DNA-binding activity and IL-18 mRNA expression in spleen lymphocytes and enhancement of contact hypersensitivity response, and these changes may be mediated by the sympathetic nervous system. These findings provide new insights into the roles of the nervous system in the aggravation of skin diseases.

Anti–citrullinated protein antibodies bind surface‐expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

Anti-citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA. ACPAs were purified from pooled ACPA-positive RA sera by cyclic citrullinated peptide-conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor alpha (TNFalpha) production and NF-kappaB DNA binding activity of the cells were measured by enzyme-linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis. ACPAs specifically enhanced TNFalpha production and increased the DNA-binding activity of NF-kappaB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface-expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S-transferase-tagged recombinant Grp78 (97.52 kd) became a 72-kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFalpha production. We clearly demonstrated that ACPAs enhance NF-kappaB activity and TNFalpha production in monocyte/macrophages via binding to surface-expressed citrullinated Grp78.

Abstract LB-168: Genistein sensitizes radio-resistant cancer cells through regulation of NF-kappaB

Abstract Background: NF-kappaB is a key transcription factor involved in the control of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. However, NF-kappaB is persistently active in a number of disease states, including cancer. It has been reported that chemo- or radio- resistant cancer cells had elevated NF-kappaB activity. Recent studies including ours have showed that genistein, a soy-derived isoflavonoid, has antitumor activity in various cancer cells. Objective: In this study, we evaluated whether genistein could sensitize ionizing radiation resistance of cervical cancer cells through regulation of NF-kappaB. Methods: Six cervical cancer cell lines (HeLa, CaSki, C33A, ME180, SiHa, HeLa229) were treated by 20 Gy radiation with 50 M genistein or not. Cell growth rate was measured by MTT assay and protein expression level was analyzed by western blotting. Protein localization was observed by confocal microscopic analysis. NF-kappaB transcriptional activity was measured by luciferase reporter gene assay. NF-kappaB DNA binding activity was evaluated by electophoretic mobility shift assay. Results: Among six cervical cancer cells, HeLa and HeLa229 cells showed radio-resistance accompanied by elevated NF-kappaB activity. Ionizing radiation induced NF-kappaB nuclear transclocation and stimulated NF-kappaB DNA binding and transcriptional activities. However, pretreatment with genistein inhibited radiation induced transcriptional activity coincident to decreased radio-resistancy. Genistein also inhibited radiation-induced NF-kappaB regulated anti-apoptotic gene expression including c-myc, Survivin, XIAP, Bcl-2, IAP-1, TRAF, A1/Bfl-1 and FLIP. Conclusion: Taken together, genistein can sensitize radio-resistant cancer cells through regulation of NF-kappaB and could be used as a radio-sensitizer for the treatment of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-168.

Abstract 550: Transcriptional regulation of the Fanconi Anemia/BRCA DNA repair pathway component FANCD2 following co-culture of bone marrow stroma with multiple myeloma cell lines

Abstract MM is an incurable B cell malignancy characterized by the acquisition of drug resistance. To this end, we have recently demonstrated that selected resistance to the melphalan correlated with a RelB/p50 mediated transactivation of FANCD2 in the 8226 LR5 MM cell line.(Oliveira et al. Cancer Res 2009) These results demonstrated a causal relationship between RelB/p50 transactivation of FANCD2, increased DNA repair, and acquired resistance to melphalan. Although numerous genetic alterations have been implicated in acquired MDR in MM, the bone marrow (BM) microenvironment has also been demonstrated to contribute to drug resistance-or environment-mediated drug resistance (EM-DR). Therefore, utilizing the HS-5 bone marrow stromal (BMS) cell line as a model of the EM-DR, we wished determine if co-culture with RPMI 8226 or 8226/LR5 affected expression of FA/BRCA DNA repair pathway components. Examination of drug sensitive, drug resistant MM, and stromal cell lines demonstrated differential enhancement of FA/BRCA DNA repair pathway protein expression. These preliminary results demonstrate that expression of FA/BRCA pathway components is regulated by cellular interactions in both MM cells and BMSC suggesting that juxtaposed bone marrow stroma influences FA/BRCA-mediated DNA repair and drug resistance. We previously demonstrated RelB/p50 specific transcriptional regulation of FANCD2 in drug selected MM cells. Here, we demonstrate that co-culture of MM cell lines with BMSCs facilitates NF-kappaB DNA binding activity (0-6hr), suggesting a similar RelB/p50 mediated- transactivation of FANCD2. To further examine this we have stably transfected HS-5 cells with luciferase reporter constructs under the control of the FANCD2 promoter. Following co-culture with RPMI 8226 or 8226 LR5 increased FANCD2 promoter activity was noted at early time points (0.5-6hours), but was diminished by 24 hours. FANCD2 promoter activity correlates with protein expression in HS-5 cells incubated with either cell line (0-6hrs). However, in HS-5 cells co-cultured with RPMI 8226 cells FANCD2 protein levels were maintained even after promoter activity had subsided. These data illustrate a novel transcriptional control of FANCD2 in under co-culture conditions. Moreover, these findings suggest that the FA/BRCA mediated DNA damage repair may be an important component of EM-DR. However, continued investigation will be needed to determine the breadth of transcriptional (and/or alternate) regulation of FAND2 and other members of the FA family. Further, future studies will examine a causal relationship between increased FANC expression and melphalan (and other drug) resistance seen in co-culture conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 550.

CK2 Modulation of NF-κB, TP53, and the Malignant Phenotype in Head and Neck Cancer by Anti-CK2 OligonucleotidesIn vitroorIn vivovia Sub–50-nm Nanocapsules

The aim of this study is to investigate the expression of CK2 subunits and CK2 effects on NF-kappaB-mediated and TP53-mediated signal activation and gene expression, the malignant phenotype, and chemosensitivity in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Protein expression of CK2 subunits was investigated by Western blot and immunohistochemistry. CK2 subunits were knocked down by small interfering RNA, and NF-kappaB activation was examined using DNA binding, Western blot, and luciferase reporter assays. Gene expression was measured by quantitative reverse transcription-PCR. Cell growth, survival, motility, and sensitivity to cisplatin were measured by MTT, flow cytometry, and migration assays. In vivo targeting of CK2alpha/alpha' in HNSCC xenograft models was achieved using anti-CK2alpha/alpha' oligodeoxynucleotide encapsulated in sub-50-nm tenfibgen nanocapsules. CK2 subunit proteins were overexpressed in HNSCC lines and tissues. Knockdown of CK2 subunits differentially inhibited IkappaBalpha degradation, NF-kappaB nuclear localization, phosphorylation, DNA binding, and reporter activity. CK2 subunits modulated gene expression and the malignant phenotype involved in cell cycle and migration, whereas CK2alpha is critical to promote proliferation, antiapoptosis, and cisplatin resistance in vitro. Furthermore, in vivo delivery of anti-CK2alpha/alpha' oligodeoxynucleotide nanocapsules significantly suppressed tumor growth in HNSCC xenograft models, in association with modulation of CK2 and NF-kappaB regulated molecules, TP53 family proteins, and induction of apoptosis. Our study reveals a novel role of CK2 in coregulating NF-kappaB activation, TP53/p63 expression, and downstream gene expression. Downregulation of CK2 in HNSCC models in vitro and in vivo shows antitumor effects as well as sensitization to cisplatin.

Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury

Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI). C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-kappaB (NF-kappaB) DNA binding activity were also evaluated. TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-kappaB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4. TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.

Critical Need for Clinical Trials: An Example of a Pilot Human Intervention Trial of a Mixture of Natural Agents Protecting Lymphocytes Against TNF-α Induced Activation of NF-κB

Humans typically consume "natural agents" that are believed to be chemoprotective and are known to decrease inflammation biomarker NF-kappaB in vitro; however, no intervention studies in humans have been done to date. This commentary documents the in vivo results as a powerful example for supporting the superiority of a complex mixture of natural agents. Human volunteers consumed two 500 mg capsules (BID) containing a mixture of natural agents for a period of 2 weeks, and blood samples were collected pre- and post-intervention. The purified lymphocytes were subjected to ex-vivo exposure to TNF-alpha or kept as untreated control. The mean NF-kappaB DNA binding activity was increased upon TNF-alpha treatment in pre-intervention samples; however, TNF-alpha was unable to induce NF-kappaB in post-intervention samples, suggesting that the mixture of four important natural agents could be useful to protect humans against oxidative stress.

Experimental study of the function and mechanism combining dihydroartemisinin and gemcitabine in treating pancreatic cancer

To investigate the anti-tumor activity of combined gemcitabine with dihydroartemisinin, and the mechanism of the anti-tumor effect of gemcitabine enhanced by dihydroartemisinin on pancreatic cancer. For cultured cells, cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis and confocal laser scanning microscope stained with Annexin V-FITC/PI. The nuclear extract for determining NF-kappaB DNA-binding activity was analyzed by EMSA, while nuclear P65 and its downstream gene expression was determined by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to agents. TUNEL assay was used to assess tumor cell apoptosis in tumor tissue. After combination of gemcitabine and dihydroartemisinin treatment, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and Panc-1 reached up to (81.1 +/- 3.9)% and (76.5 +/- 3.3)%, and the apoptosis rates were up to (53.6 +/- 3.8)% and (48.3 +/- 4.3)%, the differences were significantly (P < 0.01) compared with gemcitabine [(24.8 +/- 2.9)% and (21.8 +/- 3.5)%]. All the treatment groups inhibited the growth of pancreatic xenograft tumors in nude mice. The tumor volume and apoptosis index were (262 +/- 37) mm(3) and (50 +/- 4)% respectively in the combined treatment, compared to those of [(384 +/- 56) mm(3) and (25 +/- 3)%] in gemcitabine, the differences were significantly (P < 0.05). EMSA showed that gemcitabine alone obviously enhanced its DNA-binding activity compared to control. However, dihydroartemisinin significantly reduced its DNA-binding activity, so that abrogated the inducing effect of gemcitabine on NF-kappaB activation. Western blot assay indicated that dihydroartemisinin downregulated expression of nuclear P65, and combined treatment not only downregulated the expression of Cyclin D1, Bcl-xL and Bcl-2 while upregulated Bax, thus reduced the Bcl-2/Bax ratio, but also increased the caspase-3 activation, all of which increased apoptosis in both BxPC-3 and Panc-1 cells. Dihydroartemisinin significantly abrogated the inducing effect of gemcitabine on NF-kappaB activation and downregulated the expression of NF-kappaB targeted gene products, which may be one possible mechanism by which dihydroartemisinin augments the anti-tumor effect of gemcitabine on pancreatic cancer.

Chemical Biology Strategy Reveals Pathway-Selective Inhibitor of NF-κB Activation Induced by Protein Kinase C

Dysregulation of NF-kappaB activity contributes to many autoimmune and inflammatory diseases. At least nine pathways for NF-kappaB activation have been identified, most of which converge on the IkappaB kinases (IKKs). Although IKKs represent logical targets for potential drug discovery, chemical inhibitors of IKKs suppress all known NF-kappaB activation pathways and thus lack the selectivity required for safe use. A unique NF-kappaB activation pathway is initiated by protein kinase C (PKC) that is stimulated by antigen receptors and many growth factor receptors. Using a cell-based high-throughput screening (HTS) assay and chemical biology strategy, we identified a 2-aminobenzimidazole compound, CID-2858522, which selectively inhibits the NF-kappaB pathway induced by PKC, operating downstream of PKC but upstream of IKKbeta, without inhibiting other NF-kappaB activation pathways. In human B cells stimulated through surface immunoglobulin, CID-2858522 inhibited NF-kappaB DNA-binding activity and expression of endogenous NF-kappaB-dependent target gene, TRAF1. Altogether, as a selective chemical inhibitor of the NF-kappaB pathway induced by PKC, CID-2858522 serves as a powerful research tool and may reveal new paths toward therapeutically useful NF-kappaB inhibitors.

Cilostazol is anti‐inflammatory in BV2 microglial cells by inactivating nuclear factor‐kappaB and inhibiting mitogen‐activated protein kinases

Cilostazol is a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti-inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti-inflammatory effect of cilostazol on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs). Cilostazol suppressed production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines, interleukin-1 (IL-1), tumour necrosis factor-alpha, and monocyte chemoattractant protein-1 (MCP-1), in a concentration-dependent manner. Inhibitory effects of cilostazol were not affected by treatment with an adenylate cyclase inhibitor, SQ 22536, indicating that these actions of cilostazol were cAMP-independent. Cilostazol significantly inhibited the DNA binding and transcriptional activity of NF-kappaB. Moreover, cilostazol blocked signalling upstream of NF-kappaB activation by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but without affecting the activity of p38 MAPK. Our results demonstrate that suppression of the NF-kappaB, ERK, JNK signalling pathways may inhibit LPS-induced NO and PGE(2) production. Therefore, cilostazol may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators and cytokine production in activated microglia.

Suppression of the NF-kB signalling pathway by ergolide, sesquiterpene lactone, in HeLa cells

We have previously reported that ergolide, a sesquiterpene lactone isolated from Inula britannica, suppresses inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression by inhibiting nuclear factor-kappaB (NF-kappaB) in RAW 264.7 macrophages. In this study, we show that ergolide suppresses the DNA binding activity of NF-kappaB and nuclear translocation of NF-kappaB p65 subunit, leading to the inhibition of NF-kappaB-dependent gene transcription in 12-O-tetradecanoylphorbol 13acetate (TPA)-stimulated HeLa cells. We also show that ergolide decreases the degradation and phosphorylation of IkappaB, an inhibitory protein of NF-kappaB, and this effect is accompanied by a simultaneous reduction of IkappaB kinase (IKK) activity. However, ergolide does not inhibit in-vitro IKK activity directly, suggesting the possible involvement of upstream IKK kinases in the regulation of NF-kappaB activation. Furthermore, ergolide-mediated protein kinase Calpha (PKCalpha) inhibition is involved in reduction of NF-kappaB inhibition, as demonstrated by the observation that dominant negative PKCalpha, but not p44/42 MAPK and p38 MAPK, inhibits TPA-stimulated reporter gene expression. Taken together, our results suggest that ergolide suppresses NF-kappaB activation through the inhibition of PKCalpha-IKK activity, providing insight for PKCalpha as a molecular target for anti-inflammatory drugs.

Transcriptional upregulation of nitric oxide synthase II by nuclear factor‐κB promotes apoptotic neuronal cell death in the hippocampus following experimental status epilepticus

Whereas status epilepticus, or the condition of continuous epileptic seizures, produces a characteristic pattern of preferential neuronal cell loss in the hippocampus, the underlying mechanism is still unsettled. Based on an experimental model of temporal lobe status epilepticus, we demonstrated previously that prolonged seizures prompted an overproduction of nitric oxide (NO) by upregulation of NO synthase II (NOS II) in the hippocampal CA3 subfield, followed by the activation of mitochondrial apoptotic signaling cascade. Using the same animal model, the present study evaluated the hypothesis that transcriptional upregulation of NOS II gene by nuclear factor-kappaB (NF-kappaB) promotes apoptotic neuronal cell death in the hippocampus following status epilepticus. In Sprague-Dawley rats, significantly augmented nucleus-bound translocation of NF-kappaB p50 and p65 subunits and DNA binding activity of NF-kappaB were observed in hippocampal CA3 neurons as early as 30 min after elicitation of sustained seizure activity by microinjection of kainic acid into the CA3 subfield, followed by a progressive elevation that peaked at 90 min. In addition, application bilaterally into the hippocampal CA3 subfield of a selective NF-kappaB inhibitor, pyrrolidine dithiocarbamate or double-stranded kappaB decoy DNA significantly antagonized the activated NOS II-peroxynitrite signaling cascade (3 hr) and the associated manifestations of apoptotic cell death (7 days) in the hippocampus. We conclude that activation of NF-kappaB in hippocampal CA3 neurons upregulates NOS II gene expression following experimental temporal lobe status epilepticus, leading to apoptotic neuronal cell death in the hippocampus.

10-Hydroxy-2-decenoic acid from Royal jelly: A potential medicine for RA

Rheumatoid arthritis synovial fibroblasts (RASFs) are known to produce matrix metalloproteinases (MMPs) and cause joint destruction. The purpose of this study is to develop a potential medicine for rheumatoid arthritis (RA). To this end, first, the MMPs inhibition factor was purified from an alkali-solubilized fraction of RJ (Apis mellifera) by C18 reverse-phase column chromatography and identified as 10-hydroxy-2-decenoic acid (10H2DA) by LTQ XL analysis. Next, Experimental test 10H2DA how to inhibited the activities of MMPs: with RASFs isolated from rheumatoid tissues by enzymatic digestion, cultures in monolayers were treated with 10H2DA (0.5mM, 1mM, and 2mM) or PBS for 2h followed by stimulation with TNF-alpha (10 ng/ml) for 2h, mRNA. Protein levels of MMP-1 and MMP-3 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), the DNA-binding activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) by electrophoretic mobility shift assay (EMSA), and the protein kinase activity of p38, ERK and JNK by kinase assay. The molecular investigation revealed that the 10H2DA-mediated suppression was likely to occur through blocking p38 kinase and c-Jun N-terminal kinase-AP-1 signaling pathways. In contrast, 10H2DA had no effect on extracellular signal-regulated kinase activity, NF-kappaB DNA-binding activity and IkappaBalpha degradation. These results suggest that 10H2DA may be of potential therapeutic value in inhibiting joint destruction in RA.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Preventive Effect of a Proteasome Inhibitor on the Formation of Accelerated Atherosclerosis in Rabbits With Uremia

Inflammation plays a central role in the pathogenesis of atherosclerosis. This study investigated whether the proteasome inhibitor has the same preventive effect on the formation of accelerated atherosclerosis in rabbits with uremia compared with a NF-kappaB inhibitor. New Zealand white rabbits were subjected to five-sixths nephrectomy (chronic renal failure [CRF]) or to a sham operation. Rats in each group were randomly assigned into three subgroups (n = 24 in each group) and treated with repeated intramuscular injections of proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC for a specified period. Compared with sham rabbits, CRF rabbits displayed typical atherosclerotic changes (endothelial cell damage, intimal thickens, and appearance of foam cells). CRF rabbits had significantly higher levels of proteasome activity, NF-kappaB mRNA, protein, and DNA binding activity as well as tumor necrosis factor-a and proliferative cell nuclear antigen protein expression in aortic wall cells. CRF rabbits also showed lower levels of IkappaBalpha. Compared with CRF rabbits, CRF rabbits treatment with proteasome inhibitor MG132 showed restoration of IkappaBalpha mRNA and protein expression and decreased NF-kappaB DNA binding activity and tumor necrosis factor-a expression. Treatment with either proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC could reverse these pathologic changes in the aortic wall cells of CRF rabbits. A comparison between the inhibitory effects of the two treatments revealed no statistical difference. These results suggest that ubiquitin-proteasome activation play a pivotal role in the pathogenesis of uremia-accelerated atherosclerosis. The ubiquitin-proteasome signaling pathway in aortic cells may therefore be an important target for preventing uremia-accelerated atherosclerosis.